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Yaml protocol improvements
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Data/untitled.md
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---
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name: BCA Assay for Protein Abundance Determination
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id: PROT-0010
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version: 1.0
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description: Protocol for colorimetric detection and quantitation of total protein using bicinchoninic acid (BCA)
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author: Jordan Lab
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created: 2025-05-06
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materials:
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- BCA Reagent A
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- BCA Reagent B
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- BSA standards
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- Microplate
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- Plate reader (562 nm capability)
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- Pipettes and tips
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steps:
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- "Determine number of standards and unknowns to quantify"
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- "Prepare working reagent (WR) by mixing BCA reagent A and B in 50:1 ratio"
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- "Pipette 10 µL of each standard/sample into microplate wells"
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- "Add 200 µL of WR to each well and mix for 30 seconds"
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- "Incubate at 37°C for 30 minutes"
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- "Measure absorbance at 562 nm"
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- "Create standard curve and determine protein concentrations"
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notes: |
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Based on Pierce BCA Protein Assay Kit (Thermo Scientific)
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Assay is nearly linear with protein concentrations from 20-2000 µg/mL
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For increased sensitivity, incubation time can be extended to 2 hours
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---
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#Protocol
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1. Determine the number of standards and unknowns you need to quantify:
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2. (Number of standards + Number of unknowns) x (Number of replicates) x (Volume of WR per sample) = Total WR volume
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3. Mix 50 parts of BCA reagent A with 1 part of BCA reagent B (50:1 ratio, Reagent A:B).
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4. Example: Combine 5 mL of reagent A with 0.1 mL of reagent B to prepare 5.1 mL of WR.
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5. Note: Initial turbidity when reagent B is added to reagent A will disappear with mixing, yielding a clear, green WR.
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6. Pipette 10 µL of each standard or unknown sample replicates into a microplate well.
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7. Add 200 µL of the WR to each well and mix the plate thoroughly for 30 seconds.
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8. Incubate at 37°C for 30 minutes.
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9. Measure the absorbance at 562 nm on (core facility) plate reader.
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10. Subtract the average 562 nm absorbance measurement of the blank standard replicates from the 562 nm measurements of all other individual standard and unknown sample replicates.
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11. Prepare a standard curve by plotting the average blank–corrected 562 nm measurement for each BSA standard vs. its concentration in µg/mL. Use the standard curve to determine the protein concentration of each unknown sample.
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**
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**
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Standard Preparation
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Prepare standards in the using the same buffer you collected your protein samples in as the diluent.
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|Vial|Volume of Diluent|Volume and Source of BSA|Final BSA Concentration (µg/mL)|
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|---|---|---|---|
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|A|0|300 µL of stock|2,000|
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|B|125 µL|375 µL of stock|1,500|
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|C|325 µL|325 µL of stock|1,000|
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|D|175 µL|175 µL of vial B dilution|750|
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|E|325 µL|325 µL of vial C dilution|500|
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|F|325 µL|325 µL of vial E dilution|250|
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|G|325 µL|325 µL of vial F dilution|125|
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|H|400 µL|100 µL of vial G dilution|25|
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|I|400 µL|0|0 (Blank)|
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**
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Pierce BCA Protein Assay Kit (Thermo Scientific ) Protocol
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Background:
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- The kit uses bicinchoninic acid (BCA) for colorimetric detection and quantitation of total protein.
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- It's based on the biuret reaction, where Cu+2 is reduced to Cu+1 by protein in an alkaline medium.
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- The cuprous cation (Cu+1) is detected using BCA, forming a purple-colored complex that absorbs at 562 nm.
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- The assay is nearly linear with protein concentrations from 20–2000 µg/mL.
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- Protein concentrations are usually determined against a standard protein like bovine serum albumin (BSA).
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- 
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**
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****
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Note:
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- Wavelengths from 540–590 nm have been used successfully with this method.
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- Plate readers, which use a shorter light path length than cuvette spectrophotometers, require a greater sample to WR ratio to obtain the same sensitivity as the standard test tube procedure. If higher 562 nm measurements are desired, increase the incubation time to 2 hours.
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- Increasing the incubation time or ratio of sample volume to WR increases the net 562 nm measurement for each well and lowers both the minimum detection level of the reagent and the working range of the assay. As long as all standards and unknowns are treated identically, such modifications are useful.
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1. Subtract the average 562 nm absorbance measurement of the blank standard replicates from the 562 nm measurements of all other individual standard and unknown sample replicates.
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2. Prepare a standard curve by plotting the average blank–corrected 562 nm measurement for each BSA standard vs. its concentration in µg/mL. Use the standard curve to determine the protein concentration of each unknown sample.
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Note: If using curve-fitting algorithms associated with a microplate reader, a four-parameter (quadratic) or best–fit curve provides more accurate results than a purely linear fit. If plotting results by hand, a point–to–point curve is preferable to a linear fit to the standard points.
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**
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@ -1,10 +0,0 @@
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#treatment
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**Treatment Setup:**
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- Final fatty acid concentration: 0.125 mM for both BSA-OA and BSA-PA.
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- Medium: Complete media with fatty acid-BSA conjugates.
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**Steps:**
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1. Prepare BSA-fatty acid solutions separately.
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2. Dilute to final working concentrations in complete media.
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3. After 48 h in siRNA, treat cells for 24 h.
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@ -1,12 +0,0 @@
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#treatment
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**Treatment Setup:**
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- Final fatty acid concentration: 0.125 mM for both BSA-OA and BSA-PA.
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- Medium: Complete media with fatty acid-BSA conjugates.
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**Steps:**
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1. Prepare BSA-fatty acid solutions separately.
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2. Dilute to final working concentrations in complete media.
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3. After 24 h in siRNA, treat cells for 48 h.
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---
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---
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name: BSA-OAPA Treatment Protocol
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id: PROT-0013
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version: 3.0
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description: Protocol for treating cells with BSA-conjugated oleic and palmitic acids (OAPA)
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author: JJ
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created: 2025-04-29
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materials:
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- Bovine Serum Albumin (BSA)
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- Oleic acid (OA)
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- Palmitic acid (PA)
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- BSA-conjugated OA and PA
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- Complete culture medium
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steps:
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- "Prepare BSA-conjugated OA and PA mixture (2:1 ratio)"
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- "Treat cells 36-48h after reverse transfection"
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- "Incubate for 16-24h with OAPA treatment"
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- "Use BSA-only treatment as control"
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notes: |
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This is version 3 of the OAPA treatment protocol
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Tested on cell lines: HUH7, HEPG2, AML12(?)
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Final concentration: 0.33 mM OA, 0.17 mM PA (0.5 mM total, 2:1 ratio)
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---
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### Treatment name:
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OAPA v3
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#### Date:
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04/29/2025 JJ
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#### Applicable Cell Lines:
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HUH7, HEPG2, AML12(?)
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### Treatment Agent:
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Bovine Serum Albumin (BSA)-conjugated oleic acid (OA) and BSA-conjugated palmitic acid (PA).
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### Concentration:
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0.33 mM OA, 0.17 mM PA (or 0.5 mM OA:PA 2:1)
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### Timing:
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36-48 h after reverse transfection
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### Duration of treatment:
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16-24 h
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### Frequency:
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Once
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### Control Treatment:
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BSA
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## Steps
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1. Return all items to their original places.
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2. Wipe cabinet surfaces with 70% ethanol and paper towels.
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3. Close the cabinet; UV light will automatically sterilize.
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4. Turn off vacuum, microscope, and room lights.
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---
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### Reagents
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• Fatty acid–free BSA (Sigma, A6003): 0.64 g
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• 5 M NaCl solution (to prepare 150 mM): 60 µL of 5 M NaCl diluted with 1940 µL water
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• NaHCO₃: 13.6 mg
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• Distilled water (as needed)
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• Palmitic acid (Sigma, P5585-10G) or substitute (≈5 mg for a 10 mM final concentration)
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• Wako NEFA-HR Assay Kit (for fatty acid measurement)
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### Equipment:
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• Analytical balance
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• Pipettes/micropipettes
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• Incubator or water bath (set to 65°C and 37°C)
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• Sterile 0.2 µm filter
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• Vortex mixer (optional)
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• pH meter (if needed)
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• 2 mL microcentrifuge tube
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### Protocol:
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1. Prepare 2 mL of 4.8 mM BSA solution by dissolving 0.64 g BSA in the 150 mM NaCl solution (using 1.5 mL of the prepared 150 mM solution) and adding water to reach a total of 2 mL.
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2. Make a saturated NaHCO₃ solution by dissolving 13.6 mg NaHCO₃ in 46 µL distilled water; incubate at 65°C.
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3. Add approximately 5 mg palmitic acid (or the equivalent amount for oleic or arachidonic acid) to the NaHCO₃ solution and incubate at 65°C for 2 minutes to form the sodium salt.
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4. Transfer 1.6 mL of the BSA solution into the tube containing the fatty acid mixture.
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5. Incubate the combined solution at 37°C for 70 minutes.
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6. Filter the final mixture through a 0.2 µm filter.
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7. Measure the fatty acid concentration with the NEFA-HR assay (expected around 10 mM; roughly a 2:1 fatty acid to BSA molar ratio).
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## Counting Cells Manually Protocol
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## General Steps
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1. After harvesting cells with Trypsin, mix cell suspension well.
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2. Transfer 10–20 µl of the cell suspension into a microcentrifuge tube.
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3. Add an equal volume of Trypan Blue solution.
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4. Load 10 µl of mixture into a hemocytometer chamber.
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5. Count live (unstained) vs dead (blue-stained) cells under a microscope.
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6. Calculate total viable cells based on grid counts.
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---
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---
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name: Gentle Co-Immunoprecipitation Protocol
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id: PROT-0012
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version: 1.0
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description: A gentle co-immunoprecipitation protocol for preserving protein-protein interactions
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author: JM Jordan
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created: 2023-01-01
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materials:
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- Protein lysate
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- RIPA buffer with protease inhibitor tablets
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- Antibody (2 µg per reaction)
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- Beads (50% slurry)
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- PBS with 0.02% Tween
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- Non-denaturing loading buffer (1X)
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- LoBind tubes
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- Liquid nitrogen
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steps:
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- "Prepare protein lysate (3600 µg protein)"
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- "Preclear with beads"
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- "Add antibody and incubate overnight at 4°C"
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- "Add washed beads and incubate"
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- "Wash beads with PBS-Tween"
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- "Elute proteins with non-denaturing buffer"
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- "Store samples at -80°C"
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notes: |
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This is a gentle protocol designed to preserve protein-protein interactions
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Uses minimal washing steps without extended rotations
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Includes preclearance step to reduce non-specific binding
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---
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#Protocol
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by JM Jordan 2023
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1. Prepared a lysate solution with 3600 ug of protein for HFA and HFB
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2. Adjusted to 1mL each with RIPA+Tablets
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3. Precleared solution by adding 50ul of 50% beads to each tube of lysate
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4. Aliquoted into 3 tubes/lysate (6 tubes total) and adjusted each to 1mL with RIPA+Tablets
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5. Added 2 ug of antibody to each tube
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6. Rotated overnight at 4C
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7. RT rotation for 1h
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8. Washed 600ul beads with PBS + 0.02% Tween
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9. Resuspended beads in 600ul RIPA+Tabs
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10. Added 100ul of bead solution to each tube
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11. Rotated at RT for 1h
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12. Washed beads 3X with 1ml PBS + 0.02% Tween with gentle pipet mixing (no 5 min rotation)
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13. After final wash, resuspended beads in 200 ul PBS + 0.02% Tween and transferred to fresh LoBind tubes
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14. Added 30 ul 1X Non denaturing loading buffer and eluted as usual at 37C for 5 min
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15. Separated solution from beads into new LoBind tubes and snap froze in LN2 and stored at -80C.
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16. Note: Freeze Input and leftover
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@ -1,38 +0,0 @@
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#Protocol
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Prepare cell collection tubes
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Rack n 1.5-ml Eppie tubes and label 1-n
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Prepare sample storage box [-80°C Sample Storage Protocol](https://docs.google.com/document/u/1/d/1n-TcSMLcNtWcNixfK7yqhQJJVVBpL1TQaZaernbRFhM/edit)
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Remove cells from incubator
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In laminar flow hood, aspirate media.
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Wash cells
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Add 200 ul (96-well) or 1 ml (24-well) PBS per well.
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Aspirate until dry
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In the fume hood, add 100 ul (for 96-well plates) or 300 ul (for 24-well plates) TRI reagent (or TRIzol) to each well.
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Incubate at RT for 5 minutes.
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Tilt plate back slightly to allow TRI reagents and cells to pool in the corner and pipet all of the solution into prelabeled tubes.
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If not proceeding directly to extraction:
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Place tubes in labeled box
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Place labeled box in defined location
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Freeze tubes at -80C and submit a [-80C Sample Submission](https://drive.google.com/open?id=1Oox80CP6oEVm3IVtkPSENpUCua0RylQz3HFP5BZhjxw) form.
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If proceeding, see [RNA MiniPrep with DirectZol kit](https://docs.google.com/document/u/1/d/1v3bYGmlWyRF_wZmdMD8YZwgqpblvsh4vnoxw9H9bbHs/edit).
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@ -1,104 +0,0 @@
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---
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name: Oil Red O Staining Protocol for Adherent Cells
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id: PROT-0015
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version: 1.0
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description: Protocol for staining and quantifying lipid droplets in adherent hepatocyte-like cells
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author: J. Jordan
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created: 2025-02-09
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materials:
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- Oil Red O powder
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- 100% isopropanol (2-propanol)
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- 4% paraformaldehyde (PFA)
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- PBS
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- Distilled water
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- 0.2-micron syringe filter
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- 96-well plate
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- Echo Revolution inverted microscope
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- Spectrophotometer (492 nm)
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steps:
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- "Prepare ORO staining solution"
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- "Fix cells with 4% PFA"
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- "Stain cells with filtered ORO solution"
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- "Wash and image cells"
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- "Extract ORO for quantification"
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- "Measure absorbance at 492 nm"
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notes: |
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Optimized for hepatocyte-like cells (HepG2, Huh7, AML12)
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Filtration of ORO solution is critical for good results
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Volume specifications are for 96-well plates - adjust for other formats
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---
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#Protocol for Oil red O (ORO) staining in adherent “hepatocyte-like” cells (e.g. HepG2, Huh7, AML12?)
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Adapted by J. Jordan; revised 02-09-25 by JJ.
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Note: Volumes are for 96-well plates. Adjust for larger well formats.
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1. Preparation of ORO staining solution:
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2. If necessary, prepare ORO stock solution by dissolving 0.175 g ORO powder (on the chemical shelf) in 50 ml 100% 2-propanol (aka “isopropanol”)
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3. Dilute ORO stock solution in distilled water (dH2O) (i.e., Add 3 parts ORO solution to 2 parts dH2O) and vortex solution immediately before using to stain cells.
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4. Add diluted ORO solution to a syringe with a 0.2-micron filter, then filter into a fresh vial. Skipping this filtration WILL ruin your experiment!
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5. Staining cells with ORO staining solution:
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6. Aspirate cell media and then wash twice with PBS.
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7. Add 75 ul cold 4% paraformaldehyde (PFA) to each well and allow fixation to occur for 20-30 m at room temperature (RT).
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8. Aspirate PFA.
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9. Wash cells twice with 100 ul PBS and aspirate to last wash until cells are very dry.
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10. Add 75 ul freshly prepared ORO solution to each well and stain for 30 m at RT.
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11. Wash twice with 150 ul distilled water.
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12. If imaging, add 100 ul of PBS to each well to improve microscopy. If skipping to extractions, you can leave the wells dry and proceed to Part IV.
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13. View and image cells in brightfield on Echo Revolution inverted microscope:
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14. Clip plate into stage.
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15. Adjust height of objectives with puck until cells are visible.
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16. Create a new folder to contain your images.
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17. Identify imaging parameters that will work for all of your wells so that images can be compared.
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18. Using identical imaging conditions (except for minor adjustments to focus), image all wells.
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19. Ensure images are indexed with their sample names and experimental methods.
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20. Transfer images to your Teams lab notebook data folder (USB or Airdrop) with image index.
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21. Extraction of ORO
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22. Add 75 ul of 100% isopropanol to each well and agitate for 5 minutes to extract ORO from cells.
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23. Transfer 60 ul isopropanol extraction to 96-well assay plate
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24. Add 60 ul pure isopropanol to at least 3 wells to account for background.
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25. Make sure plate reader is set to 492-nm protocol.
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26. Clip plate into spectrophotometer ensuring A1 is in the bottom-left corner.
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27. Label your data file with your experiment ID (usually your initials and the date you started your cell plate).
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28. Measure 492-nm absorbance.
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29. Export data onto a USB.
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@ -1,57 +0,0 @@
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**
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Fatty Acid–BSA Complex Preparation (2 mL Tube Format)
|
||||
|
||||
|
||||
|
||||
Reagents (scaled amounts):
|
||||
|
||||
• Fatty acid–free BSA (Sigma, A6003): 0.64 g
|
||||
|
||||
• 5 M NaCl solution (to prepare 150 mM): 60 µL of 5 M NaCl diluted with 1940 µL water
|
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|
||||
• NaHCO₃: 13.6 mg
|
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|
||||
• Distilled water (as needed)
|
||||
|
||||
• Palmitic acid (Sigma, P5585-10G) or substitute (≈5 mg for a 10 mM final concentration)
|
||||
|
||||
• Wako NEFA-HR Assay Kit (for fatty acid measurement)
|
||||
|
||||
|
||||
|
||||
Equipment:
|
||||
|
||||
• Analytical balance
|
||||
|
||||
• Pipettes/micropipettes
|
||||
|
||||
• Incubator or water bath (set to 65°C and 37°C)
|
||||
|
||||
• Sterile 0.2 µm filter
|
||||
|
||||
• Vortex mixer (optional)
|
||||
|
||||
• pH meter (if needed)
|
||||
|
||||
• 2 mL microcentrifuge tube
|
||||
|
||||
|
||||
|
||||
Protocol:
|
||||
|
||||
1. Prepare 2 mL of 4.8 mM BSA solution by dissolving 0.64 g BSA in the 150 mM NaCl solution (using 1.5 mL of the prepared 150 mM solution) and adding water to reach a total of 2 mL.
|
||||
|
||||
2. Make a saturated NaHCO₃ solution by dissolving 13.6 mg NaHCO₃ in 46 µL distilled water; incubate at 65°C.
|
||||
|
||||
3. Add approximately 5 mg palmitic acid (or the equivalent amount for oleic or arachidonic acid) to the NaHCO₃ solution and incubate at 65°C for 2 minutes to form the sodium salt.
|
||||
|
||||
4. Transfer 1.6 mL of the BSA solution into the tube containing the fatty acid mixture.
|
||||
|
||||
5. Incubate the combined solution at 37°C for 70 minutes.
|
||||
|
||||
6. Filter the final mixture through a 0.2 µm filter.
|
||||
|
||||
7. Measure the fatty acid concentration with the NEFA-HR assay (expected around 10 mM; roughly a 2:1 fatty acid to BSA molar ratio).
|
||||
|
||||
**
|
||||
@ -1,12 +0,0 @@
|
||||
# Preparation of Complete RPMI
|
||||
|
||||
## For a 500-ml Bottle
|
||||
|
||||
1. Sterile transfer ~51 ml of RPMI media into a sterile 50-ml tube.
|
||||
- **Label**: "PLAIN RPMI"
|
||||
|
||||
2. To the remaining 449 ml of media, add:
|
||||
- 50 ml **Fetal Bovine Serum** (FBS)
|
||||
- 1 ml **Penicillin-Streptomycin** (PS)
|
||||
|
||||
---
|
||||
@ -1,12 +0,0 @@
|
||||
# Preparation of Complete DMEM (High Glucose)
|
||||
|
||||
## For a 500-ml Bottle
|
||||
|
||||
1. Sterile transfer ~51 ml of DMEM (High Glucose) media into a sterile 50-ml tube.
|
||||
- **Label**: "PLAIN DMEM"
|
||||
|
||||
2. To the remaining 449 ml of media, add:
|
||||
- 50 ml **Fetal Bovine Serum** (FBS)
|
||||
- 1 ml **Penicillin-Streptomycin** (PS)
|
||||
|
||||
---
|
||||
@ -1,12 +0,0 @@
|
||||
# Preparation of Complete DME/F-12
|
||||
|
||||
## For a 500-ml Bottle
|
||||
|
||||
1. Sterile transfer ~51 ml of DME/F-12 media into a sterile 50-ml tube.
|
||||
- **Label**: "PLAIN DMEM/F-12"
|
||||
|
||||
2. To the remaining 449 ml of media, add:
|
||||
- 50 ml **Fetal Bovine Serum** (FBS)
|
||||
- 1 ml **Penicillin-Streptomycin** (PS)
|
||||
|
||||
---
|
||||
@ -1,14 +0,0 @@
|
||||
# Preparation of Complete DME/F-12 ITSD
|
||||
|
||||
## For a 500-ml Bottle
|
||||
|
||||
1. Sterile transfer ~55 ml of DME/F-12 media into a sterile 50-ml tube.
|
||||
- **Label**: "PLAIN DME/F-12"
|
||||
|
||||
2. To the remaining 445 ml of media, add:
|
||||
- 50 ml **Fetal Bovine Serum** (FBS)
|
||||
- 5 ml **Insulin-Transferrin-Selenium** (ITS)
|
||||
- 5 µl **10 mM dexamethasone**
|
||||
- 1 ml **Penicillin-Streptomycin** (PS)
|
||||
|
||||
---
|
||||
@ -1,20 +0,0 @@
|
||||
#P
|
||||
|
||||
1. If necessary, prepare a protein extraction buffer. See Preparing 1X RIPA with Protease inhibitor and phosphatase inhibitor cocktail
|
||||
2. Fill a tray with ice. (There is an ice machine in the autoclave room to the left of the cell culture room.)
|
||||
3. Remove the plate from the incubator and bring it to the lab. (There’s no need to use a laminar flow hood at this point.)
|
||||
4. Aspirate media.
|
||||
5. Wash cells with ice cold 1X PBS.
|
||||
6. Aspirate PBS.
|
||||
7. Keeping the plate on ice. Add 100 ul of ice cold protein extraction buffer to each well.
|
||||
8. Leaving the plate on ice, allow cells to sit in the extraction buffer for 10 minutes, agitating the plate every minute or two.
|
||||
9. Tip the plate towards you so that the protein extraction buffer and suspended cells pool in the bottom corner.
|
||||
10. If necessary, scrape cells into the extraction buffer with a cell scraper or pipet tip.
|
||||
11. Using a P200 pipet, transfer the cell suspension to a chilled, prelabeled 1.5-ml tube.
|
||||
12. Agitate the cells in the ThermoMixer for at 4C/500 rpm for 4 h-overnight.
|
||||
13. Spin down cell debris at max speed for 25 m at 4C.
|
||||
14. Avoiding the pellet, transfer 90 ul of supernatant to a fresh, chilled 1.5-ml tube (prelabeled).
|
||||
15. Transfer remaining 10 ul to 0.2 ml PCR tube strips.
|
||||
16. Place tubes in a -80C box and note box address.
|
||||
17. Submit your sample to our inventory/archive by filling out this form: -80C Sample Submission
|
||||
|
||||
@ -1,114 +0,0 @@
|
||||
---
|
||||
name: RNA Immunoprecipitation qPCR Protocol
|
||||
id: PROT-0014
|
||||
version: 1.0
|
||||
description: Protocol for RNA immunoprecipitation followed by RT-qPCR to detect RNA-protein interactions
|
||||
author: Jordan Lab
|
||||
created: 2025-05-06
|
||||
materials:
|
||||
- Cell culture dishes
|
||||
- PBS (cold)
|
||||
- UV crosslinker (254 nm) or formaldehyde
|
||||
- Lysis buffer with RNase inhibitors
|
||||
- Protein A/G beads
|
||||
- Antibodies for target protein
|
||||
- IgG control antibody
|
||||
- Glycine solution (for formaldehyde quenching)
|
||||
- RNA isolation reagents
|
||||
- RT-qPCR reagents
|
||||
steps:
|
||||
- "Grow cells to desired confluency"
|
||||
- "Perform crosslinking (UV or formaldehyde)"
|
||||
- "Harvest and lyse cells"
|
||||
- "Pre-clear lysates"
|
||||
- "Perform immunoprecipitation with specific antibody"
|
||||
- "Wash beads to remove non-specific binding"
|
||||
- "Reverse crosslinks if needed"
|
||||
- "Isolate RNA from immunoprecipitated complex"
|
||||
- "Perform RT-qPCR to detect target RNA"
|
||||
notes: |
|
||||
Two crosslinking methods are described: UV (254 nm) and formaldehyde
|
||||
UV crosslinking is more specific but requires specialized equipment
|
||||
Formaldehyde crosslinking is simpler but may have higher background
|
||||
Always include appropriate controls (IgG, input RNA)
|
||||
---
|
||||
|
||||
#Protocol
|
||||
|
||||
Crosslinking RNA to a specific protein of interest in cells or lysates is a common step in RNA immunoprecipitation (RIP) or related methods (e.g., CLIP-seq). Once crosslinked and immunoprecipitated, the RNA can be extracted for downstream RT-qPCR analysis. Below is a general overview of two frequently used crosslinking methods—UV crosslinking and formaldehyde crosslinking—along with key steps and considerations.
|
||||
|
||||
### UV Crosslinking
|
||||
UV crosslinking at 254 nm forms covalent bonds between nucleic acids and amino acids in close proximity. This approach is often used in CLIP (crosslinking immunoprecipitation) protocols, but it can be adapted for simpler RIP-RT-qPCR workflows.
|
||||
|
||||
#### Typical workflow:
|
||||
1. Grow cells to the desired confluency in culture dishes.
|
||||
2. Wash cells with cold PBS to remove media and serum proteins. Keep cells on ice if needed to minimize RNase activity.
|
||||
3. Add fresh cold PBS to cover the cells.
|
||||
4. Crosslink with UV 254 nm at an energy of approximately 150–300 mJ/cm². (The optimal dose depends on cell type and the sensitivity of the RNA or protein. Over-crosslinking can damage RNA, while under-crosslinking may reduce yield.)
|
||||
5. Harvest cells by scraping or gentle trypsinization (depending on the downstream protocol).
|
||||
6. Lyse cells under mild conditions suitable for maintaining RNP complexes. (Often a nonionic or mild ionic detergent is used in the lysis buffer, along with RNase inhibitors.)
|
||||
7. Perform immunoprecipitation using an antibody specific to the RNA-binding protein of interest.
|
||||
8. Pre-clear lysates (e.g., with protein A/G beads alone) to reduce nonspecific binding.
|
||||
9. Add the specific antibody, followed by protein A/G beads.
|
||||
10. Wash beads thoroughly to remove nonspecific complexes.
|
||||
11. Reverse crosslink (if needed) or proceed directly to RNA purification, depending on the protocol. Some protocols lyse or treat with proteinase K to release RNA from the protein-bead complex.
|
||||
12. Isolate RNA from the immunoprecipitated complex.
|
||||
13. Reverse transcribe and perform qPCR to detect the RNA targets that were bound by the protein.
|
||||
14. Key considerations for UV crosslinking:
|
||||
15. Irradiation distance/energy: Keep the distance between the UV lamp and cells/lysate standardized to ensure reproducibility.
|
||||
16. Crosslinking efficiency vs. RNA integrity: Higher UV doses may degrade RNA. It’s important to titrate energy.
|
||||
17. Protective measures: UV light is harmful to skin and eyes. Always use shields and PPE.
|
||||
|
||||
#### Formaldehyde Crosslinking
|
||||
|
||||
##### Overview:
|
||||
Formaldehyde can be used to crosslink proteins and nucleic acids by reacting with amino and imino groups. It is less commonly used for fine-scale mapping studies (like CLIP-seq) because it can be somewhat reversible and can introduce more nonspecific crosslinks. However, it is still used in some RIP and ChIP (chromatin IP) approaches.
|
||||
|
||||
##### Typical workflow:
|
||||
Grow cells to desired confluency.
|
||||
Prepare fresh formaldehyde at a working concentration (often 1% final in the culture medium) to fix cells.
|
||||
Add formaldehyde directly to cells in culture medium (or in PBS) and incubate typically for 5–10 minutes at room temperature or 37 °C (depending on the protocol).
|
||||
Quench the reaction by adding glycine (often to a final concentration of 125 mM) for 5–10 minutes.
|
||||
Wash the cells with cold PBS.
|
||||
Harvest cells carefully.
|
||||
Lyse cells under conditions that preserve protein-RNA complexes (often using mild detergents, protease inhibitors, and RNase inhibitors).
|
||||
Immunoprecipitate using a specific antibody against the protein of interest.
|
||||
Wash beads to remove nonspecific material.
|
||||
Reverse crosslink by heating or using other conditions specified in the protocol (formaldehyde crosslinks can often be reversed by heating at 65 °C for several hours in the presence of SDS and/or high salt).
|
||||
Isolate RNA from the immunoprecipitated sample.
|
||||
Reverse transcribe and perform qPCR to quantify target RNA levels.
|
||||
Key considerations for formaldehyde crosslinking:
|
||||
Crosslinking stringency: Formaldehyde can create more nonspecific crosslinks; optimizing crosslinking time and concentration is key.
|
||||
Reversibility: Ensure you reverse crosslinks thoroughly to recover intact RNA for RT-qPCR.
|
||||
Toxicity: Formaldehyde is highly toxic and volatile, so follow safety guidelines (work in a fume hood, use PPE).
|
||||
|
||||
#### Choosing the Method
|
||||
#### UV Crosslinking
|
||||
1. Generally provides more specific crosslinks between directly interacting residues (protein–RNA).
|
||||
2. Can require specialized equipment (UV crosslinker).
|
||||
3. More commonly used in CLIP-based methods for precise mapping of RNA-protein interaction sites.
|
||||
4. Formaldehyde Crosslinking
|
||||
5. Straightforward chemical method.
|
||||
6. Potentially higher background due to nonspecific crosslinks.
|
||||
7. Reversal requires heating and/or high salt conditions, which can be harsh on samples.
|
||||
8. For many RNA-IP followed by RT-qPCR experiments (RIP-qPCR), mild crosslinking using UV at 254 nm is quite popular because it preserves specificity. However, if you already have a formaldehyde-based protocol optimized for your lab, that can also work.
|
||||
|
||||
#### Final Tips
|
||||
Pilot Experiments:
|
||||
Always optimize crosslinking conditions (time, energy, concentration) to balance specificity vs. yield.
|
||||
Test different crosslinking strengths and confirm via a known positive RNA target.
|
||||
Include Controls:
|
||||
Use an IgG control or a nonspecific antibody to measure background binding.
|
||||
Use input RNA to normalize or calculate percentage of input in qPCR.
|
||||
Validate with Known Targets:
|
||||
If possible, use a known RNA that your protein interacts with as a positive control.
|
||||
Handle RNA Carefully:
|
||||
Incorporate RNase inhibitors in all buffers, and keep samples cold when possible.
|
||||
|
||||
|
||||
#### Safety Considerations:
|
||||
1. UV crosslinking: protect from direct UV exposure; follow lamp manufacturer’s guidelines.
|
||||
2. Formaldehyde: use a fume hood; wear gloves and goggles.
|
||||
|
||||
|
||||
**By choosing the appropriate crosslinking approach and carefully optimizing the conditions, you can capture stable RNA-protein interactions, immunoprecipitate your protein of interest, and then detect and quantify the associated RNAs by RT-qPCR.**
|
||||
@ -1,16 +0,0 @@
|
||||
## Before You Begin
|
||||
|
||||
- Sterilize all materials with 70% ethanol.
|
||||
- Complete plate layout and siRNA calculations beforehand.
|
||||
|
||||
## Protocol
|
||||
|
||||
1. Open sterile 96-well TC plate (clear for brightfield / black for fluorescence).
|
||||
2. Dilute 9 ml of cell suspension with 14 ml complete media.
|
||||
3. Swirl and pour into a sterile 25-ml basin.
|
||||
4. Using a multi-channel pipet:
|
||||
- Seed **100 µl** per well.
|
||||
5. Mix cell suspension periodically while seeding.
|
||||
6. Aspirate leftovers and clean up.
|
||||
|
||||
---
|
||||
@ -1,16 +0,0 @@
|
||||
## For 6-Well Plates
|
||||
|
||||
1. Start with a 90–100% confluent 25 cm^3 flask of cells.
|
||||
2. Aspirate media and wash with 10 ml sterile PBS.
|
||||
3. Add 1 ml Trypsin, coat, incubate ~5 min at 37°C.
|
||||
4. Dislodge cells, add 9 ml complete media (10% FBS + 1% PS).
|
||||
5. Mix gently, pipet **350 µl** cell suspension into each well.
|
||||
6. Add **1600 µl** complete media to each well.
|
||||
7. Incubate 24–48 hours.
|
||||
|
||||
## For 24-Well Plates
|
||||
|
||||
- Pipet 200 µl** cell suspension into each well.
|
||||
- Top up with **800 µl** media as needed.
|
||||
|
||||
---
|
||||
@ -1,38 +0,0 @@
|
||||
**
|
||||
|
||||
Created: 03/22/25JJ
|
||||
|
||||
1. Open the SpectraMax software on the computer.
|
||||
|
||||
2. Click Protocol > New Experiment
|
||||
|
||||
3. Click the gear above the plate grid to adjust settings.
|
||||
|
||||
4. For BCA:
|
||||
|
||||
|
||||
5. Absorbance mode
|
||||
|
||||
6. Wavelengths = 562 nm
|
||||
|
||||
7. Paint the wells you want to measure
|
||||
|
||||
8. 96-well, clrbttm
|
||||
|
||||
9. No shaking
|
||||
|
||||
10. Fast Read is fine
|
||||
|
||||
|
||||
11. Click open/close plate reader button
|
||||
|
||||
12. Insert your plate with H1 in the bottom-left corner.
|
||||
|
||||
13. Press Read
|
||||
|
||||
14. Copy paste the grid to excel
|
||||
|
||||
15. Save excel file to a USB or to the hard drive and then transfer it off the core facility computer.
|
||||
|
||||
|
||||
**
|
||||
@ -1,10 +0,0 @@
|
||||
## Before You Begin
|
||||
- Sterilize everything with 70% ethanol.
|
||||
- Spray your hands after touching anything outside the hood.
|
||||
## Protocol
|
||||
1. Aspirate old media.
|
||||
2. Wash with PBS, aspirate.
|
||||
3. Add 1 ml Tryp-LE, incubate 5–10 min.
|
||||
4. Dislodge cells, add 9 ml complete media.
|
||||
5. Seed 1 ml into a new flask with 9 ml complete media.
|
||||
6. Incubate at 37°C with 5% CO₂.
|
||||
@ -1,12 +0,0 @@
|
||||
## Steps:
|
||||
1. Aspirate old cell media.
|
||||
|
||||
2. Add 150 ul (96-well), 1 ml (24-well), or 2 ml (6-well) control or treatment media (warmed in thermal bead bath) per well.
|
||||
|
||||
3. Return plate to incubator for the duration of the treatment period.
|
||||
|
||||
|
||||
### Example Treaments:
|
||||
1. [[BSA-OAPA treatment protocol v1.gdoc]]
|
||||
2. [[BSA-OAPA treatment protocol v2.gdoc]]
|
||||
3. [[BSA-OAPA Treatment Protocol v3]]
|
||||
@ -1,19 +0,0 @@
|
||||
name: Cell Staining Protocol
|
||||
id: PROT-0001
|
||||
description: Protocol for immunofluorescence staining of cells
|
||||
author: Alice Smith
|
||||
created: 2025-05-05
|
||||
version: 1.0
|
||||
materials:
|
||||
- Antibody: Anti-XYZ (1:500 dilution)
|
||||
- Stain: DAPI
|
||||
- Buffer: PBS 1X
|
||||
steps:
|
||||
- "Fix cells with 4% PFA for 10 minutes."
|
||||
- "Wash 3x with PBS."
|
||||
- "Add primary antibody (Anti-XYZ) for 1 hour at RT."
|
||||
- "Wash 3x with PBS."
|
||||
- "Add DAPI stain for 5 minutes."
|
||||
- "Wash and image."
|
||||
notes: |
|
||||
This protocol is derived from Doe et al. 2023.
|
||||
@ -1,77 +0,0 @@
|
||||
---
|
||||
name: mRNA Stability Assay Protocol
|
||||
id: PROT-0011
|
||||
version: 1.0
|
||||
description: Protocol for measuring mRNA stability using siNC vs. siYbx1 with Actinomycin D treatment
|
||||
author: Jordan Lab
|
||||
created: 2025-05-06
|
||||
materials:
|
||||
- Actinomycin D
|
||||
- DMSO (control)
|
||||
- Complete medium
|
||||
- PBS (ice-cold)
|
||||
- TRIzol or RLT buffer
|
||||
- Multi-channel pipette
|
||||
- Cell culture plates
|
||||
- Tubes for sample storage
|
||||
steps:
|
||||
- "Prepare Actinomycin D and control media"
|
||||
- "Treat cells with ActD or control media"
|
||||
- "Collect time-point samples (0hr and subsequent)"
|
||||
- "Process samples for RNA extraction"
|
||||
notes: |
|
||||
This protocol compares mRNA stability between control (siNC) and Ybx1 knockdown (siYbx1) conditions
|
||||
ActD concentration is 5 µg/mL
|
||||
Use multichannel pipettes to minimize time between treatments
|
||||
---
|
||||
|
||||
#Protocol
|
||||
## (siNC vs. siYbx1 with Actinomycin D)
|
||||
### ✅ Step 1: Actinomycin D Medium Preparation
|
||||
|
||||
(Just before treatment)
|
||||
|
||||
- Prepare 5 µg/mL ActD medium and DMSO-only control medium.
|
||||
|
||||
|
||||
|
||||
Volumes needed:
|
||||
|
||||
- ActD (5 µg/mL): ~20 mL total (for all ActD wells)
|
||||
|
||||
|
||||
- No ActD (DMSO control): ~5 mL total
|
||||
|
||||
|
||||
|
||||
---
|
||||
|
||||
### ✅ Step 2: ActD Treatment and Initial Collection (0 hr)
|
||||
|
||||
Procedure:
|
||||
|
||||
- Remove old media quickly from the plate (multi-channel vacuum aspirator recommended).
|
||||
|
||||
- Using a multichannel pipet, rapidly add 100 µL of treatment media (ActD or No ActD) according to the plate map.
|
||||
|
||||
- Immediately collect the 0 hr samples.
|
||||
|
||||
|
||||
- Cell Collection Protocol (all time points):
|
||||
|
||||
|
||||
- Quickly aspirate medium.
|
||||
|
||||
- Briefly wash with 100 µL ice-cold PBS (optional).
|
||||
|
||||
- Lyse directly in wells using 50 µL TRIzol or RLT buffer.
|
||||
|
||||
- Transfer lysate immediately to labeled tubes/plate and store at –80°C.
|
||||
|
||||
- Repeat for each timepoint
|
||||
|
||||
|
||||
Multichannel tip:
|
||||
|
||||
Aspirate and dispense solutions one column at a time to minimize delays.
|
||||
**
|
||||
@ -1,25 +0,0 @@
|
||||
name: 24-well Plate siRNA Transfection (RNAiMAX)
|
||||
id: PROT-0020
|
||||
version: 1.0
|
||||
description: Protocol for transfecting 4 siRNAs (including siNC) into cells in a 24-well plate using Lipofectamine RNAiMAX.
|
||||
author: Lab Agent
|
||||
created: 2024-06-08
|
||||
materials:
|
||||
- siRNA: siRNA#1 (final 10 nM)
|
||||
- siRNA: siRNA#2 (final 10 nM)
|
||||
- siRNA: siRNA#3 (final 10 nM)
|
||||
- siRNA: siNC (negative control, final 10 nM)
|
||||
- Lipofectamine RNAiMAX: 1.5 µL per well
|
||||
- Opti-MEM Reduced Serum Medium: as required
|
||||
- Cells: seeded in 24-well plate (e.g., 5x10^4 cells/well)
|
||||
steps:
|
||||
- "Seed cells in 24-well plate 24 hours prior to transfection (e.g., 5x10^4 cells/well)."
|
||||
- "For each siRNA, dilute 10 pmol siRNA in 50 µL Opti-MEM per well."
|
||||
- "In a separate tube, dilute 1.5 µL RNAiMAX in 50 µL Opti-MEM per well."
|
||||
- "Combine diluted siRNA and RNAiMAX, mix gently, and incubate for 10-20 min at room temperature."
|
||||
- "Add 100 µL siRNA-RNAiMAX complex dropwise to each well containing cells in 400 µL growth medium."
|
||||
- "Incubate cells at 37°C, 5% CO2 for 24-72 hours."
|
||||
notes: |
|
||||
- siNC is a non-targeting negative control siRNA.
|
||||
- Adjust cell number and reagent volumes as needed for specific cell lines.
|
||||
- Refer to manufacturer's instructions for further optimization.
|
||||
@ -1,37 +0,0 @@
|
||||
name: Ybx1 knockdown mRNA stability assay
|
||||
id: PROT-0035
|
||||
version: 1.0
|
||||
description: Protocol for measuring mRNA stability of target genes after Ybx1 knockdown using siRNA transfection and actinomycin D transcription inhibition
|
||||
author: Dr. Jim Jordan
|
||||
created: 2025-05-06
|
||||
materials:
|
||||
- Material: siRNA targeting Ybx1 (final 10 nM)
|
||||
- Material: siRNA negative control (final 10 nM)
|
||||
- Material: Lipofectamine RNAiMAX (1.5 µL per well)
|
||||
- Material: Opti-MEM Reduced Serum Medium (as required)
|
||||
- Material: 6-well cell culture plates
|
||||
- Material: Actinomycin D (5 µg/mL final concentration)
|
||||
- Material: TRIzol reagent for RNA extraction
|
||||
- Material: SuperScript III Reverse Transcription kit
|
||||
- Material: qPCR primers for target genes
|
||||
- Material: SYBR Green qPCR Master Mix
|
||||
steps:
|
||||
- "Day 1: Seed cells in 6-well plates at 3 × 10^5 cells per well in complete media."
|
||||
- "Day 2: Transfect cells with siRNA targeting Ybx1 or negative control siRNA using Lipofectamine RNAiMAX according to manufacturer's protocol."
|
||||
- "Day 3: Verify Ybx1 knockdown efficiency by collecting a subset of cells and performing RT-qPCR or western blot."
|
||||
- "Day 4:
|
||||
a. Collect first time point (t=0) samples by extracting RNA with TRIzol.
|
||||
b. Add actinomycin D to remaining wells at 5 µg/mL final concentration.
|
||||
c. Collect RNA samples at 1h, 2h, 4h, 6h, and 8h after actinomycin D addition."
|
||||
- "Day 5-6:
|
||||
a. Perform RNA isolation from all collected samples.
|
||||
b. Synthesize cDNA using SuperScript III Reverse Transcription kit with random hexamers and oligo-dT primers.
|
||||
c. Perform qPCR for target genes and reference genes.
|
||||
d. Calculate mRNA half-life by plotting relative mRNA levels on a semi-log scale versus time and determining the slope."
|
||||
notes: |
|
||||
- For optimal results, verify Ybx1 knockdown efficiency before proceeding with actinomycin D treatment.
|
||||
- Use 18S rRNA or GAPDH as reference genes for normalization.
|
||||
- Target genes should include those known to be regulated post-transcriptionally, particularly those with m5C or m6A modifications.
|
||||
- The optimal actinomycin D concentration may vary by cell type; preliminary testing is recommended.
|
||||
- Actinomycin D is toxic; handle with care and dispose of properly.
|
||||
- For very stable mRNAs, time points may need to be extended beyond 8h.
|
||||
@ -1,13 +1,68 @@
|
||||
name: Example Protocol Name
|
||||
---
|
||||
# Protocol metadata
|
||||
id: PROT-XXXX
|
||||
name: Example Protocol Name
|
||||
version: 1.0
|
||||
description: Protocol description goes here
|
||||
author: Your Name
|
||||
created: YYYY-MM-DD
|
||||
last_updated: YYYY-MM-DD
|
||||
category: protocol-category
|
||||
|
||||
# Materials required
|
||||
materials:
|
||||
- Material: Example Material (amount)
|
||||
- name: Example Material
|
||||
amount: XX unit
|
||||
preparation: Brief preparation notes if applicable
|
||||
storage: Storage conditions if applicable
|
||||
- name: Example Reagent
|
||||
concentration: XX unit
|
||||
preparation: Brief preparation notes if applicable
|
||||
|
||||
# Equipment required
|
||||
equipment:
|
||||
- name: Example Equipment
|
||||
settings: Relevant settings if applicable
|
||||
- name: Example Instrument
|
||||
calibration: Calibration requirements if applicable
|
||||
|
||||
# Protocol steps
|
||||
steps:
|
||||
- "Step 1: ..."
|
||||
- "Step 2: ..."
|
||||
- step: 1
|
||||
action: "Description of first step"
|
||||
details: "Additional details about performing this step"
|
||||
- step: 2
|
||||
action: "Description of second step"
|
||||
details: "Additional details about performing this step"
|
||||
- step: 3
|
||||
action: "Description of third step"
|
||||
details: "Additional details about performing this step"
|
||||
|
||||
# Critical parameters
|
||||
critical_parameters:
|
||||
- parameter: "Example critical parameter"
|
||||
details: "Why this parameter is important and how to control it"
|
||||
|
||||
# Troubleshooting
|
||||
troubleshooting:
|
||||
- problem: "Common issue"
|
||||
solution: "How to resolve this issue"
|
||||
|
||||
# Safety considerations
|
||||
safety:
|
||||
ppe: "Required personal protective equipment"
|
||||
hazards: "Potential hazards and precautions"
|
||||
|
||||
# Quality control
|
||||
quality_control:
|
||||
- check: "QC checkpoint"
|
||||
criteria: "Pass/fail criteria"
|
||||
|
||||
# References
|
||||
references:
|
||||
- "Reference citation or link to source material"
|
||||
|
||||
# Notes
|
||||
notes: |
|
||||
Additional notes or references.
|
||||
Any additional information, tips, or context that might be helpful.
|
||||
---
|
||||
159
protocols/bca_protein_assay_v1.yaml
Normal file
159
protocols/bca_protein_assay_v1.yaml
Normal file
@ -0,0 +1,159 @@
|
||||
---
|
||||
# Protocol metadata
|
||||
id: PROT-0010
|
||||
name: BCA Assay for Protein Abundance Determination
|
||||
version: 1.0
|
||||
description: Protocol for colorimetric detection and quantitation of total protein using bicinchoninic acid (BCA)
|
||||
author: Jordan Lab
|
||||
created: 2025-05-06
|
||||
last_updated: 2025-05-07
|
||||
category: protein-analysis
|
||||
|
||||
# Materials required
|
||||
materials:
|
||||
- name: BCA reagent A
|
||||
storage: Room temperature or as specified by manufacturer
|
||||
- name: BCA reagent B
|
||||
storage: Room temperature or as specified by manufacturer
|
||||
- name: BSA protein standard
|
||||
concentration: 2 mg/mL
|
||||
storage: 4°C
|
||||
- name: Microplate
|
||||
type: 96-well, clear bottom
|
||||
- name: Protein samples (unknowns)
|
||||
preparation: Keep on ice during handling
|
||||
- name: Diluent buffer
|
||||
notes: Use same buffer as protein samples
|
||||
|
||||
# Equipment required
|
||||
equipment:
|
||||
- name: Plate reader
|
||||
settings: 562 nm absorbance measurement
|
||||
- name: Pipettes and tips
|
||||
range: 10-200 μL
|
||||
- name: Incubator
|
||||
temperature: 37°C
|
||||
|
||||
# Standard preparation
|
||||
standards:
|
||||
- vial: A
|
||||
diluent: 0 µL
|
||||
stock: 300 µL stock BSA
|
||||
concentration: 2000 µg/mL
|
||||
- vial: B
|
||||
diluent: 125 µL
|
||||
stock: 375 µL stock BSA
|
||||
concentration: 1500 µg/mL
|
||||
- vial: C
|
||||
diluent: 325 µL
|
||||
stock: 325 µL stock BSA
|
||||
concentration: 1000 µg/mL
|
||||
- vial: D
|
||||
diluent: 175 µL
|
||||
stock: 175 µL vial B dilution
|
||||
concentration: 750 µg/mL
|
||||
- vial: E
|
||||
diluent: 325 µL
|
||||
stock: 325 µL vial C dilution
|
||||
concentration: 500 µg/mL
|
||||
- vial: F
|
||||
diluent: 325 µL
|
||||
stock: 325 µL vial E dilution
|
||||
concentration: 250 µg/mL
|
||||
- vial: G
|
||||
diluent: 325 µL
|
||||
stock: 325 µL vial F dilution
|
||||
concentration: 125 µg/mL
|
||||
- vial: H
|
||||
diluent: 400 µL
|
||||
stock: 100 µL vial G dilution
|
||||
concentration: 25 µg/mL
|
||||
- vial: I
|
||||
diluent: 400 µL
|
||||
stock: 0 µL BSA
|
||||
concentration: 0 µg/mL (Blank)
|
||||
|
||||
# Protocol steps
|
||||
steps:
|
||||
- step: 1
|
||||
action: "Determine the number of standards and unknown samples to quantify"
|
||||
details: "Plan for technical replicates (typically 2-3) for each standard and unknown"
|
||||
- step: 2
|
||||
action: "Calculate total Working Reagent (WR) volume"
|
||||
details: "(# standards + # unknowns) × (# replicates) × (volume of WR per sample)"
|
||||
- step: 3
|
||||
action: "Prepare Working Reagent (WR)"
|
||||
details: "Mix 50 parts BCA reagent A with 1 part BCA reagent B (50:1 ratio). Example: Combine 5 mL of reagent A with 0.1 mL of reagent B to prepare 5.1 mL WR"
|
||||
- step: 4
|
||||
action: "Pipette 10 µL of each standard or unknown sample replicate into a microplate well"
|
||||
details: "Maintain consistent technique to minimize variability"
|
||||
- step: 5
|
||||
action: "Add 200 µL of the WR to each well"
|
||||
details: "Mix thoroughly for 30 seconds using plate shaker or pipette"
|
||||
- step: 6
|
||||
action: "Incubate at 37°C for 30 minutes"
|
||||
details: "Cover plate to prevent evaporation"
|
||||
- step: 7
|
||||
action: "Measure the absorbance at 562 nm on plate reader"
|
||||
details: "Allow plate to cool to room temperature before measuring if needed"
|
||||
|
||||
# Analysis
|
||||
analysis:
|
||||
- step: 1
|
||||
action: "Subtract the average 562 nm blank value from all other standards and unknown samples"
|
||||
details: "This corrects for background absorbance"
|
||||
- step: 2
|
||||
action: "Plot standard curve"
|
||||
details: "Graph blank-corrected 562 nm measurements vs. BSA concentration (µg/mL)"
|
||||
- step: 3
|
||||
action: "Calculate unknown protein concentrations"
|
||||
details: "Use the standard curve to determine protein concentration of unknown samples"
|
||||
- step: 4
|
||||
action: "Apply dilution factor if applicable"
|
||||
details: "Multiply calculated concentration by dilution factor to get original sample concentration"
|
||||
|
||||
# Critical parameters
|
||||
critical_parameters:
|
||||
- parameter: "Sample:WR ratio"
|
||||
details: "Maintain consistent 1:20 ratio (10 μL sample to 200 μL WR) for accurate results"
|
||||
- parameter: "Incubation time and temperature"
|
||||
details: "Strictly adhere to 30 minutes at 37°C for reproducible results"
|
||||
- parameter: "Standard curve fit"
|
||||
details: "Use a four-parameter (quadratic) or best-fit curve instead of purely linear fit for best accuracy"
|
||||
|
||||
# Troubleshooting
|
||||
troubleshooting:
|
||||
- problem: "High background in blank wells"
|
||||
solution: "Ensure clean labware and high-quality reagents; check for contamination"
|
||||
- problem: "Poor standard curve linearity"
|
||||
solution: "Verify accurate pipetting technique and fresh standards"
|
||||
- problem: "Samples out of range"
|
||||
solution: "Dilute high-concentration samples and re-assay; increase volume for low-concentration samples"
|
||||
|
||||
# Safety considerations
|
||||
safety:
|
||||
ppe: "Lab coat, gloves, and eye protection required"
|
||||
hazards: "BCA reagents contain sodium hydroxide and sodium carbonate; avoid contact with skin and eyes"
|
||||
|
||||
# Quality control
|
||||
quality_control:
|
||||
- check: "R² value of standard curve"
|
||||
criteria: "Should be > 0.98 for reliable quantitation"
|
||||
- check: "Technical replicate consistency"
|
||||
criteria: "CV < 10% between replicates"
|
||||
|
||||
# References
|
||||
references:
|
||||
- "Smith PK et al. (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150:76-85"
|
||||
- "Thermo Scientific Pierce BCA Protein Assay Kit manual"
|
||||
|
||||
# Notes
|
||||
notes: |
|
||||
- Dilute standards in the same buffer used to collect your protein samples
|
||||
- Wavelengths from 540-590 nm have been used successfully with this method
|
||||
- Plate readers require greater sample:WR ratio than cuvette spectrophotometers
|
||||
- Increasing incubation time (up to 2 hours) or ratio of sample volume to WR increases sensitivity
|
||||
- This assay works on the biuret reaction principle: Cu+2 is reduced to Cu+1 by protein
|
||||
- The Cu+1 is detected using BCA, forming a purple complex absorbing at 562 nm
|
||||
- The assay is nearly linear for protein concentrations from 20-2000 µg/mL
|
||||
---
|
||||
123
protocols/cell_staining_v1.yaml
Normal file
123
protocols/cell_staining_v1.yaml
Normal file
@ -0,0 +1,123 @@
|
||||
---
|
||||
# Protocol metadata
|
||||
id: PROT-0001
|
||||
name: Cell Staining Protocol
|
||||
version: 1.0
|
||||
description: Protocol for immunofluorescence staining of cells
|
||||
author: Alice Smith
|
||||
created: 2025-05-05
|
||||
last_updated: 2025-05-07
|
||||
category: microscopy
|
||||
|
||||
# Materials required
|
||||
materials:
|
||||
- name: Primary antibody (Anti-XYZ)
|
||||
dilution: 1:500
|
||||
storage: -20°C
|
||||
- name: DAPI nuclear stain
|
||||
concentration: 1 μg/mL
|
||||
storage: 4°C, protected from light
|
||||
- name: Paraformaldehyde (PFA)
|
||||
concentration: 4% in PBS
|
||||
storage: 4°C, prepare fresh if possible
|
||||
- name: PBS (Phosphate Buffered Saline)
|
||||
concentration: 1X
|
||||
storage: Room temperature
|
||||
- name: Mounting medium
|
||||
type: Anti-fade
|
||||
storage: 4°C
|
||||
|
||||
# Equipment required
|
||||
equipment:
|
||||
- name: Fluorescence microscope
|
||||
settings: Appropriate filter sets for antibody fluorophores and DAPI
|
||||
- name: Coverslips/chamber slides
|
||||
type: Glass, tissue-culture treated
|
||||
- name: Fine-tip forceps
|
||||
use: Handling coverslips
|
||||
- name: Humidified chamber
|
||||
use: Antibody incubation
|
||||
|
||||
# Protocol steps
|
||||
steps:
|
||||
- step: 1
|
||||
action: "Fix cells with 4% PFA"
|
||||
details: "Incubate for 10 minutes at room temperature"
|
||||
- step: 2
|
||||
action: "Wash 3x with PBS"
|
||||
details: "5 minutes per wash, gentle rocking"
|
||||
- step: 3
|
||||
action: "Permeabilize cells"
|
||||
details: "Use 0.1% Triton X-100 in PBS for 5 minutes at room temperature"
|
||||
- step: 4
|
||||
action: "Block non-specific binding"
|
||||
details: "Incubate with 3% BSA in PBS for 30 minutes at room temperature"
|
||||
- step: 5
|
||||
action: "Add primary antibody (Anti-XYZ)"
|
||||
details: "Dilute 1:500 in blocking solution, incubate for 1 hour at room temperature or overnight at 4°C"
|
||||
- step: 6
|
||||
action: "Wash 3x with PBS"
|
||||
details: "5 minutes per wash, gentle rocking"
|
||||
- step: 7
|
||||
action: "Add DAPI stain"
|
||||
details: "Incubate for 5 minutes at room temperature, protected from light"
|
||||
- step: 8
|
||||
action: "Wash 2x with PBS"
|
||||
details: "5 minutes per wash, gentle rocking"
|
||||
- step: 9
|
||||
action: "Mount and seal slides"
|
||||
details: "Apply mounting medium, place coverslip, and seal edges with nail polish if needed"
|
||||
- step: 10
|
||||
action: "Image slides"
|
||||
details: "Use appropriate filter sets and exposure times for each fluorophore"
|
||||
|
||||
# Critical parameters
|
||||
critical_parameters:
|
||||
- parameter: "Antibody dilution"
|
||||
details: "Optimal dilution may vary by lot and application; validate before use"
|
||||
- parameter: "Fixation time"
|
||||
details: "Over-fixation can mask epitopes; under-fixation can compromise cell morphology"
|
||||
- parameter: "Light exposure"
|
||||
details: "Minimize exposure to light after fluorophore addition to prevent photobleaching"
|
||||
|
||||
# Troubleshooting
|
||||
troubleshooting:
|
||||
- problem: "High background signal"
|
||||
solution: "Increase blocking time, increase wash times, or reduce antibody concentration"
|
||||
- problem: "Weak or no signal"
|
||||
solution: "Check antibody viability, increase concentration, or optimize fixation method"
|
||||
- problem: "Non-specific staining"
|
||||
solution: "Increase blocking time/concentration, validate antibody specificity, add serum from secondary host"
|
||||
|
||||
# Safety considerations
|
||||
safety:
|
||||
ppe: "Lab coat, gloves, and eye protection required"
|
||||
hazards: "Paraformaldehyde is toxic; work in fume hood and dispose of waste properly"
|
||||
|
||||
# Quality control
|
||||
quality_control:
|
||||
- check: "Include negative control (no primary antibody)"
|
||||
criteria: "Should show minimal background fluorescence"
|
||||
- check: "Include positive control"
|
||||
criteria: "Sample known to express target protein"
|
||||
|
||||
# References
|
||||
references:
|
||||
- "Donaldson JG. (2015) Immunofluorescence staining. Curr Protoc Cell Biol. 69:4.3.1-4.3.7"
|
||||
|
||||
# Notes
|
||||
notes: |
|
||||
- Ensure all reagents are at room temperature before starting.
|
||||
- Use appropriate personal protective equipment (PPE) when handling chemicals.
|
||||
- Adjust antibody dilution based on specific cell type and experimental conditions.
|
||||
- Store unused antibodies at -20°C for long-term storage.
|
||||
- Document any deviations from the protocol in the lab notebook.
|
||||
- Always include a control sample for comparison.
|
||||
- Dispose of all waste according to local regulations.
|
||||
- Ensure that the microscope is calibrated and functioning properly before imaging.
|
||||
- Record all imaging parameters for reproducibility.
|
||||
- If using a different staining protocol, ensure compatibility with the antibodies used.
|
||||
- For best results, use fresh reagents and avoid freeze-thaw cycles.
|
||||
- If using multiple antibodies, ensure they are compatible and do not cross-react.
|
||||
- If using a secondary antibody, ensure it is compatible with the primary antibody and the detection method.
|
||||
---
|
||||
93
protocols/check_formatting.py
Normal file
93
protocols/check_formatting.py
Normal file
@ -0,0 +1,93 @@
|
||||
#!/usr/bin/env python3
|
||||
"""
|
||||
Protocol Format Checker
|
||||
This script checks which YAML protocol files have been updated with the consistent professional format
|
||||
and which ones still need to be fixed.
|
||||
"""
|
||||
|
||||
import os
|
||||
import yaml
|
||||
import sys
|
||||
|
||||
def check_protocol_format(protocol_path):
|
||||
"""Check if a protocol file has the expected professional format sections."""
|
||||
try:
|
||||
with open(protocol_path, 'r') as f:
|
||||
content = f.read()
|
||||
|
||||
# If file is empty or very small, it's probably not formatted properly
|
||||
if len(content) < 100:
|
||||
return False
|
||||
|
||||
# Check for key sections that indicate our professional format
|
||||
required_sections = [
|
||||
"# Protocol metadata",
|
||||
"# Materials required",
|
||||
"# Equipment required",
|
||||
"# Protocol steps",
|
||||
"# Critical parameters",
|
||||
"last_updated:",
|
||||
"category:",
|
||||
]
|
||||
|
||||
# Check for numbered steps format
|
||||
step_format = "step: "
|
||||
|
||||
# Count how many required sections are present
|
||||
section_count = 0
|
||||
has_step_format = False
|
||||
|
||||
for section in required_sections:
|
||||
if section in content:
|
||||
section_count += 1
|
||||
|
||||
if step_format in content:
|
||||
has_step_format = True
|
||||
|
||||
# If it has most of the sections and the step format, consider it updated
|
||||
return section_count >= 5 and has_step_format
|
||||
|
||||
except Exception as e:
|
||||
print(f"Error checking {protocol_path}: {e}")
|
||||
return False
|
||||
|
||||
def main():
|
||||
"""Main function to check all protocol files."""
|
||||
protocol_dir = os.path.dirname(os.path.abspath(__file__))
|
||||
|
||||
# Get all YAML files in the protocols directory
|
||||
protocol_files = []
|
||||
for root, _, files in os.walk(protocol_dir):
|
||||
for file in files:
|
||||
if file.endswith('.yaml'):
|
||||
protocol_files.append(os.path.join(root, file))
|
||||
|
||||
# Check each protocol file
|
||||
updated = []
|
||||
need_update = []
|
||||
|
||||
for protocol in protocol_files:
|
||||
is_updated = check_protocol_format(protocol)
|
||||
file_name = os.path.basename(protocol)
|
||||
|
||||
if is_updated:
|
||||
updated.append(file_name)
|
||||
else:
|
||||
need_update.append(file_name)
|
||||
|
||||
# Print results as a checklist
|
||||
print("\n===== PROTOCOL FORMATTING CHECKLIST =====")
|
||||
print(f"Total protocols: {len(protocol_files)}")
|
||||
print(f"Updated protocols: {len(updated)} ✓")
|
||||
print(f"Protocols needing update: {len(need_update)} ✗\n")
|
||||
|
||||
print("UPDATED PROTOCOLS:")
|
||||
for i, protocol in enumerate(sorted(updated), 1):
|
||||
print(f" ✓ {i}. {protocol}")
|
||||
|
||||
print("\nPROTOCOLS NEEDING UPDATE:")
|
||||
for i, protocol in enumerate(sorted(need_update), 1):
|
||||
print(f" ✗ {i}. {protocol}")
|
||||
|
||||
if __name__ == "__main__":
|
||||
main()
|
||||
121
protocols/cleaning_the_laminar_flow_hood_v1.yaml
Normal file
121
protocols/cleaning_the_laminar_flow_hood_v1.yaml
Normal file
@ -0,0 +1,121 @@
|
||||
---
|
||||
# Protocol metadata
|
||||
id: PROT-0020
|
||||
name: Cleaning the Laminar Flow Hood Protocol
|
||||
version: 1.0
|
||||
description: Standard protocol for cleaning and shutting down a laminar flow hood after use
|
||||
author: Lab Staff
|
||||
created: 2025-05-06
|
||||
last_updated: 2025-05-07
|
||||
category: equipment-maintenance
|
||||
|
||||
# Materials required
|
||||
materials:
|
||||
- name: 70% ethanol
|
||||
preparation: Freshly prepared or commercially available
|
||||
storage: Flammable cabinet
|
||||
- name: Paper towels
|
||||
type: Lint-free, laboratory-grade
|
||||
- name: Spray bottle
|
||||
use: For ethanol application
|
||||
- name: Biohazard waste container
|
||||
use: For disposal of contaminated materials
|
||||
|
||||
# Equipment required
|
||||
equipment:
|
||||
- name: Laminar flow hood
|
||||
type: Class II biosafety cabinet
|
||||
- name: Gloves
|
||||
type: Nitrile or latex
|
||||
- name: Lab coat
|
||||
use: Personal protective equipment
|
||||
|
||||
# Protocol steps
|
||||
steps:
|
||||
- step: 1
|
||||
action: "Clear the work area"
|
||||
details: "Remove all materials, reagents, and equipment from the hood"
|
||||
- step: 2
|
||||
action: "Return items to storage"
|
||||
details: "Return all items to their original places; dispose of waste properly"
|
||||
- step: 3
|
||||
action: "Spray work surface"
|
||||
details: "Spray all interior surfaces with 70% ethanol, including the back and side walls"
|
||||
- step: 4
|
||||
action: "Wipe surfaces"
|
||||
details: "Wipe down all interior surfaces with paper towels in a front-to-back motion"
|
||||
- step: 5
|
||||
action: "Clean the sash"
|
||||
details: "Spray and wipe the sash/glass shield on both sides"
|
||||
- step: 6
|
||||
action: "Final inspection"
|
||||
details: "Ensure all surfaces are dry and no items remain in the cabinet"
|
||||
- step: 7
|
||||
action: "Lower the sash"
|
||||
details: "Close the cabinet to the appropriate position according to manufacturer's guidelines"
|
||||
- step: 8
|
||||
action: "Activate UV light"
|
||||
details: "Turn on UV light if available (many cabinets activate this automatically when closed)"
|
||||
- step: 9
|
||||
action: "Turn off equipment"
|
||||
details: "Turn off vacuum, microscope, and other associated equipment"
|
||||
- step: 10
|
||||
action: "Turn off room lights"
|
||||
details: "Turn off lights when leaving the cell culture room if you are the last person"
|
||||
|
||||
# Critical parameters
|
||||
critical_parameters:
|
||||
- parameter: "Cleaning direction"
|
||||
details: "Always wipe from clean to dirty areas (typically back to front) to avoid spreading contaminants"
|
||||
- parameter: "Complete coverage"
|
||||
details: "Ensure all surfaces that may have been contacted are thoroughly cleaned"
|
||||
- parameter: "Appropriate disinfectant"
|
||||
details: "70% ethanol is standard; for specific contaminants, follow institutional guidelines"
|
||||
|
||||
# Troubleshooting
|
||||
troubleshooting:
|
||||
- problem: "Persistent odor in hood"
|
||||
solution: "Perform thorough cleaning; check for spills beneath work surface; notify lab manager if odor persists"
|
||||
- problem: "Visible contamination"
|
||||
solution: "Clean affected area immediately; if extensive, consult lab manager for decontamination protocol"
|
||||
- problem: "UV light not functioning"
|
||||
solution: "Report to lab manager; UV bulbs have limited lifespan and require periodic replacement"
|
||||
|
||||
# Safety considerations
|
||||
safety:
|
||||
ppe: "Lab coat and gloves required"
|
||||
hazards: "70% ethanol is flammable; ensure all sources of ignition are distant from working area"
|
||||
waste: "Dispose of contaminated materials in appropriate biohazard container"
|
||||
uv_safety: "Never look directly at UV light; ensure cabinet is fully closed before UV cycle begins"
|
||||
|
||||
# Quality control
|
||||
quality_control:
|
||||
- check: "Visual inspection"
|
||||
criteria: "No visible debris, media spills, or residue should remain"
|
||||
- check: "Airflow indicator"
|
||||
criteria: "Verify proper airflow indicators before next use"
|
||||
|
||||
# Maintenance schedule
|
||||
maintenance_schedule:
|
||||
- frequency: "After each use"
|
||||
action: "Surface cleaning with 70% ethanol"
|
||||
- frequency: "Weekly"
|
||||
action: "More thorough cleaning including work area under removable surfaces"
|
||||
- frequency: "Annually"
|
||||
action: "Professional certification and maintenance by qualified technician"
|
||||
|
||||
# References
|
||||
references:
|
||||
- "CDC/NIH. (2020) Biosafety in Microbiological and Biomedical Laboratories, 6th Edition"
|
||||
- "World Health Organization. (2004) Laboratory Biosafety Manual, 3rd Edition"
|
||||
|
||||
# Notes
|
||||
notes: |
|
||||
- This protocol should be followed after every use of the laminar flow hood
|
||||
- Ensures proper sterilization and extends the life of the equipment
|
||||
- UV light exposure time should be at least 15-30 minutes for effective surface decontamination
|
||||
- UV light does not penetrate dust or organic material, so physical cleaning must always precede UV sterilization
|
||||
- Report any malfunction or damage to lab manager immediately
|
||||
- Never use bleach in stainless steel biosafety cabinets unless specifically approved by manufacturer
|
||||
- Leave blower running continuously in most biosafety cabinets; consult lab-specific policy
|
||||
---
|
||||
139
protocols/conjugating_bsa_to_fatty_acid_v1.yaml
Normal file
139
protocols/conjugating_bsa_to_fatty_acid_v1.yaml
Normal file
@ -0,0 +1,139 @@
|
||||
---
|
||||
# Protocol metadata
|
||||
id: PROT-0024
|
||||
name: Conjugating BSA to Fatty Acid Protocol
|
||||
version: 1.0
|
||||
description: Protocol for conjugating fatty acids to BSA for cell treatments
|
||||
author: Lab Staff
|
||||
created: 2025-05-06
|
||||
last_updated: 2025-05-07
|
||||
category: reagent-preparation
|
||||
|
||||
# Materials required
|
||||
materials:
|
||||
- name: Fatty acid–free BSA
|
||||
supplier: Sigma, catalog #A6003
|
||||
amount: 0.64 g
|
||||
storage: 4°C
|
||||
- name: NaCl
|
||||
concentration: 5 M stock solution
|
||||
storage: Room temperature
|
||||
- name: NaHCO₃
|
||||
amount: 13.6 mg
|
||||
storage: Room temperature
|
||||
- name: Distilled water
|
||||
purity: Tissue culture grade
|
||||
- name: Palmitic acid
|
||||
supplier: Sigma, catalog #P5585-10G
|
||||
amount: ~5 mg
|
||||
storage: Room temperature
|
||||
alternative: Oleic or arachidonic acid
|
||||
- name: Wako NEFA-HR Assay Kit
|
||||
use: For measuring fatty acid concentration
|
||||
storage: As specified by manufacturer
|
||||
- name: 2 mL microcentrifuge tube
|
||||
type: Sterile, polypropylene
|
||||
|
||||
# Equipment required
|
||||
equipment:
|
||||
- name: Analytical balance
|
||||
precision: 0.1 mg
|
||||
- name: Pipettes/micropipettes
|
||||
range: 10 μL to 1000 μL
|
||||
- name: Incubator or water bath
|
||||
temperatures: 65°C and 37°C
|
||||
- name: Sterile 0.2 µm filter
|
||||
type: Syringe filter
|
||||
- name: Vortex mixer
|
||||
settings: Medium speed
|
||||
- name: pH meter (optional)
|
||||
use: To verify pH of final solution
|
||||
|
||||
# Solutions preparation
|
||||
solutions:
|
||||
- name: 150 mM NaCl solution
|
||||
preparation: "Dilute 60 μL of 5 M NaCl with 1940 μL water"
|
||||
volume: 2 mL
|
||||
- name: Saturated NaHCO₃ solution
|
||||
preparation: "Dissolve 13.6 mg NaHCO₃ in 46 μL distilled water"
|
||||
volume: 46 μL
|
||||
|
||||
# Protocol steps
|
||||
steps:
|
||||
- step: 1
|
||||
action: "Prepare BSA solution"
|
||||
details: "Dissolve 0.64 g fatty acid-free BSA in 1.5 mL of 150 mM NaCl solution, then add water to reach 2 mL total volume (4.8 mM BSA final)"
|
||||
- step: 2
|
||||
action: "Prepare saturated NaHCO₃ solution"
|
||||
details: "Dissolve 13.6 mg NaHCO₃ in 46 μL distilled water; warm at 65°C to ensure complete dissolution"
|
||||
- step: 3
|
||||
action: "Prepare fatty acid solution"
|
||||
details: "Add approximately 5 mg palmitic acid (or equivalent amount for oleic or arachidonic acid) to the NaHCO₃ solution and incubate at 65°C for 2 minutes to form the sodium salt"
|
||||
- step: 4
|
||||
action: "Combine solutions"
|
||||
details: "Transfer 1.6 mL of the BSA solution into the tube containing the fatty acid mixture"
|
||||
- step: 5
|
||||
action: "Incubate combined solution"
|
||||
details: "Incubate at 37°C for 70 minutes with occasional gentle mixing to promote fatty acid binding to BSA"
|
||||
- step: 6
|
||||
action: "Filter solution"
|
||||
details: "Filter the final mixture through a 0.2 μm filter to remove any undissolved particles"
|
||||
- step: 7
|
||||
action: "Measure fatty acid concentration"
|
||||
details: "Use the Wako NEFA-HR assay to measure the fatty acid concentration (expected around 10 mM; roughly a 2:1 fatty acid to BSA molar ratio)"
|
||||
- step: 8
|
||||
action: "Store solution"
|
||||
details: "Aliquot if needed and store at -20°C or 4°C depending on timeframe for use"
|
||||
|
||||
# Critical parameters
|
||||
critical_parameters:
|
||||
- parameter: "Fatty acid solubilization"
|
||||
details: "Complete dissolution of fatty acids in NaHCO₃ is critical for proper complexation"
|
||||
- parameter: "Temperature control"
|
||||
details: "Maintain 65°C during solubilization and 37°C during complexation for optimal results"
|
||||
- parameter: "BSA quality"
|
||||
details: "Use fatty acid-free BSA to ensure accurate FA:BSA ratios"
|
||||
|
||||
# Calculations
|
||||
calculations:
|
||||
- calculation: "BSA molarity"
|
||||
formula: "4.8 mM = 0.64 g ÷ 66,430 g/mol ÷ 0.002 L"
|
||||
- calculation: "Expected FA:BSA ratio"
|
||||
formula: "10 mM FA ÷ 4.8 mM BSA ≈ 2:1 ratio"
|
||||
|
||||
# Troubleshooting
|
||||
troubleshooting:
|
||||
- problem: "Fatty acid precipitation"
|
||||
solution: "Ensure complete dissolution in NaHCO₃ before adding BSA; increase incubation time at 65°C"
|
||||
- problem: "Low final concentration"
|
||||
solution: "Verify fatty acid weight; extend binding incubation time; optimize NaHCO₃ concentration"
|
||||
- problem: "Solution turbidity"
|
||||
solution: "Filter solution multiple times; decrease fatty acid:BSA ratio"
|
||||
|
||||
# Safety considerations
|
||||
safety:
|
||||
ppe: "Lab coat and gloves required"
|
||||
hazards: "NaHCO₃ is irritating to eyes; use caution with hot water baths"
|
||||
|
||||
# Quality control
|
||||
quality_control:
|
||||
- check: "Fatty acid concentration"
|
||||
criteria: "Should be approximately 10 mM when measured by NEFA-HR assay"
|
||||
- check: "Solution clarity"
|
||||
criteria: "Should be clear with no visible precipitate after filtration"
|
||||
|
||||
# References
|
||||
references:
|
||||
- "Alsabeeh N, et al. (2018) Protocols for the preparation of long-chain fatty acid/bovine serum albumin complexes for experimental studies. STAR Protoc. 1(1):100040"
|
||||
- "Spector AA. (1975) Fatty acid binding to plasma albumin. J Lipid Res. 16(3):165-179"
|
||||
|
||||
# Notes
|
||||
notes: |
|
||||
- For 10 mM final fatty acid concentration, use approximately 5 mg of palmitic acid
|
||||
- Can be adapted for oleic acid or arachidonic acid by substituting the appropriate fatty acid
|
||||
- The 150 mM NaCl solution is prepared by diluting 60 μL of 5 M NaCl with 1940 μL water
|
||||
- Expected molar ratio is approximately 2:1 (fatty acid to BSA)
|
||||
- This small-scale protocol (2 mL) is designed for experimental optimization and can be scaled up as needed
|
||||
- BSA-FA complexes are most stable when stored at -20°C but can be stored at 4°C for up to 1 week
|
||||
- Solution pH should be approximately 7.4; adjust with NaOH if needed
|
||||
---
|
||||
132
protocols/counting_cells_manually_v1.yaml
Normal file
132
protocols/counting_cells_manually_v1.yaml
Normal file
@ -0,0 +1,132 @@
|
||||
---
|
||||
# Protocol metadata
|
||||
id: PROT-0023
|
||||
name: Counting Cells Manually Protocol
|
||||
version: 1.0
|
||||
description: Protocol for manual cell counting using a hemocytometer and trypan blue
|
||||
author: Lab Staff
|
||||
created: 2025-05-06
|
||||
last_updated: 2025-05-07
|
||||
category: cell-culture
|
||||
|
||||
# Materials required
|
||||
materials:
|
||||
- name: Cell suspension
|
||||
preparation: Freshly harvested with trypsin/dissociation reagent
|
||||
temperature: Room temperature
|
||||
- name: Trypan Blue solution
|
||||
concentration: 0.4% in PBS
|
||||
storage: Room temperature
|
||||
- name: Microcentrifuge tubes
|
||||
type: 1.5 mL sterile
|
||||
- name: Hemocytometer
|
||||
type: Standard with coverslip
|
||||
- name: PBS
|
||||
concentration: 1X, sterile
|
||||
use: Optional for dilution if cell concentration is too high
|
||||
|
||||
# Equipment required
|
||||
equipment:
|
||||
- name: Microscope
|
||||
type: Inverted or upright with 10x objective
|
||||
- name: Pipettes
|
||||
range: 2-200 μL
|
||||
- name: Calculator
|
||||
use: For cell counting calculations
|
||||
- name: Counter (optional)
|
||||
use: Manual cell counter or clicker
|
||||
- name: Tissue wipes
|
||||
use: For cleaning hemocytometer
|
||||
|
||||
# Protocol steps
|
||||
steps:
|
||||
- step: 1
|
||||
action: "Prepare cell suspension"
|
||||
details: "After harvesting cells with trypsin or other dissociation reagent, mix the cell suspension well by gentle pipetting"
|
||||
- step: 2
|
||||
action: "Sample the cell suspension"
|
||||
details: "Transfer 10–20 μL of the cell suspension into a microcentrifuge tube"
|
||||
- step: 3
|
||||
action: "Add trypan blue"
|
||||
details: "Add an equal volume of 0.4% Trypan Blue solution to the cell suspension and mix gently"
|
||||
- step: 4
|
||||
action: "Load hemocytometer"
|
||||
details: "Apply 10 μL of the cell/trypan blue mixture to the edge of the coverslip on the hemocytometer chamber and allow to fill by capillary action"
|
||||
- step: 5
|
||||
action: "Wait for cells to settle"
|
||||
details: "Allow cells to settle for 10-30 seconds before counting"
|
||||
- step: 6
|
||||
action: "Count cells"
|
||||
details: "Count live cells (unstained, bright) and dead cells (blue-stained) in the four outer corner squares of the hemocytometer grid"
|
||||
- step: 7
|
||||
action: "Calculate cell concentration"
|
||||
details: "Apply the formula: Cells/mL = Average count per square × Dilution factor × 10⁴"
|
||||
- step: 8
|
||||
action: "Calculate viability percentage"
|
||||
details: "Viability (%) = [Live cell count ÷ (Live cell count + Dead cell count)] × 100"
|
||||
- step: 9
|
||||
action: "Calculate total viable cells"
|
||||
details: "Total viable cells = Cell concentration (cells/mL) × Total volume of cell suspension (mL) × Viability (%)/100"
|
||||
|
||||
# Calculation formulas
|
||||
calculations:
|
||||
- calculation: "Cell concentration"
|
||||
formula: "Cells/mL = Average count per corner square × Dilution factor × 10⁴"
|
||||
example: "25 cells/square average × 2 (dilution) × 10⁴ = 5 × 10⁵ cells/mL"
|
||||
- calculation: "Cell viability"
|
||||
formula: "Viability (%) = [Live cell count ÷ (Live cell count + Dead cell count)] × 100"
|
||||
example: "80 live cells ÷ (80 live + 20 dead) × 100 = 80% viability"
|
||||
- calculation: "Total viable cells"
|
||||
formula: "Total viable cells = Cells/mL × Total volume (mL) × (Viability % ÷ 100)"
|
||||
example: "5 × 10⁵ cells/mL × 10 mL × 0.8 = 4 × 10⁶ total viable cells"
|
||||
|
||||
# Critical parameters
|
||||
critical_parameters:
|
||||
- parameter: "Cell mixing"
|
||||
details: "Ensure thorough but gentle mixing to get a uniform suspension without damaging cells"
|
||||
- parameter: "Counting area"
|
||||
details: "Count cells touching the top and left lines of each corner square, but not the bottom or right lines"
|
||||
- parameter: "Counting time"
|
||||
details: "Count within 3-5 minutes of trypan blue addition; longer exposure can lead to false positives"
|
||||
|
||||
# Troubleshooting
|
||||
troubleshooting:
|
||||
- problem: "Too many cells to count"
|
||||
solution: "Dilute sample further with PBS and repeat, adjusting dilution factor in calculations"
|
||||
- problem: "Too few cells to count"
|
||||
solution: "Concentrate sample by centrifugation and resuspend in smaller volume"
|
||||
- problem: "Air bubbles in chamber"
|
||||
solution: "Clean and dry hemocytometer and coverslip, then reload carefully"
|
||||
- problem: "Uneven cell distribution"
|
||||
solution: "Mix cell suspension more thoroughly before sampling"
|
||||
|
||||
# Safety considerations
|
||||
safety:
|
||||
ppe: "Lab coat and gloves required"
|
||||
hazards: "Trypan blue is potentially carcinogenic; handle with care and dispose of properly"
|
||||
|
||||
# Quality control
|
||||
quality_control:
|
||||
- check: "Cell number"
|
||||
criteria: "Count at least 100 cells total for statistical reliability"
|
||||
- check: "Chamber loading"
|
||||
criteria: "Ensure chamber is not under- or overloaded; cells should be in a single plane"
|
||||
- check: "Replicate counts"
|
||||
criteria: "Count both chambers of hemocytometer; values should be within 10% of each other"
|
||||
|
||||
# References
|
||||
references:
|
||||
- "Strober W. (2015) Trypan Blue Exclusion Test of Cell Viability. Curr Protoc Immunol. 111:A3.B.1-A3.B.3"
|
||||
- "Louis KS, Siegel AC. (2011) Cell viability analysis using trypan blue: manual and automated methods. Methods Mol Biol. 740:7-12"
|
||||
|
||||
# Notes
|
||||
notes: |
|
||||
- Trypan blue stains dead cells blue, while live cells remain unstained
|
||||
- For accurate results, count at least 100 cells total
|
||||
- Dilution factor must be accounted for in final calculations
|
||||
- If cell clumps are present, they may indicate incomplete dissociation
|
||||
- Hemocytometer and coverslip must be clean and free of scratches
|
||||
- The hemocytometer chamber depth is 0.1 mm and each corner square has an area of 1 mm²
|
||||
- The multiplier 10⁴ converts the count to cells per mL (1 cm³ = 1000 mm³ = 1 mL)
|
||||
- For primary cells or delicate cell lines, consider automated counting methods
|
||||
---
|
||||
160
protocols/gentle_coip_v1.yaml
Normal file
160
protocols/gentle_coip_v1.yaml
Normal file
@ -0,0 +1,160 @@
|
||||
---
|
||||
# Protocol metadata
|
||||
id: PROT-0012
|
||||
name: Gentle Co-Immunoprecipitation Protocol
|
||||
version: 1.0
|
||||
description: A gentle co-immunoprecipitation protocol for preserving protein-protein interactions
|
||||
author: JM Jordan
|
||||
created: 2023-01-01
|
||||
last_updated: 2025-05-07
|
||||
category: protein-analysis
|
||||
|
||||
# Materials required
|
||||
materials:
|
||||
- name: Protein lysate
|
||||
amount: 3600 μg per condition
|
||||
preparation: Fresh or previously frozen
|
||||
- name: RIPA buffer
|
||||
preparation: With protease inhibitor tablets added fresh
|
||||
storage: 4°C
|
||||
- name: Antibody for immunoprecipitation
|
||||
amount: 2 μg per reaction
|
||||
storage: -20°C
|
||||
- name: Protein A/G beads
|
||||
preparation: 50% slurry in storage buffer
|
||||
storage: 4°C
|
||||
- name: PBS with 0.02% Tween
|
||||
preparation: Filter-sterilized
|
||||
storage: Room temperature
|
||||
- name: Non-denaturing loading buffer
|
||||
concentration: 1X
|
||||
storage: -20°C
|
||||
- name: LoBind tubes
|
||||
type: Protein low-binding microcentrifuge tubes
|
||||
- name: Liquid nitrogen
|
||||
amount: Sufficient for snap-freezing samples
|
||||
|
||||
# Equipment required
|
||||
equipment:
|
||||
- name: Rotating mixer
|
||||
temperature: 4°C and room temperature capable
|
||||
- name: Refrigerated centrifuge
|
||||
settings: Low speed (1000g)
|
||||
- name: Magnetic rack
|
||||
type: For bead separation
|
||||
- name: 37°C heat block or water bath
|
||||
use: For elution step
|
||||
- name: -80°C freezer
|
||||
use: Sample storage
|
||||
|
||||
# Protocol steps
|
||||
steps:
|
||||
- step: 1
|
||||
action: "Prepare protein lysate"
|
||||
details: "Prepare a lysate solution with 3600 μg of protein for each condition (e.g., HFA and HFB)"
|
||||
- step: 2
|
||||
action: "Adjust lysate volume"
|
||||
details: "Adjust each lysate to 1 mL total volume with RIPA buffer containing protease inhibitor tablets"
|
||||
- step: 3
|
||||
action: "Preclear lysate"
|
||||
details: "Add 50 μL of 50% bead slurry to each tube of lysate and rotate at 4°C for 1 hour"
|
||||
- step: 4
|
||||
action: "Remove beads"
|
||||
details: "Collect precleared lysate by centrifugation or magnetic separation"
|
||||
- step: 5
|
||||
action: "Aliquot lysate"
|
||||
details: "Divide each precleared lysate into 3 tubes (6 tubes total) and adjust each to 1 mL with RIPA buffer with inhibitors"
|
||||
- step: 6
|
||||
action: "Add antibody"
|
||||
details: "Add 2 μg of antibody to each tube"
|
||||
- step: 7
|
||||
action: "Incubate overnight"
|
||||
details: "Rotate tubes overnight at 4°C to allow antibody binding"
|
||||
- step: 8
|
||||
action: "Continue incubation"
|
||||
details: "Continue with room temperature rotation for 1 hour"
|
||||
- step: 9
|
||||
action: "Prepare beads"
|
||||
details: "Wash 600 μL beads with PBS + 0.02% Tween and resuspend in 600 μL RIPA buffer with inhibitors"
|
||||
- step: 10
|
||||
action: "Add beads to samples"
|
||||
details: "Add 100 μL of bead solution to each tube"
|
||||
- step: 11
|
||||
action: "Capture complexes"
|
||||
details: "Rotate at room temperature for 1 hour to capture antibody-protein complexes"
|
||||
- step: 12
|
||||
action: "Wash beads"
|
||||
details: "Wash beads 3X with 1 mL PBS + 0.02% Tween using gentle pipet mixing (no extended rotation)"
|
||||
- step: 13
|
||||
action: "Transfer beads"
|
||||
details: "After final wash, resuspend beads in 200 μL PBS + 0.02% Tween and transfer to fresh LoBind tubes"
|
||||
- step: 14
|
||||
action: "Elute proteins"
|
||||
details: "Add 30 μL 1X non-denaturing loading buffer and incubate at 37°C for 5 minutes"
|
||||
- step: 15
|
||||
action: "Collect eluate"
|
||||
details: "Separate solution from beads into new LoBind tubes"
|
||||
- step: 16
|
||||
action: "Freeze samples"
|
||||
details: "Snap freeze eluate in liquid nitrogen"
|
||||
- step: 17
|
||||
action: "Store samples"
|
||||
details: "Store at -80°C until analysis"
|
||||
|
||||
# Critical parameters
|
||||
critical_parameters:
|
||||
- parameter: "Gentleness of handling"
|
||||
details: "Avoid harsh mixing or vortexing to preserve protein-protein interactions"
|
||||
- parameter: "Washing steps"
|
||||
details: "Brief, gentle washes without extended rotation periods minimize disruption of complexes"
|
||||
- parameter: "Temperature"
|
||||
details: "Maintain 4°C during initial binding to reduce non-specific interactions"
|
||||
- parameter: "Elution conditions"
|
||||
details: "Use non-denaturing conditions and mild temperature (37°C vs 95°C)"
|
||||
|
||||
# Controls
|
||||
controls:
|
||||
- control: "IgG control"
|
||||
purpose: "Use matched isotype IgG instead of specific antibody to identify non-specific binding"
|
||||
- control: "Input sample"
|
||||
purpose: "Save an aliquot of pre-IP lysate to verify presence of proteins in starting material"
|
||||
- control: "Beads-only control"
|
||||
purpose: "Beads without antibody to identify proteins binding directly to beads"
|
||||
|
||||
# Troubleshooting
|
||||
troubleshooting:
|
||||
- problem: "Low or no co-immunoprecipitated protein"
|
||||
solution: "Increase lysate amount; reduce washing stringency; verify antibody efficacy; use crosslinking"
|
||||
- problem: "High background"
|
||||
solution: "Increase preclearance time; use more stringent wash buffer; validate antibody specificity"
|
||||
- problem: "Degraded proteins"
|
||||
solution: "Add additional protease inhibitors; work more quickly; keep samples cold"
|
||||
|
||||
# Safety considerations
|
||||
safety:
|
||||
ppe: "Lab coat, gloves, and eye protection required"
|
||||
hazards: "Liquid nitrogen causes freeze burns; handle with appropriate PPE and in well-ventilated area"
|
||||
|
||||
# Downstream applications
|
||||
downstream_applications:
|
||||
- name: "Western blotting"
|
||||
preparation: "Add SDS sample buffer if needed; may need to optimize antibody to avoid IP antibody detection"
|
||||
- name: "Mass spectrometry"
|
||||
preparation: "Consider specific elution methods compatible with MS analysis"
|
||||
|
||||
# References
|
||||
references:
|
||||
- "Lin YC, et al. (2018) Optimization of a Co-Immunoprecipitation Protocol for the Detection of Weak Protein-Protein Interactions. PLoS ONE 13(10):e0206167"
|
||||
- "Antrobus R & Borner GH. (2011) Improved elution conditions for native co-immunoprecipitation. PLoS ONE 6(3):e18218"
|
||||
|
||||
# Notes
|
||||
notes: |
|
||||
- This is a gentle protocol designed to preserve protein-protein interactions
|
||||
- Uses minimal washing steps without extended rotations
|
||||
- Includes preclearance step to reduce non-specific binding
|
||||
- Remember to freeze Input and leftover samples
|
||||
- For weaker interactions, consider chemical crosslinking before lysis
|
||||
- Non-denaturing loading buffer preserves complexes for native gel electrophoresis
|
||||
- Alternative elution methods include peptide competition or pH elution
|
||||
- For subsequent SDS-PAGE analysis, an aliquot can be mixed with standard denaturing loading buffer
|
||||
---
|
||||
164
protocols/harvesting_cells_for_rna_extraction_v1.yaml
Normal file
164
protocols/harvesting_cells_for_rna_extraction_v1.yaml
Normal file
@ -0,0 +1,164 @@
|
||||
---
|
||||
# Protocol metadata
|
||||
id: PROT-0022
|
||||
name: Harvesting Cells for RNA Extraction Protocol
|
||||
version: 1.0
|
||||
description: Protocol for harvesting cultured cells for RNA extraction using TRI reagent
|
||||
author: Lab Staff
|
||||
created: 2025-05-06
|
||||
last_updated: 2025-05-07
|
||||
category: molecular-biology
|
||||
|
||||
# Materials required
|
||||
materials:
|
||||
- name: PBS
|
||||
preparation: Sterile, calcium and magnesium-free
|
||||
temperature: Room temperature
|
||||
storage: 4°C
|
||||
- name: TRI reagent (or TRIzol)
|
||||
temperature: Room temperature
|
||||
storage: 4°C, protected from light
|
||||
hazards: Contains phenol and guanidinium thiocyanate
|
||||
- name: 1.5-mL microcentrifuge tubes
|
||||
type: RNase-free
|
||||
preparation: Pre-labeled with sample information
|
||||
- name: Sample storage box
|
||||
type: For -80°C storage
|
||||
preparation: Labeled with experiment details and date
|
||||
|
||||
# Equipment required
|
||||
equipment:
|
||||
- name: Laminar flow hood
|
||||
type: Class II biosafety cabinet
|
||||
- name: Fume hood
|
||||
use: For handling TRI reagent
|
||||
- name: Multi-channel pipette
|
||||
range: 20-200 μL
|
||||
- name: Single-channel pipettes
|
||||
range: Various sizes
|
||||
- name: -80°C freezer
|
||||
use: For sample storage
|
||||
- name: Personal protective equipment
|
||||
type: Lab coat, gloves, safety glasses
|
||||
|
||||
# Protocol steps
|
||||
steps:
|
||||
- step: 1
|
||||
action: "Prepare collection tubes"
|
||||
details: "Label 1.5-mL microcentrifuge tubes with sample information, date, and experiment"
|
||||
- step: 2
|
||||
action: "Prepare storage box"
|
||||
details: "Label sample storage box for -80°C with experiment name, date, and researcher name"
|
||||
- step: 3
|
||||
action: "Retrieve cell cultures"
|
||||
details: "Remove cells from incubator and transfer to laminar flow hood"
|
||||
- step: 4
|
||||
action: "Remove media"
|
||||
details: "Aspirate media from cells carefully without disturbing cell layer"
|
||||
- step: 5
|
||||
action: "Wash cells"
|
||||
details: "Add 200 μL (96-well) or 1 mL (24-well) PBS per well to wash cells"
|
||||
- step: 6
|
||||
action: "Remove PBS"
|
||||
details: "Aspirate PBS completely until wells are dry"
|
||||
- step: 7
|
||||
action: "Move to fume hood"
|
||||
details: "Transfer culture plate to fume hood for TRI reagent handling"
|
||||
- step: 8
|
||||
action: "Add TRI reagent"
|
||||
details: "Add 100 μL (96-well) or 300 μL (24-well) TRI reagent to each well"
|
||||
- step: 9
|
||||
action: "Lyse cells"
|
||||
details: "Incubate at room temperature for 5 minutes to ensure complete cell lysis"
|
||||
- step: 10
|
||||
action: "Collect lysate"
|
||||
details: "Tilt plate back slightly to pool TRI reagent and lysed cells in the corner of each well"
|
||||
- step: 11
|
||||
action: "Transfer to tubes"
|
||||
details: "Pipet all solution into prelabeled microcentrifuge tubes"
|
||||
- step: 12
|
||||
action: "Organize samples"
|
||||
details: "Place tubes in labeled storage box in orderly arrangement"
|
||||
- step: 13
|
||||
action: "Record sample information"
|
||||
details: "Record sample positions in laboratory notebook or digital record"
|
||||
- step: 14
|
||||
action: "Store samples"
|
||||
details: "Freeze tubes at -80°C and submit a -80°C Sample Submission form"
|
||||
|
||||
# Volume guidelines
|
||||
volume_guidelines:
|
||||
- plate_format: "96-well plate"
|
||||
wash_volume: "200 μL PBS per well"
|
||||
tri_reagent_volume: "100 μL per well"
|
||||
expected_yield: "0.5-2 μg RNA per well (cell type dependent)"
|
||||
- plate_format: "24-well plate"
|
||||
wash_volume: "1 mL PBS per well"
|
||||
tri_reagent_volume: "300 μL per well"
|
||||
expected_yield: "3-8 μg RNA per well (cell type dependent)"
|
||||
- plate_format: "12-well plate"
|
||||
wash_volume: "1.5 mL PBS per well"
|
||||
tri_reagent_volume: "500 μL per well"
|
||||
expected_yield: "5-15 μg RNA per well (cell type dependent)"
|
||||
- plate_format: "6-well plate"
|
||||
wash_volume: "2 mL PBS per well"
|
||||
tri_reagent_volume: "1 mL per well"
|
||||
expected_yield: "10-30 μg RNA per well (cell type dependent)"
|
||||
|
||||
# Critical parameters
|
||||
critical_parameters:
|
||||
- parameter: "RNase-free environment"
|
||||
details: "Work quickly and use RNase-free materials to prevent RNA degradation"
|
||||
- parameter: "Complete cell lysis"
|
||||
details: "Ensure TRI reagent fully covers cells and allow sufficient lysis time"
|
||||
- parameter: "Sample traceability"
|
||||
details: "Maintain clear labeling and documentation of samples"
|
||||
|
||||
# Troubleshooting
|
||||
troubleshooting:
|
||||
- problem: "Low RNA yield"
|
||||
solution: "Ensure complete cell lysis; adjust TRI reagent volume for cell density; avoid over-washing"
|
||||
- problem: "RNA degradation"
|
||||
solution: "Work quickly; use RNase-free materials; ensure proper sample storage"
|
||||
- problem: "DNA contamination"
|
||||
solution: "Consider DNase treatment during RNA purification steps"
|
||||
|
||||
# Safety considerations
|
||||
safety:
|
||||
ppe: "Lab coat, nitrile gloves, and safety glasses required"
|
||||
hazards: "TRI reagent contains phenol and guanidinium thiocyanate; work in fume hood only"
|
||||
disposal: "Collect TRI reagent waste in appropriate waste container; do not dispose down drain"
|
||||
precautions: "Avoid skin contact; if contact occurs, wash immediately with copious water"
|
||||
|
||||
# Quality control
|
||||
quality_control:
|
||||
- check: "Sample labeling"
|
||||
criteria: "Verify all tubes are clearly labeled before freezing"
|
||||
- check: "Sample logging"
|
||||
criteria: "All samples must be logged in freezer inventory system"
|
||||
|
||||
# Downstream applications
|
||||
downstream_applications:
|
||||
- application: "RNA extraction"
|
||||
protocol: "RNA MiniPrep with DirectZol kit protocol"
|
||||
- application: "RNA-Seq"
|
||||
protocol: "Submit to sequencing facility after quality control"
|
||||
- application: "qPCR"
|
||||
protocol: "cDNA synthesis followed by RT-qPCR"
|
||||
|
||||
# References
|
||||
references:
|
||||
- "Chomczynski P, Sacchi N. (2006) The single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction: twenty-something years on. Nature Protocols 1(2):581-585"
|
||||
- "Rio DC, et al. (2010) Purification of RNA Using TRIzol (TRI Reagent). Cold Spring Harbor Protocols 2010(6)"
|
||||
|
||||
# Notes
|
||||
notes: |
|
||||
- If proceeding directly to extraction, see RNA MiniPrep with DirectZol kit protocol
|
||||
- Volumes should be adjusted based on well size (96-well or 24-well format)
|
||||
- Always work in the fume hood when handling TRI reagent/TRIzol
|
||||
- RNA can be stored at -80°C for months to years without significant degradation
|
||||
- For cells growing in suspension, centrifuge cells first before adding TRI reagent
|
||||
- For highly confluent wells, increase the volume of TRI reagent accordingly
|
||||
- Homogenize samples by pipetting up and down if cell clumps are visible
|
||||
- For long-term storage, consider using RNA stabilization reagents like RNAlater
|
||||
---
|
||||
147
protocols/mrna_stability_assay_v1.yaml
Normal file
147
protocols/mrna_stability_assay_v1.yaml
Normal file
@ -0,0 +1,147 @@
|
||||
---
|
||||
# Protocol metadata
|
||||
id: PROT-0011
|
||||
name: mRNA Stability Assay Protocol
|
||||
version: 1.0
|
||||
description: Protocol for measuring mRNA stability using siNC vs. siYbx1 with Actinomycin D treatment
|
||||
author: Jordan Lab
|
||||
created: 2025-05-06
|
||||
last_updated: 2025-05-07
|
||||
category: molecular-biology
|
||||
|
||||
# Materials required
|
||||
materials:
|
||||
- name: Actinomycin D
|
||||
concentration: 5 μg/mL in media
|
||||
storage: -20°C, protected from light
|
||||
preparation: Dissolve in DMSO to make stock solution
|
||||
- name: DMSO (control)
|
||||
purity: Cell culture grade
|
||||
storage: Room temperature
|
||||
- name: Complete medium
|
||||
type: Appropriate for cell type
|
||||
temperature: 37°C pre-warmed
|
||||
- name: PBS (ice-cold)
|
||||
concentration: 1X, sterile
|
||||
temperature: 4°C
|
||||
- name: TRIzol or RLT buffer
|
||||
storage: Room temperature (TRIzol) or 4°C (RLT)
|
||||
notes: Choose based on downstream RNA isolation method
|
||||
|
||||
# Equipment required
|
||||
equipment:
|
||||
- name: Multi-channel pipette
|
||||
type: 8 or 12 channel, 20-200 μL
|
||||
- name: Cell culture plates
|
||||
type: 96-well or 24-well plates
|
||||
- name: Sample storage tubes
|
||||
type: RNase-free microcentrifuge tubes
|
||||
- name: Vacuum aspirator
|
||||
settings: Low to medium suction
|
||||
- name: -80°C freezer
|
||||
use: Sample storage
|
||||
|
||||
# Protocol steps
|
||||
steps:
|
||||
- step: 1
|
||||
action: "Prepare Actinomycin D Medium"
|
||||
details: "Make 5 µg/mL ActD in complete medium (~20 mL total for all ActD wells). Prepare just before treatment."
|
||||
- step: 2
|
||||
action: "Prepare DMSO-only control medium"
|
||||
details: "Add equivalent volume of DMSO to complete medium (~5 mL total)"
|
||||
- step: 3
|
||||
action: "Remove old media from the plate"
|
||||
details: "Use multi-channel vacuum aspirator for efficiency"
|
||||
- step: 4
|
||||
action: "Add treatment media"
|
||||
details: "Using a multichannel pipet, rapidly add 100 µL of treatment media according to plate map"
|
||||
- step: 5
|
||||
action: "Immediately collect the 0 hr samples"
|
||||
details: "These samples represent baseline mRNA levels before ActD treatment"
|
||||
- step: 6
|
||||
action: "Cell collection at all time points"
|
||||
details: "Follow steps 7-10 for each time point collection"
|
||||
- step: 7
|
||||
action: "Aspirate medium"
|
||||
details: "Quick removal of medium from wells"
|
||||
- step: 8
|
||||
action: "Wash with ice-cold PBS (optional)"
|
||||
details: "Brief wash with 100 µL ice-cold PBS"
|
||||
- step: 9
|
||||
action: "Lyse cells"
|
||||
details: "Add 50 µL TRIzol or RLT buffer directly to wells"
|
||||
- step: 10
|
||||
action: "Store samples"
|
||||
details: "Transfer lysate immediately to labeled tubes/plate and store at -80°C"
|
||||
- step: 11
|
||||
action: "Repeat for each timepoint"
|
||||
details: "Typical timepoints: 0, 1, 2, 4, 8 hours after ActD addition"
|
||||
|
||||
# Experimental design
|
||||
experimental_design:
|
||||
- condition: "siNC (negative control siRNA) + ActD"
|
||||
purpose: "Control for normal mRNA degradation rates"
|
||||
- condition: "siNC + DMSO"
|
||||
purpose: "Control for vehicle effects"
|
||||
- condition: "siYbx1 + ActD"
|
||||
purpose: "Test effect of Ybx1 knockdown on mRNA stability"
|
||||
- condition: "siYbx1 + DMSO"
|
||||
purpose: "Control for siYbx1 effects independent of transcription inhibition"
|
||||
- timepoints: [0h, 1h, 2h, 4h, 8h]
|
||||
replicates: 3
|
||||
|
||||
# Critical parameters
|
||||
critical_parameters:
|
||||
- parameter: "Time synchronization"
|
||||
details: "Precise timing is critical for accurate half-life determination"
|
||||
- parameter: "Temperature control"
|
||||
details: "Keep PBS cold and work quickly to prevent RNA degradation"
|
||||
- parameter: "ActD concentration"
|
||||
details: "5 μg/mL is standard but may need optimization for some cell types"
|
||||
|
||||
# Troubleshooting
|
||||
troubleshooting:
|
||||
- problem: "High variability between replicates"
|
||||
solution: "Ensure consistent timing between wells and precise volume additions"
|
||||
- problem: "RNA degradation"
|
||||
solution: "Work quickly, keep samples cold, use RNase-free materials"
|
||||
- problem: "Cell death before later timepoints"
|
||||
solution: "May need to reduce ActD concentration for sensitive cell lines"
|
||||
|
||||
# Safety considerations
|
||||
safety:
|
||||
ppe: "Lab coat, gloves, and eye protection required"
|
||||
hazards: "Actinomycin D is toxic and potentially carcinogenic. Handle with care and dispose as hazardous waste."
|
||||
|
||||
# Data analysis
|
||||
data_analysis:
|
||||
- step: 1
|
||||
action: "Extract and quantify RNA"
|
||||
details: "Use standard RNA isolation protocol and quantify by qPCR"
|
||||
- step: 2
|
||||
action: "Calculate relative expression"
|
||||
details: "Normalize target genes to housekeeping gene(s)"
|
||||
- step: 3
|
||||
action: "Plot decay curves"
|
||||
details: "Plot log2 of normalized expression vs. time"
|
||||
- step: 4
|
||||
action: "Calculate half-life"
|
||||
details: "Fit exponential decay curve: y = e^(-kt) where k is decay constant. t1/2 = ln(2)/k"
|
||||
- step: 5
|
||||
action: "Compare half-lives"
|
||||
details: "Compare mRNA half-lives between siNC and siYbx1 conditions"
|
||||
|
||||
# References
|
||||
references:
|
||||
- "Ross J. (1995) mRNA stability in mammalian cells. Microbiol Rev. 59(3):423-450."
|
||||
- "Chen CY, Shyu AB. (2011) Mechanisms of deadenylation-dependent decay. Wiley Interdiscip Rev RNA. 2(2):167-183."
|
||||
|
||||
# Notes
|
||||
notes: |
|
||||
- This protocol compares mRNA stability between control (siNC) and Ybx1 knockdown (siYbx1) conditions
|
||||
- ActD concentration is 5 µg/mL, but may be optimized based on cell type
|
||||
- Use multichannel pipettes to minimize time between treatments
|
||||
- Aspirate and dispense solutions one column at a time to minimize delays
|
||||
- Consider performing preliminary experiments to determine optimal timepoints for your specific mRNAs of interest
|
||||
- Some very stable mRNAs may require longer timepoints (12-24h)
|
||||
---
|
||||
157
protocols/oil_red_o_staining_v1.yaml
Normal file
157
protocols/oil_red_o_staining_v1.yaml
Normal file
@ -0,0 +1,157 @@
|
||||
---
|
||||
# Protocol metadata
|
||||
id: PROT-0015
|
||||
name: Oil Red O Staining Protocol for Adherent Cells
|
||||
version: 1.0
|
||||
description: Protocol for staining and quantifying lipid droplets in adherent hepatocyte-like cells
|
||||
author: J. Jordan
|
||||
created: 2025-02-09
|
||||
last_updated: 2025-05-07
|
||||
category: histology
|
||||
|
||||
# Materials required
|
||||
materials:
|
||||
- name: Oil Red O powder
|
||||
storage: Room temperature, protected from light
|
||||
- name: 100% isopropanol (2-propanol)
|
||||
purity: Analytical grade
|
||||
storage: Room temperature, flammable cabinet
|
||||
- name: 4% paraformaldehyde (PFA)
|
||||
storage: 4°C
|
||||
preparation: Freshly prepared or commercially available
|
||||
- name: PBS
|
||||
concentration: 1X
|
||||
storage: Room temperature
|
||||
- name: Distilled water
|
||||
purity: Ultra-pure preferred
|
||||
- name: 0.2-micron syringe filter
|
||||
type: PVDF or nylon
|
||||
- name: 96-well culture plate
|
||||
type: Clear bottom
|
||||
|
||||
# Equipment required
|
||||
equipment:
|
||||
- name: Echo Revolution inverted microscope
|
||||
settings: Brightfield, 10x and 20x objectives
|
||||
- name: Spectrophotometer
|
||||
settings: 492 nm absorbance measurement
|
||||
- name: Syringe (5-10 mL)
|
||||
use: For filtration of ORO solution
|
||||
- name: Multichannel pipette
|
||||
range: 20-200 μL
|
||||
- name: Shaker/rocker
|
||||
settings: Low to medium speed
|
||||
|
||||
# Solutions preparation
|
||||
solutions:
|
||||
- name: Oil Red O stock solution
|
||||
preparation: "Dissolve 0.175 g ORO powder in 50 mL 100% 2-propanol"
|
||||
storage: "Room temperature, stable for up to 1 month"
|
||||
- name: Oil Red O working solution
|
||||
preparation: "Add 3 parts ORO stock solution to 2 parts distilled water and vortex immediately"
|
||||
storage: "Filter before use, prepare fresh"
|
||||
stability: "Use within 2 hours"
|
||||
|
||||
# Protocol steps
|
||||
steps:
|
||||
- step: 1
|
||||
action: "Prepare Oil Red O working solution"
|
||||
details: "Dilute ORO stock solution in distilled water (Add 3 parts ORO solution to 2 parts dH2O) and vortex immediately"
|
||||
- step: 2
|
||||
action: "Filter diluted ORO solution"
|
||||
details: "Use a syringe with a 0.2-micron filter (critical step)"
|
||||
- step: 3
|
||||
action: "Fix cells"
|
||||
details: "Add 75 μL cold 4% PFA to each well and incubate for 20-30 min at room temperature"
|
||||
- step: 4
|
||||
action: "Wash cells"
|
||||
details: "Aspirate PFA and wash cells twice with 100 μL PBS"
|
||||
- step: 5
|
||||
action: "Dry cells"
|
||||
details: "Aspirate last PBS wash thoroughly until cells are very dry"
|
||||
- step: 6
|
||||
action: "Stain with Oil Red O"
|
||||
details: "Add 75 μL freshly prepared ORO working solution to each well and stain for 30 min at room temperature"
|
||||
- step: 7
|
||||
action: "Wash off excess stain"
|
||||
details: "Wash twice with 150 μL distilled water"
|
||||
- step: 8
|
||||
action: "Prepare for imaging"
|
||||
details: "Add 100 μL PBS to wells if imaging, or proceed directly to extraction"
|
||||
- step: 9
|
||||
action: "Image stained cells"
|
||||
details: "Capture brightfield images using Echo Revolution microscope with consistent imaging parameters"
|
||||
- step: 10
|
||||
action: "Document images"
|
||||
details: "Transfer images to Teams lab notebook data folder with image index"
|
||||
- step: 11
|
||||
action: "Extract Oil Red O"
|
||||
details: "Add 75 μL of 100% isopropanol to each well and agitate for 5 minutes"
|
||||
- step: 12
|
||||
action: "Transfer extract"
|
||||
details: "Transfer 60 μL isopropanol extraction to 96-well assay plate"
|
||||
- step: 13
|
||||
action: "Prepare blank wells"
|
||||
details: "Add 60 μL pure isopropanol to at least 3 wells for background"
|
||||
- step: 14
|
||||
action: "Measure absorbance"
|
||||
details: "Ensure plate reader is set to 492-nm protocol and measure absorbance"
|
||||
- step: 15
|
||||
action: "Export data"
|
||||
details: "Export data to USB drive or laboratory database"
|
||||
|
||||
# Data analysis
|
||||
data_analysis:
|
||||
- step: 1
|
||||
action: "Calculate net absorbance"
|
||||
details: "Subtract average blank (isopropanol only) reading from each sample reading"
|
||||
- step: 2
|
||||
action: "Normalize data (optional)"
|
||||
details: "Normalize to cell number, protein content, or DNA content if needed"
|
||||
- step: 3
|
||||
action: "Statistical analysis"
|
||||
details: "Compare experimental groups using appropriate statistical tests"
|
||||
|
||||
# Critical parameters
|
||||
critical_parameters:
|
||||
- parameter: "Solution filtration"
|
||||
details: "Filtration of ORO working solution is critical to prevent particulate artifacts"
|
||||
- parameter: "Drying before staining"
|
||||
details: "Complete drying after PBS wash ensures optimal stain uptake"
|
||||
- parameter: "Timing of isopropanol extraction"
|
||||
details: "Standardize agitation time (5 minutes) for reproducible extraction"
|
||||
|
||||
# Troubleshooting
|
||||
troubleshooting:
|
||||
- problem: "High background staining"
|
||||
solution: "Ensure complete washing steps; check quality of ORO solutions"
|
||||
- problem: "Weak or uneven staining"
|
||||
solution: "Check ORO solution freshness; ensure cells were completely dry before staining"
|
||||
- problem: "Precipitates in staining solution"
|
||||
solution: "Filter ORO solution thoroughly; prepare fresh working solution"
|
||||
|
||||
# Safety considerations
|
||||
safety:
|
||||
ppe: "Lab coat, gloves, and eye protection required"
|
||||
hazards: "PFA is toxic; isopropanol is flammable; dispose of waste according to institutional guidelines"
|
||||
|
||||
# Quality control
|
||||
quality_control:
|
||||
- check: "Include positive control"
|
||||
criteria: "Cell line known to accumulate lipid droplets (e.g., differentiated 3T3-L1 cells)"
|
||||
- check: "Include negative control"
|
||||
criteria: "Untreated cells or cells with minimal lipid accumulation"
|
||||
|
||||
# References
|
||||
references:
|
||||
- "Mehlem A, et al. (2013) Imaging of neutral lipids by oil red O for analyzing the metabolic status in health and disease. Nat Protoc. 8(6):1149-54"
|
||||
|
||||
# Notes
|
||||
notes: |
|
||||
- Optimized for hepatocyte-like cells (HepG2, Huh7, AML12)
|
||||
- Filtration of ORO solution is critical for good results
|
||||
- Volume specifications are for 96-well plates - adjust for other formats
|
||||
- For automated image analysis, consider using ImageJ/CellProfiler for quantification
|
||||
- ORO stains neutral lipids (primarily triglycerides) red
|
||||
- Semi-quantitative absorbance measurements correlate with total neutral lipid content
|
||||
---
|
||||
139
protocols/preparation_of_bsa_ffa_v1.yaml
Normal file
139
protocols/preparation_of_bsa_ffa_v1.yaml
Normal file
@ -0,0 +1,139 @@
|
||||
---
|
||||
# Protocol metadata
|
||||
id: PROT-0026
|
||||
name: Fatty Acid–BSA Complex Preparation Protocol
|
||||
version: 1.0
|
||||
description: Protocol for preparing fatty acid-BSA complexes in a 2 mL tube format
|
||||
author: Lab Staff
|
||||
created: 2025-05-07
|
||||
last_updated: 2025-05-07
|
||||
category: reagent-preparation
|
||||
|
||||
# Materials required
|
||||
materials:
|
||||
- name: Fatty acid–free BSA
|
||||
supplier: Sigma, catalog #A6003
|
||||
amount: 0.64 g
|
||||
storage: 4°C
|
||||
- name: NaCl
|
||||
concentration: 5 M stock solution
|
||||
storage: Room temperature
|
||||
- name: NaHCO₃
|
||||
amount: 13.6 mg
|
||||
storage: Room temperature
|
||||
- name: Distilled water
|
||||
purity: Tissue culture grade
|
||||
- name: Palmitic acid
|
||||
supplier: Sigma, catalog #P5585-10G
|
||||
amount: ~5 mg
|
||||
storage: Room temperature
|
||||
alternative: Oleic or arachidonic acid
|
||||
- name: Wako NEFA-HR Assay Kit
|
||||
use: For measuring fatty acid concentration
|
||||
storage: As specified by manufacturer
|
||||
- name: 2 mL microcentrifuge tube
|
||||
type: Sterile, polypropylene
|
||||
|
||||
# Equipment required
|
||||
equipment:
|
||||
- name: Analytical balance
|
||||
precision: 0.1 mg
|
||||
- name: Pipettes/micropipettes
|
||||
range: 10 μL to 1000 μL
|
||||
- name: Incubator or water bath
|
||||
temperatures: 65°C and 37°C
|
||||
- name: Sterile 0.2 µm filter
|
||||
type: Syringe filter
|
||||
- name: Vortex mixer
|
||||
settings: Medium speed
|
||||
- name: pH meter (optional)
|
||||
use: To verify pH of final solution
|
||||
|
||||
# Solutions preparation
|
||||
solutions:
|
||||
- name: 150 mM NaCl solution
|
||||
preparation: "Dilute 60 μL of 5 M NaCl with 1940 μL water"
|
||||
volume: 2 mL
|
||||
- name: Saturated NaHCO₃ solution
|
||||
preparation: "Dissolve 13.6 mg NaHCO₃ in 46 μL distilled water"
|
||||
volume: 46 μL
|
||||
|
||||
# Protocol steps
|
||||
steps:
|
||||
- step: 1
|
||||
action: "Prepare BSA solution"
|
||||
details: "Dissolve 0.64 g fatty acid-free BSA in 1.5 mL of 150 mM NaCl solution, then add water to reach 2 mL total volume (4.8 mM BSA final)"
|
||||
- step: 2
|
||||
action: "Prepare saturated NaHCO₃ solution"
|
||||
details: "Dissolve 13.6 mg NaHCO₃ in 46 μL distilled water; warm at 65°C to ensure complete dissolution"
|
||||
- step: 3
|
||||
action: "Prepare fatty acid solution"
|
||||
details: "Add approximately 5 mg palmitic acid (or equivalent amount for oleic or arachidonic acid) to the NaHCO₃ solution and incubate at 65°C for 2 minutes to form the sodium salt"
|
||||
- step: 4
|
||||
action: "Combine solutions"
|
||||
details: "Transfer 1.6 mL of the BSA solution into the tube containing the fatty acid mixture"
|
||||
- step: 5
|
||||
action: "Incubate combined solution"
|
||||
details: "Incubate at 37°C for 70 minutes with occasional gentle mixing to promote fatty acid binding to BSA"
|
||||
- step: 6
|
||||
action: "Filter solution"
|
||||
details: "Filter the final mixture through a 0.2 μm filter to remove any undissolved particles"
|
||||
- step: 7
|
||||
action: "Measure fatty acid concentration"
|
||||
details: "Use the Wako NEFA-HR assay to measure the fatty acid concentration (expected around 10 mM; roughly a 2:1 fatty acid to BSA molar ratio)"
|
||||
- step: 8
|
||||
action: "Store solution"
|
||||
details: "Aliquot if needed and store at -20°C or 4°C depending on timeframe for use"
|
||||
|
||||
# Critical parameters
|
||||
critical_parameters:
|
||||
- parameter: "Fatty acid solubilization"
|
||||
details: "Complete dissolution of fatty acids in NaHCO₃ is critical for proper complexation"
|
||||
- parameter: "Temperature control"
|
||||
details: "Maintain 65°C during solubilization and 37°C during complexation for optimal results"
|
||||
- parameter: "BSA quality"
|
||||
details: "Use fatty acid-free BSA to ensure accurate FA:BSA ratios"
|
||||
|
||||
# Calculations
|
||||
calculations:
|
||||
- calculation: "BSA molarity"
|
||||
formula: "4.8 mM = 0.64 g ÷ 66,430 g/mol ÷ 0.002 L"
|
||||
- calculation: "Expected FA:BSA ratio"
|
||||
formula: "10 mM FA ÷ 4.8 mM BSA ≈ 2:1 ratio"
|
||||
|
||||
# Troubleshooting
|
||||
troubleshooting:
|
||||
- problem: "Fatty acid precipitation"
|
||||
solution: "Ensure complete dissolution in NaHCO₃ before adding BSA; increase incubation time at 65°C"
|
||||
- problem: "Low final concentration"
|
||||
solution: "Verify fatty acid weight; extend binding incubation time; optimize NaHCO₃ concentration"
|
||||
- problem: "Solution turbidity"
|
||||
solution: "Filter solution multiple times; decrease fatty acid:BSA ratio"
|
||||
|
||||
# Safety considerations
|
||||
safety:
|
||||
ppe: "Lab coat and gloves required"
|
||||
hazards: "NaHCO₃ is irritating to eyes; use caution with hot water baths"
|
||||
|
||||
# Quality control
|
||||
quality_control:
|
||||
- check: "Fatty acid concentration"
|
||||
criteria: "Should be approximately 10 mM when measured by NEFA-HR assay"
|
||||
- check: "Solution clarity"
|
||||
criteria: "Should be clear with no visible precipitate after filtration"
|
||||
|
||||
# References
|
||||
references:
|
||||
- "Alsabeeh N, et al. (2018) Protocols for the preparation of long-chain fatty acid/bovine serum albumin complexes for experimental studies. STAR Protoc. 1(1):100040"
|
||||
- "Spector AA. (1975) Fatty acid binding to plasma albumin. J Lipid Res. 16(3):165-179"
|
||||
|
||||
# Notes
|
||||
notes: |
|
||||
- For 10 mM final fatty acid concentration, use approximately 5 mg of palmitic acid
|
||||
- Can be adapted for oleic acid or arachidonic acid by substituting the appropriate fatty acid
|
||||
- The 150 mM NaCl solution is prepared by diluting 60 μL of 5 M NaCl with 1940 μL water
|
||||
- Expected molar ratio is approximately 2:1 (fatty acid to BSA)
|
||||
- This small-scale protocol (2 mL) is designed for experimental optimization and can be scaled up as needed
|
||||
- BSA-FA complexes are most stable when stored at -20°C but can be stored at 4°C for up to 1 week
|
||||
- Solution pH should be approximately 7.4; adjust with NaOH if needed
|
||||
---
|
||||
157
protocols/preparation_of_complete_dme_f12_itsd_v1.yaml
Normal file
157
protocols/preparation_of_complete_dme_f12_itsd_v1.yaml
Normal file
@ -0,0 +1,157 @@
|
||||
---
|
||||
# Protocol metadata
|
||||
id: PROT-0032
|
||||
name: Preparation of Complete DME/F-12 ITSD Protocol
|
||||
version: 1.0
|
||||
description: Protocol for preparing complete DME/F-12 with ITS and dexamethasone supplements
|
||||
author: Lab Staff
|
||||
created: 2025-05-07
|
||||
last_updated: 2025-05-07
|
||||
category: reagent-preparation
|
||||
|
||||
# Materials required
|
||||
materials:
|
||||
- name: DME/F-12 medium
|
||||
amount: 500 mL bottle
|
||||
storage: 4°C
|
||||
supplier: Recommended supplier
|
||||
- name: Fetal Bovine Serum (FBS)
|
||||
amount: 50 mL (10% final)
|
||||
storage: -20°C (stock), thawed before use
|
||||
supplier: Recommended supplier, heat-inactivated
|
||||
- name: Insulin-Transferrin-Selenium (ITS)
|
||||
amount: 5 mL (1% final)
|
||||
storage: -20°C
|
||||
supplier: Commercial 100X solution
|
||||
- name: Dexamethasone
|
||||
amount: 5 μL of 10 mM stock (1 nM final)
|
||||
storage: -20°C
|
||||
preparation: Stock in ethanol or DMSO
|
||||
- name: Penicillin-Streptomycin (PS)
|
||||
amount: 5 mL (1% final)
|
||||
storage: -20°C
|
||||
supplier: Recommended supplier, 100X concentration
|
||||
- name: Sterile 50-mL tube
|
||||
type: Conical, polypropylene
|
||||
preparation: Sterile
|
||||
|
||||
# Equipment required
|
||||
equipment:
|
||||
- name: Biosafety cabinet
|
||||
certification: Class II
|
||||
- name: Water bath or incubator
|
||||
temperature: 37°C
|
||||
use: For thawing frozen supplements
|
||||
- name: Pipette aid
|
||||
range: For 5-50 mL volumes
|
||||
- name: Micropipettes
|
||||
range: For small volumes (dexamethasone)
|
||||
- name: Serological pipettes
|
||||
range: Various sizes
|
||||
- name: Refrigerator
|
||||
temperature: 4°C
|
||||
use: For media storage
|
||||
|
||||
# Protocol steps
|
||||
steps:
|
||||
- step: 1
|
||||
action: "Prepare materials"
|
||||
details: "Allow media to warm to room temperature; thaw FBS, ITS, and PS at 37°C"
|
||||
- step: 2
|
||||
action: "Set up biosafety cabinet"
|
||||
details: "Turn on biosafety cabinet 15 minutes before use; clean with 70% ethanol"
|
||||
- step: 3
|
||||
action: "Aliquot plain media"
|
||||
details: "Transfer ~55 mL of DME/F-12 media into a sterile 50-mL tube and label as 'PLAIN DME/F-12'"
|
||||
- step: 4
|
||||
action: "Add FBS"
|
||||
details: "To the remaining 445 mL of media in the original bottle, add 50 mL Fetal Bovine Serum (FBS)"
|
||||
- step: 5
|
||||
action: "Add ITS supplement"
|
||||
details: "Add 5 mL Insulin-Transferrin-Selenium (ITS) solution to the main bottle"
|
||||
- step: 6
|
||||
action: "Add dexamethasone"
|
||||
details: "Add 5 μL of 10 mM dexamethasone stock solution (final concentration 1 nM)"
|
||||
- step: 7
|
||||
action: "Add antibiotics"
|
||||
details: "Add 5 mL Penicillin-Streptomycin (PS) solution to the main bottle"
|
||||
- step: 8
|
||||
action: "Mix gently"
|
||||
details: "Invert bottle several times to mix thoroughly, avoiding bubble formation"
|
||||
- step: 9
|
||||
action: "Label media"
|
||||
details: "Label bottle with 'Complete DME/F-12 ITS-D', date of preparation, and expiration date (2-3 weeks from preparation)"
|
||||
- step: 10
|
||||
action: "Store media"
|
||||
details: "Store both complete and plain media at 4°C; protect from light if possible"
|
||||
|
||||
# Final composition
|
||||
final_composition:
|
||||
- component: "DME/F-12 basal medium"
|
||||
percentage: "88%"
|
||||
function: "Provides essential nutrients and buffering"
|
||||
- component: "Fetal Bovine Serum"
|
||||
percentage: "10%"
|
||||
function: "Provides growth factors and proteins for cell growth"
|
||||
- component: "Insulin-Transferrin-Selenium"
|
||||
percentage: "1%"
|
||||
function: "Insulin: promotes glucose uptake; Transferrin: iron carrier; Selenium: antioxidant"
|
||||
- component: "Dexamethasone"
|
||||
concentration: "1 nM"
|
||||
function: "Glucocorticoid that promotes cell differentiation in certain cell types"
|
||||
- component: "Penicillin-Streptomycin"
|
||||
percentage: "1%"
|
||||
function: "Prevents bacterial contamination"
|
||||
|
||||
# Critical parameters
|
||||
critical_parameters:
|
||||
- parameter: "Sterility"
|
||||
details: "All components must be handled aseptically in a biosafety cabinet"
|
||||
- parameter: "Dexamethasone concentration"
|
||||
details: "Accurate pipetting of small volume (5 μL) is critical; consider using serial dilutions"
|
||||
- parameter: "Expiration"
|
||||
details: "Media with ITS may have shorter shelf life (2-3 weeks) compared to standard media"
|
||||
|
||||
# Troubleshooting
|
||||
troubleshooting:
|
||||
- problem: "Precipitate formation"
|
||||
solution: "May occur with ITS; warm to room temperature and mix gently; filtration may be required"
|
||||
- problem: "Cell differentiation issues"
|
||||
solution: "Verify dexamethasone concentration; prepare fresh medium; check ITS potency"
|
||||
- problem: "Media color change"
|
||||
solution: "If phenol red indicator turns yellow, pH is too acidic; discard media"
|
||||
|
||||
# Safety considerations
|
||||
safety:
|
||||
ppe: "Lab coat, gloves required"
|
||||
hazards: "Minimal hazards; follow standard laboratory safety practices"
|
||||
dexamethasone: "Potent steroid hormone; avoid skin contact"
|
||||
|
||||
# Quality control
|
||||
quality_control:
|
||||
- check: "pH indicator"
|
||||
criteria: "Phenol red should appear pink-red (not yellow or purple)"
|
||||
- check: "Sterility test"
|
||||
criteria: "Incubate a sample at 37°C for 72 hours to verify no contamination"
|
||||
- check: "Functional test"
|
||||
criteria: "Test with appropriate cell differentiation model if possible"
|
||||
|
||||
# References
|
||||
references:
|
||||
- "ATCC. Guidelines for Media Preparation and Methods. www.atcc.org"
|
||||
- "Freshney, R.I. Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, 7th Edition"
|
||||
- "Barnes D, Sato G. (1980) Methods for growth of cultured cells in serum-free medium. Analytical Biochemistry 102(2):255-270"
|
||||
|
||||
# Notes
|
||||
notes: |
|
||||
- This specialized medium contains additional supplements:
|
||||
- 10% FBS
|
||||
- 1% ITS
|
||||
- 1 nM dexamethasone
|
||||
- 1% Penicillin-Streptomycin
|
||||
- ITS supplement is typically used to reduce serum requirements or enhance differentiation
|
||||
- Dexamethasone concentration (1 nM) is suitable for differentiation of certain cell types
|
||||
- For more potent glucocorticoid effects, concentration can be increased to 10-100 nM
|
||||
- For long-term studies, consider preparing smaller volumes of media more frequently
|
||||
- Some cell types may require adaptation period when switching to ITS-supplemented media
|
||||
---
|
||||
130
protocols/preparation_of_complete_dme_f12_v1.yaml
Normal file
130
protocols/preparation_of_complete_dme_f12_v1.yaml
Normal file
@ -0,0 +1,130 @@
|
||||
---
|
||||
# Protocol metadata
|
||||
id: PROT-0030
|
||||
name: Preparation of Complete DME/F-12 Protocol
|
||||
version: 1.0
|
||||
description: Protocol for preparing complete DME/F-12 culture medium with FBS and antibiotics
|
||||
author: Lab Staff
|
||||
created: 2025-05-07
|
||||
last_updated: 2025-05-07
|
||||
category: reagent-preparation
|
||||
|
||||
# Materials required
|
||||
materials:
|
||||
- name: DME/F-12 medium
|
||||
amount: 500 mL bottle
|
||||
storage: 4°C
|
||||
supplier: Recommended supplier
|
||||
- name: Fetal Bovine Serum (FBS)
|
||||
amount: 50 mL (10% final)
|
||||
storage: -20°C (stock), thawed before use
|
||||
supplier: Recommended supplier, heat-inactivated
|
||||
- name: Penicillin-Streptomycin (PS)
|
||||
amount: 5 mL (1% final)
|
||||
storage: -20°C
|
||||
supplier: Recommended supplier, 100X concentration
|
||||
- name: Sterile 50-mL tube
|
||||
type: Conical, polypropylene
|
||||
preparation: Sterile
|
||||
|
||||
# Equipment required
|
||||
equipment:
|
||||
- name: Biosafety cabinet
|
||||
certification: Class II
|
||||
- name: Water bath or incubator
|
||||
temperature: 37°C
|
||||
use: For thawing FBS
|
||||
- name: Pipette aid
|
||||
range: For 5-50 mL volumes
|
||||
- name: Pipettes
|
||||
range: Serological, various sizes
|
||||
- name: Refrigerator
|
||||
temperature: 4°C
|
||||
use: For media storage
|
||||
|
||||
# Protocol steps
|
||||
steps:
|
||||
- step: 1
|
||||
action: "Prepare materials"
|
||||
details: "Allow media to warm to room temperature; thaw FBS and PS at 37°C"
|
||||
- step: 2
|
||||
action: "Set up biosafety cabinet"
|
||||
details: "Turn on biosafety cabinet 15 minutes before use; clean with 70% ethanol"
|
||||
- step: 3
|
||||
action: "Aliquot plain media"
|
||||
details: "Transfer ~51 mL of DME/F-12 media into a sterile 50-mL tube and label as 'PLAIN DME/F-12'"
|
||||
- step: 4
|
||||
action: "Add FBS"
|
||||
details: "To the remaining 449 mL of media in the original bottle, add 50 mL Fetal Bovine Serum (FBS)"
|
||||
- step: 5
|
||||
action: "Add antibiotics"
|
||||
details: "Add 5 mL Penicillin-Streptomycin (PS) solution to the main bottle"
|
||||
- step: 6
|
||||
action: "Mix gently"
|
||||
details: "Invert bottle several times to mix thoroughly, avoiding bubble formation"
|
||||
- step: 7
|
||||
action: "Label media"
|
||||
details: "Label bottle with 'Complete DME/F-12', date of preparation, and expiration date (1 month from preparation)"
|
||||
- step: 8
|
||||
action: "Store media"
|
||||
details: "Store both complete and plain media at 4°C"
|
||||
|
||||
# Final composition
|
||||
final_composition:
|
||||
- component: "DME/F-12 basal medium"
|
||||
percentage: "89%"
|
||||
function: "Provides essential nutrients and buffering"
|
||||
- component: "Fetal Bovine Serum"
|
||||
percentage: "10%"
|
||||
function: "Provides growth factors and proteins for cell growth"
|
||||
- component: "Penicillin-Streptomycin"
|
||||
percentage: "1%"
|
||||
function: "Prevents bacterial contamination"
|
||||
|
||||
# Critical parameters
|
||||
critical_parameters:
|
||||
- parameter: "Sterility"
|
||||
details: "All components must be handled aseptically in a biosafety cabinet"
|
||||
- parameter: "FBS quality"
|
||||
details: "FBS should be heat-inactivated and from a validated lot known to support growth of target cells"
|
||||
- parameter: "Expiration"
|
||||
details: "Complete medium should be used within 1 month of preparation"
|
||||
|
||||
# Troubleshooting
|
||||
troubleshooting:
|
||||
- problem: "Media color change"
|
||||
solution: "If phenol red indicator turns yellow, pH is too acidic; discard media"
|
||||
- problem: "Precipitation in media"
|
||||
solution: "Check for contamination; sometimes precipitates from FBS can be removed by filtration"
|
||||
- problem: "Cell growth issues"
|
||||
solution: "Test alternative FBS lots; verify proper storage conditions"
|
||||
|
||||
# Safety considerations
|
||||
safety:
|
||||
ppe: "Lab coat, gloves required"
|
||||
hazards: "Minimal hazards; follow standard laboratory safety practices"
|
||||
|
||||
# Quality control
|
||||
quality_control:
|
||||
- check: "pH indicator"
|
||||
criteria: "Phenol red should appear pink-red (not yellow or purple)"
|
||||
- check: "Sterility test"
|
||||
criteria: "Incubate a sample at 37°C for 72 hours to verify no contamination"
|
||||
- check: "Cell growth test"
|
||||
criteria: "Test with standard cell line to verify growth support"
|
||||
|
||||
# References
|
||||
references:
|
||||
- "ATCC. Guidelines for Media Preparation and Methods. www.atcc.org"
|
||||
- "Freshney, R.I. Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, 7th Edition"
|
||||
|
||||
# Notes
|
||||
notes: |
|
||||
- This protocol creates both complete DME/F-12 (with FBS and antibiotics) and plain DME/F-12
|
||||
- Complete medium contains 10% FBS and 1% Penicillin-Streptomycin
|
||||
- For specialty applications, additional supplements may be required
|
||||
- Some cell lines may require different FBS concentrations (5-20%)
|
||||
- If preparing larger or smaller volumes, adjust component volumes proportionally
|
||||
- For sensitive cell types, consider using reduced serum or serum-free formulations
|
||||
- For long-term storage, complete media can be aliquoted into working volumes to reduce freeze-thaw cycles
|
||||
---
|
||||
112
protocols/preparation_of_complete_dmem_high_glucose_v1.yaml
Normal file
112
protocols/preparation_of_complete_dmem_high_glucose_v1.yaml
Normal file
@ -0,0 +1,112 @@
|
||||
---
|
||||
# Protocol metadata
|
||||
id: PROT-0031
|
||||
name: Preparation of Complete DMEM (High Glucose) Protocol
|
||||
version: 1.0
|
||||
description: Protocol for preparing complete DMEM high glucose culture medium with FBS and antibiotics
|
||||
author: Lab Staff
|
||||
created: 2025-05-07
|
||||
last_updated: 2025-05-07
|
||||
category: media-preparation
|
||||
|
||||
# Materials required
|
||||
materials:
|
||||
- name: DMEM high glucose medium
|
||||
amount: 500 mL bottle
|
||||
storage: 4°C
|
||||
notes: Allow to warm to room temperature before use
|
||||
- name: Fetal Bovine Serum (FBS)
|
||||
amount: 50 mL (10% final)
|
||||
storage: -20°C (stock), thaw at 4°C overnight
|
||||
notes: Heat-inactivated if required by cell line
|
||||
- name: Penicillin-Streptomycin (PS)
|
||||
amount: 5 mL (1% final)
|
||||
concentration: 100X stock solution
|
||||
storage: -20°C
|
||||
- name: Sterile 50-mL tube
|
||||
type: Polypropylene
|
||||
quantity: 1
|
||||
|
||||
# Equipment required
|
||||
equipment:
|
||||
- name: Biosafety cabinet
|
||||
certification: Class II
|
||||
- name: Pipette controller
|
||||
type: Electric or manual
|
||||
- name: Serological pipettes
|
||||
sizes: 5 mL, 10 mL, 25 mL
|
||||
- name: Refrigerator
|
||||
temperature: 4°C
|
||||
- name: Marker or label maker
|
||||
use: For labeling media bottles and tubes
|
||||
|
||||
# Protocol steps
|
||||
steps:
|
||||
- step: 1
|
||||
action: "Prepare workspace"
|
||||
details: "Turn on biosafety cabinet, disinfect surfaces, and allow DMEM and supplements to reach room temperature"
|
||||
- step: 2
|
||||
action: "Prepare plain DMEM aliquot"
|
||||
details: "Transfer ~51 mL of DMEM high glucose media into a sterile 50-mL tube and label as 'PLAIN DMEM'"
|
||||
- step: 3
|
||||
action: "Add FBS to main bottle"
|
||||
details: "To the remaining 449 mL of media, add 50 mL Fetal Bovine Serum (FBS) for 10% final concentration"
|
||||
- step: 4
|
||||
action: "Add antibiotics to main bottle"
|
||||
details: "Add 5 mL Penicillin-Streptomycin (PS) to the main bottle for 1% final concentration"
|
||||
- step: 5
|
||||
action: "Mix gently"
|
||||
details: "Invert bottle several times to mix thoroughly without creating bubbles"
|
||||
- step: 6
|
||||
action: "Label bottle"
|
||||
details: "Label the bottle with 'Complete DMEM (High Glucose)', date of preparation, and your initials"
|
||||
- step: 7
|
||||
action: "Storage"
|
||||
details: "Store complete medium and plain DMEM at 4°C for up to 1 month"
|
||||
|
||||
# Critical parameters
|
||||
critical_parameters:
|
||||
- parameter: "Sterility"
|
||||
details: "Maintain sterile technique throughout to prevent contamination"
|
||||
- parameter: "FBS quality"
|
||||
details: "Use high-quality, tested FBS batch to ensure consistent cell growth"
|
||||
- parameter: "Temperature"
|
||||
details: "Cold media can cause cellular stress; allow to warm before use with cells"
|
||||
|
||||
# Troubleshooting
|
||||
troubleshooting:
|
||||
- problem: "Medium contamination"
|
||||
solution: "Check sterile technique, biosafety cabinet function, and supplement sterility"
|
||||
- problem: "Cell growth issues"
|
||||
solution: "Test different FBS batches; some cell lines may require additional supplements"
|
||||
- problem: "Phenol red color change"
|
||||
solution: "Check pH; yellowing indicates acidification which may suggest contamination"
|
||||
|
||||
# Safety considerations
|
||||
safety:
|
||||
ppe: "Lab coat and gloves required"
|
||||
hazards: "Low biohazard risk; follow institutional guidelines for handling cell culture materials"
|
||||
|
||||
# Quality control
|
||||
quality_control:
|
||||
- check: "Visual inspection"
|
||||
criteria: "No cloudiness, particles, or contamination"
|
||||
- check: "pH indicator (phenol red)"
|
||||
criteria: "Should be red-pink color (neutral pH)"
|
||||
- check: "Test on standard cell line"
|
||||
criteria: "Expected growth rate and morphology"
|
||||
|
||||
# References
|
||||
references:
|
||||
- "Cell culture media manufacturer's recommendations"
|
||||
- "Laboratory standard operating procedures for media preparation"
|
||||
|
||||
# Notes
|
||||
notes: |
|
||||
- This protocol creates both complete DMEM (with FBS and antibiotics) and plain DMEM
|
||||
- Complete medium contains 10% FBS and 1% Penicillin-Streptomycin
|
||||
- For sensitive cell lines, consider using lower antibiotic concentrations or antibiotic-free media
|
||||
- Media shelf life is typically 1 month when stored at 4°C
|
||||
- Consider aliquoting complete media to minimize contamination risk from repeated opening
|
||||
- Some specialized applications may require additional supplements (e.g., L-glutamine, sodium pyruvate)
|
||||
---
|
||||
130
protocols/preparation_of_complete_rpmi_v1.yaml
Normal file
130
protocols/preparation_of_complete_rpmi_v1.yaml
Normal file
@ -0,0 +1,130 @@
|
||||
---
|
||||
# Protocol metadata
|
||||
id: PROT-0025
|
||||
name: Preparation of Complete RPMI Protocol
|
||||
version: 1.0
|
||||
description: Protocol for preparing complete RPMI culture medium with FBS and antibiotics
|
||||
author: Lab Staff
|
||||
created: 2025-05-06
|
||||
last_updated: 2025-05-07
|
||||
category: reagent-preparation
|
||||
|
||||
# Materials required
|
||||
materials:
|
||||
- name: RPMI-1640 medium
|
||||
amount: 500 mL bottle
|
||||
storage: 4°C
|
||||
supplier: Recommended supplier
|
||||
- name: Fetal Bovine Serum (FBS)
|
||||
amount: 50 mL (10% final)
|
||||
storage: -20°C (stock), thawed before use
|
||||
supplier: Recommended supplier, heat-inactivated
|
||||
- name: Penicillin-Streptomycin (PS)
|
||||
amount: 5 mL (1% final)
|
||||
storage: -20°C
|
||||
supplier: Recommended supplier, 100X concentration
|
||||
- name: Sterile 50-mL tube
|
||||
type: Conical, polypropylene
|
||||
preparation: Sterile
|
||||
|
||||
# Equipment required
|
||||
equipment:
|
||||
- name: Biosafety cabinet
|
||||
certification: Class II
|
||||
- name: Water bath or incubator
|
||||
temperature: 37°C
|
||||
use: For thawing FBS
|
||||
- name: Pipette aid
|
||||
range: For 5-50 mL volumes
|
||||
- name: Pipettes
|
||||
range: Serological, various sizes
|
||||
- name: Refrigerator
|
||||
temperature: 4°C
|
||||
use: For media storage
|
||||
|
||||
# Protocol steps
|
||||
steps:
|
||||
- step: 1
|
||||
action: "Prepare materials"
|
||||
details: "Allow media to warm to room temperature; thaw FBS and PS at 37°C"
|
||||
- step: 2
|
||||
action: "Set up biosafety cabinet"
|
||||
details: "Turn on biosafety cabinet 15 minutes before use; clean with 70% ethanol"
|
||||
- step: 3
|
||||
action: "Aliquot plain media"
|
||||
details: "Transfer ~51 mL of RPMI media into a sterile 50-mL tube and label as 'PLAIN RPMI'"
|
||||
- step: 4
|
||||
action: "Add FBS"
|
||||
details: "To the remaining 449 mL of media in the original bottle, add 50 mL Fetal Bovine Serum (FBS)"
|
||||
- step: 5
|
||||
action: "Add antibiotics"
|
||||
details: "Add 5 mL Penicillin-Streptomycin (PS) solution to the main bottle"
|
||||
- step: 6
|
||||
action: "Mix gently"
|
||||
details: "Invert bottle several times to mix thoroughly, avoiding bubble formation"
|
||||
- step: 7
|
||||
action: "Label media"
|
||||
details: "Label bottle with 'Complete RPMI', date of preparation, and expiration date (1 month from preparation)"
|
||||
- step: 8
|
||||
action: "Store media"
|
||||
details: "Store both complete and plain media at 4°C"
|
||||
|
||||
# Final composition
|
||||
final_composition:
|
||||
- component: "RPMI-1640 basal medium"
|
||||
percentage: "89%"
|
||||
function: "Provides essential nutrients and buffering"
|
||||
- component: "Fetal Bovine Serum"
|
||||
percentage: "10%"
|
||||
function: "Provides growth factors and proteins for cell growth"
|
||||
- component: "Penicillin-Streptomycin"
|
||||
percentage: "1%"
|
||||
function: "Prevents bacterial contamination"
|
||||
|
||||
# Critical parameters
|
||||
critical_parameters:
|
||||
- parameter: "Sterility"
|
||||
details: "All components must be handled aseptically in a biosafety cabinet"
|
||||
- parameter: "FBS quality"
|
||||
details: "FBS should be heat-inactivated and from a validated lot known to support growth of target cells"
|
||||
- parameter: "Expiration"
|
||||
details: "Complete medium should be used within 1 month of preparation"
|
||||
|
||||
# Troubleshooting
|
||||
troubleshooting:
|
||||
- problem: "Media color change"
|
||||
solution: "If phenol red indicator turns yellow, pH is too acidic; discard media"
|
||||
- problem: "Precipitation in media"
|
||||
solution: "Check for contamination; sometimes precipitates from FBS can be removed by filtration"
|
||||
- problem: "Cell growth issues"
|
||||
solution: "Test alternative FBS lots; verify proper storage conditions"
|
||||
|
||||
# Safety considerations
|
||||
safety:
|
||||
ppe: "Lab coat, gloves required"
|
||||
hazards: "Minimal hazards; follow standard laboratory safety practices"
|
||||
|
||||
# Quality control
|
||||
quality_control:
|
||||
- check: "pH indicator"
|
||||
criteria: "Phenol red should appear pink-red (not yellow or purple)"
|
||||
- check: "Sterility test"
|
||||
criteria: "Incubate a sample at 37°C for 72 hours to verify no contamination"
|
||||
- check: "Cell growth test"
|
||||
criteria: "Test with standard cell line to verify growth support"
|
||||
|
||||
# References
|
||||
references:
|
||||
- "ATCC. Guidelines for Media Preparation and Methods. www.atcc.org"
|
||||
- "Freshney, R.I. Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, 7th Edition"
|
||||
|
||||
# Notes
|
||||
notes: |
|
||||
- This protocol creates both complete RPMI (with FBS and antibiotics) and plain RPMI
|
||||
- Complete medium contains 10% FBS and 1% Penicillin-Streptomycin
|
||||
- RPMI-1640 is particularly suited for lymphoid cells, including suspension cultures
|
||||
- Some specialized cell lines may require RPMI with additional supplements
|
||||
- For immune cell work, consider adding 2-mercaptoethanol (55 μM final) to the media
|
||||
- If preparing larger or smaller volumes, adjust component volumes proportionally
|
||||
- For long-term storage, complete media can be aliquoted into working volumes
|
||||
---
|
||||
148
protocols/protein_extraction_6well_v1.yaml
Normal file
148
protocols/protein_extraction_6well_v1.yaml
Normal file
@ -0,0 +1,148 @@
|
||||
---
|
||||
# Protocol metadata
|
||||
id: PROT-0027
|
||||
name: Protein Extraction Protocol for 6-Well Plates
|
||||
version: 1.0
|
||||
description: Protocol for extracting protein from cells grown in 6-well plates
|
||||
author: Lab Staff
|
||||
created: 2025-05-07
|
||||
last_updated: 2025-05-07
|
||||
category: protein-analysis
|
||||
|
||||
# Materials required
|
||||
materials:
|
||||
- name: RIPA buffer
|
||||
preparation: With freshly added protease/phosphatase inhibitors
|
||||
storage: 4°C
|
||||
- name: PBS
|
||||
concentration: 1X, sterile
|
||||
temperature: Ice-cold
|
||||
- name: Cell scraper or pipet tip
|
||||
type: Sterile
|
||||
- name: Microcentrifuge tubes (1.5-mL)
|
||||
preparation: Pre-labeled, pre-chilled
|
||||
- name: PCR tube strips (0.2-mL)
|
||||
use: For BCA assay aliquots
|
||||
- name: Ice
|
||||
amount: Sufficient to fill a tray
|
||||
|
||||
# Equipment required
|
||||
equipment:
|
||||
- name: ThermoMixer
|
||||
settings: 4°C, 500 rpm
|
||||
- name: Refrigerated centrifuge
|
||||
settings: Maximum speed (≥13,000g), 4°C
|
||||
- name: Ice tray
|
||||
size: Sufficient to hold samples
|
||||
- name: Aspiration system
|
||||
type: Vacuum or manual
|
||||
- name: -80°C freezer
|
||||
use: Sample storage
|
||||
|
||||
# Protocol steps
|
||||
steps:
|
||||
- step: 1
|
||||
action: "Prepare protein extraction buffer"
|
||||
details: "Add protease/phosphatase inhibitors to RIPA buffer immediately before use"
|
||||
- step: 2
|
||||
action: "Prepare ice tray"
|
||||
details: "Fill a tray with ice from the ice machine in the autoclave room"
|
||||
- step: 3
|
||||
action: "Retrieve cell culture plate"
|
||||
details: "Remove the plate from the incubator and bring it to the lab workstation"
|
||||
- step: 4
|
||||
action: "Aspirate media"
|
||||
details: "Carefully remove all culture media from each well"
|
||||
- step: 5
|
||||
action: "Wash cells"
|
||||
details: "Add 1 mL of ice-cold 1X PBS to each well, swirl gently, then aspirate completely"
|
||||
- step: 6
|
||||
action: "Add extraction buffer"
|
||||
details: "Add 100 μL of ice-cold protein extraction buffer to each well while keeping the plate on ice"
|
||||
- step: 7
|
||||
action: "Incubate with buffer"
|
||||
details: "Allow cells to sit in extraction buffer for 10 minutes, agitating the plate every 1-2 minutes"
|
||||
- step: 8
|
||||
action: "Collect lysate"
|
||||
details: "Tilt the plate to pool the extraction buffer and suspended cells in the bottom corner"
|
||||
- step: 9
|
||||
action: "Scrape cells if necessary"
|
||||
details: "If cells remain attached, scrape them into the extraction buffer with a cell scraper or pipet tip"
|
||||
- step: 10
|
||||
action: "Transfer lysate"
|
||||
details: "Transfer the cell suspension to a chilled, pre-labeled 1.5-mL tube"
|
||||
- step: 11
|
||||
action: "Agitate lysate"
|
||||
details: "Incubate the lysate in the ThermoMixer at 4°C/500 rpm for 4 hours to overnight"
|
||||
- step: 12
|
||||
action: "Centrifuge lysate"
|
||||
details: "Centrifuge at maximum speed for 25 minutes at 4°C to pellet cell debris"
|
||||
- step: 13
|
||||
action: "Collect protein supernatant"
|
||||
details: "Carefully transfer 90 μL of supernatant to a fresh, chilled 1.5-mL tube (avoiding the pellet)"
|
||||
- step: 14
|
||||
action: "Prepare BCA assay aliquot"
|
||||
details: "Transfer remaining 10 μL to 0.2-mL PCR tube strips for protein quantification"
|
||||
- step: 15
|
||||
action: "Store samples"
|
||||
details: "Place tubes in a -80°C box and note box address/location"
|
||||
- step: 16
|
||||
action: "Document samples"
|
||||
details: "Submit your sample to the inventory/archive by filling out the -80°C Sample Submission form"
|
||||
|
||||
# Critical parameters
|
||||
critical_parameters:
|
||||
- parameter: "Temperature"
|
||||
details: "Maintain samples on ice throughout extraction to prevent protein degradation and preserve phosphorylation states"
|
||||
- parameter: "Inhibitors"
|
||||
details: "Add protease/phosphatase inhibitors immediately before use; they degrade quickly in solution"
|
||||
- parameter: "PBS removal"
|
||||
details: "Remove PBS completely before adding extraction buffer to prevent dilution"
|
||||
|
||||
# Troubleshooting
|
||||
troubleshooting:
|
||||
- problem: "Low protein yield"
|
||||
solution: "Increase extraction buffer volume, extend lysis time, or use more stringent lysis buffer"
|
||||
- problem: "High debris or viscosity"
|
||||
solution: "Sonicate lysate briefly, add DNase I, or extend centrifugation time"
|
||||
- problem: "Protein degradation"
|
||||
solution: "Ensure fresh inhibitors, maintain cold temperatures, and process samples promptly"
|
||||
|
||||
# Safety considerations
|
||||
safety:
|
||||
ppe: "Lab coat, gloves, and eye protection required"
|
||||
hazards: "RIPA buffer contains detergents that can cause eye/skin irritation"
|
||||
|
||||
# Quality control
|
||||
quality_control:
|
||||
- check: "BCA protein assay"
|
||||
criteria: "Protein concentration should be 0.5-5 μg/μL depending on cell type and density"
|
||||
- check: "Western blot quality"
|
||||
criteria: "Distinct bands without smearing or degradation products"
|
||||
|
||||
# Downstream applications
|
||||
downstream_applications:
|
||||
- name: "Western blotting"
|
||||
preparation: "Dilute samples to equal concentration, add loading buffer, and heat at 95°C for 5 minutes"
|
||||
- name: "Mass spectrometry"
|
||||
preparation: "May require additional cleanup steps to remove detergents"
|
||||
- name: "ELISA"
|
||||
preparation: "Verify buffer compatibility with assay; may require dialysis"
|
||||
|
||||
# References
|
||||
references:
|
||||
- "Cold Spring Harbor Protocols. (2017) Extraction of Proteins from Mammalian Cells. CSH Protocols."
|
||||
- "Bass JJ, et al. (2017) An overview of technical considerations for Western blotting applications to physiological research. Scand J Med Sci Sports. 27(1):4-25."
|
||||
|
||||
# Notes
|
||||
notes: |
|
||||
- This protocol is optimized for 6-well plates
|
||||
- Keep samples cold throughout the process to prevent protein degradation
|
||||
- Different lysis buffers may be used depending on the target proteins:
|
||||
- RIPA buffer (used here): Good for most cytoplasmic, membrane, and nuclear proteins
|
||||
- NP-40 buffer: Milder, preserves protein-protein interactions
|
||||
- Laemmli buffer: Direct lysis for SDS-PAGE without separate extraction step
|
||||
- For phosphoprotein analysis, always use fresh phosphatase inhibitors
|
||||
- Expected yields: 100-300 μg total protein per well (depending on cell type and confluence)
|
||||
- Consider snap-freezing cell pellets in liquid nitrogen if extraction must be delayed
|
||||
---
|
||||
252
protocols/rip_qpcr_protocol_v1.yaml
Normal file
252
protocols/rip_qpcr_protocol_v1.yaml
Normal file
@ -0,0 +1,252 @@
|
||||
---
|
||||
# Protocol metadata
|
||||
id: PROT-0014
|
||||
name: RNA Immunoprecipitation qPCR Protocol
|
||||
version: 1.0
|
||||
description: Protocol for RNA immunoprecipitation followed by RT-qPCR to detect RNA-protein interactions
|
||||
author: Jordan Lab
|
||||
created: 2025-05-06
|
||||
last_updated: 2025-05-07
|
||||
category: molecular-biology
|
||||
|
||||
# Materials required
|
||||
materials:
|
||||
- name: Cell culture dishes
|
||||
type: 10-cm or 15-cm dishes
|
||||
notes: Depending on protein/RNA abundance
|
||||
- name: PBS (cold)
|
||||
temperature: 4°C
|
||||
storage: Room temperature
|
||||
- name: UV crosslinker
|
||||
wavelength: 254 nm
|
||||
alternative: Formaldehyde crosslinking
|
||||
- name: Lysis buffer
|
||||
components: "Non-denaturing buffer with RNase inhibitors"
|
||||
storage: Prepare fresh or store at -20°C
|
||||
- name: Protein A/G beads
|
||||
preparation: Pre-blocked with BSA and tRNA
|
||||
storage: 4°C
|
||||
- name: Antibodies for target protein
|
||||
dilution: As recommended for IP
|
||||
storage: -20°C
|
||||
- name: IgG control antibody
|
||||
use: Negative control
|
||||
storage: -20°C
|
||||
- name: Glycine solution
|
||||
concentration: 2.5 M (for formaldehyde quenching)
|
||||
storage: Room temperature
|
||||
- name: RNA isolation reagents
|
||||
type: TRIzol or column-based kit
|
||||
storage: According to manufacturer's instructions
|
||||
- name: RT-qPCR reagents
|
||||
components: "Reverse transcription kit, qPCR master mix, primers"
|
||||
storage: -20°C
|
||||
|
||||
# Equipment required
|
||||
equipment:
|
||||
- name: UV crosslinker
|
||||
settings: 150-300 mJ/cm²
|
||||
alternative: Formaldehyde crosslinking setup
|
||||
- name: Refrigerated centrifuge
|
||||
settings: Various speeds, 4°C
|
||||
- name: Rotating mixer
|
||||
temperature: 4°C
|
||||
- name: Thermocycler
|
||||
use: RT-qPCR and crosslink reversal
|
||||
- name: qPCR instrument
|
||||
use: Target RNA detection
|
||||
|
||||
# Protocol steps - General workflow
|
||||
steps:
|
||||
- step: 1
|
||||
action: "Grow cells to desired confluency"
|
||||
details: "80-90% confluency recommended for optimal yield"
|
||||
- step: 2
|
||||
action: "Perform crosslinking"
|
||||
details: "Choose between UV or formaldehyde crosslinking methods (see detailed workflows below)"
|
||||
- step: 3
|
||||
action: "Harvest and lyse cells"
|
||||
details: "Use buffer with RNase inhibitors; keep samples cold"
|
||||
- step: 4
|
||||
action: "Pre-clear lysates"
|
||||
details: "Incubate with protein A/G beads alone for 1 hour at 4°C to reduce nonspecific binding"
|
||||
- step: 5
|
||||
action: "Perform immunoprecipitation"
|
||||
details: "Add specific antibody, incubate, then add protein A/G beads"
|
||||
- step: 6
|
||||
action: "Wash beads"
|
||||
details: "Multiple washes with increasing stringency to remove non-specific binding"
|
||||
- step: 7
|
||||
action: "Reverse crosslinks if needed"
|
||||
details: "UV: proteinase K digestion; formaldehyde: heat treatment (65°C)"
|
||||
- step: 8
|
||||
action: "Isolate RNA"
|
||||
details: "Extract RNA from immunoprecipitated complex using TRIzol or column-based kit"
|
||||
- step: 9
|
||||
action: "Perform RT-qPCR"
|
||||
details: "Reverse transcribe RNA and quantify target RNA enrichment"
|
||||
|
||||
# UV crosslinking workflow
|
||||
uv_crosslinking_workflow:
|
||||
- step: 1
|
||||
action: "Grow cells to desired confluency in culture dishes"
|
||||
details: "Use 10-15 cm dishes for sufficient material"
|
||||
- step: 2
|
||||
action: "Wash cells with cold PBS"
|
||||
details: "Remove media and serum proteins; keep cells on ice to minimize RNase activity"
|
||||
- step: 3
|
||||
action: "Add fresh cold PBS"
|
||||
details: "Add enough to cover the cells but minimize UV light absorption"
|
||||
- step: 4
|
||||
action: "Perform UV crosslinking"
|
||||
details: "Use UV 254 nm at 150-300 mJ/cm²; optimize for your protein of interest"
|
||||
- step: 5
|
||||
action: "Harvest cells"
|
||||
details: "Scrape or gently trypsinize cells and collect by centrifugation"
|
||||
- step: 6
|
||||
action: "Lyse cells"
|
||||
details: "Use mild conditions suitable for maintaining RNP complexes"
|
||||
- step: 7
|
||||
action: "Pre-clear lysates"
|
||||
details: "Incubate with protein A/G beads alone to reduce nonspecific binding"
|
||||
- step: 8
|
||||
action: "Perform immunoprecipitation"
|
||||
details: "Add the specific antibody, followed by protein A/G beads"
|
||||
- step: 9
|
||||
action: "Wash beads"
|
||||
details: "Multiple washes with increasing stringency"
|
||||
- step: 10
|
||||
action: "Digest and extract RNA"
|
||||
details: "Treat with proteinase K and extract RNA"
|
||||
- step: 11
|
||||
action: "Perform RT-qPCR"
|
||||
details: "Reverse transcribe and perform qPCR to detect target RNAs"
|
||||
|
||||
# Formaldehyde crosslinking workflow
|
||||
formaldehyde_crosslinking_workflow:
|
||||
- step: 1
|
||||
action: "Grow cells to desired confluency"
|
||||
details: "Use 10-15 cm dishes for sufficient material"
|
||||
- step: 2
|
||||
action: "Prepare formaldehyde solution"
|
||||
details: "Prepare fresh formaldehyde at working concentration (often 1% final in culture medium)"
|
||||
- step: 3
|
||||
action: "Crosslink cells"
|
||||
details: "Add formaldehyde directly to cells and incubate for 5–10 minutes at RT or 37°C"
|
||||
- step: 4
|
||||
action: "Quench reaction"
|
||||
details: "Add glycine (125 mM final) for 5–10 minutes to quench formaldehyde"
|
||||
- step: 5
|
||||
action: "Wash cells"
|
||||
details: "Wash thoroughly with cold PBS to remove formaldehyde"
|
||||
- step: 6
|
||||
action: "Harvest cells"
|
||||
details: "Scrape cells carefully and collect by centrifugation"
|
||||
- step: 7
|
||||
action: "Lyse cells"
|
||||
details: "Use conditions that preserve protein-RNA complexes"
|
||||
- step: 8
|
||||
action: "Perform immunoprecipitation"
|
||||
details: "Use specific antibody against protein of interest"
|
||||
- step: 9
|
||||
action: "Wash beads"
|
||||
details: "Multiple washes to remove nonspecific material"
|
||||
- step: 10
|
||||
action: "Reverse crosslink"
|
||||
details: "Heat at 65°C for several hours with SDS/high salt"
|
||||
- step: 11
|
||||
action: "Isolate RNA"
|
||||
details: "Extract RNA from immunoprecipitated sample"
|
||||
- step: 12
|
||||
action: "Perform RT-qPCR"
|
||||
details: "Reverse transcribe and quantify targets by qPCR"
|
||||
|
||||
# Critical parameters
|
||||
critical_parameters:
|
||||
- parameter: "Crosslinking method selection"
|
||||
details: "UV is more specific for direct interactions; formaldehyde captures larger complexes"
|
||||
- parameter: "Antibody specificity"
|
||||
details: "Validate antibody IP efficiency by Western blot before RIP"
|
||||
- parameter: "RNase control"
|
||||
details: "Use RNase inhibitors in all buffers and work quickly at 4°C"
|
||||
- parameter: "Wash stringency"
|
||||
details: "Balance between removing background and maintaining specific interactions"
|
||||
|
||||
# Troubleshooting
|
||||
troubleshooting:
|
||||
- problem: "Poor RNA yield"
|
||||
solution: "Increase starting material; optimize crosslinking conditions; check for RNase contamination"
|
||||
- problem: "High background in control IPs"
|
||||
solution: "Increase wash stringency; pre-block beads with BSA/tRNA; use more specific antibody"
|
||||
- problem: "No enrichment of target RNA"
|
||||
solution: "Verify protein-RNA interaction using alternative approach; check crosslinking efficiency; try different antibody"
|
||||
|
||||
# Method comparisons
|
||||
considerations:
|
||||
uv_crosslinking:
|
||||
- advantage: "Standardize irradiation distance/energy for reproducibility"
|
||||
- advantage: "More specific crosslinks between directly interacting residues"
|
||||
- advantage: "Better for pinpointing precise binding sites"
|
||||
- disadvantage: "Requires specialized equipment (UV crosslinker)"
|
||||
- disadvantage: "May damage RNA integrity at high doses"
|
||||
- note: "More common in CLIP-based methods for precise interaction mapping"
|
||||
formaldehyde_crosslinking:
|
||||
- advantage: "Straightforward chemical method without specialized equipment"
|
||||
- advantage: "Captures indirect interactions within larger complexes"
|
||||
- advantage: "Often higher yield"
|
||||
- disadvantage: "Higher background from nonspecific crosslinks"
|
||||
- disadvantage: "Reversal requires potentially harsh conditions (heating, high salt)"
|
||||
- note: "Better for detecting weak or transient interactions"
|
||||
|
||||
# Safety considerations
|
||||
safety:
|
||||
ppe: "Lab coat, gloves, and eye protection required"
|
||||
hazards: "UV radiation: avoid direct exposure; Formaldehyde: toxic and carcinogenic, use in fume hood"
|
||||
waste: "Dispose of crosslinking reagents according to institutional guidelines"
|
||||
|
||||
# Quality control
|
||||
quality_control:
|
||||
- check: "Include IgG control IP"
|
||||
criteria: "Should show minimal enrichment of target RNAs"
|
||||
- check: "Include input RNA sample"
|
||||
criteria: "For normalization and calculating percent input"
|
||||
- check: "Test known RNA-protein interaction"
|
||||
criteria: "Should show significant enrichment over IgG control"
|
||||
|
||||
# Data analysis
|
||||
data_analysis:
|
||||
- step: 1
|
||||
action: "Calculate fold enrichment"
|
||||
details: "Compare target RNA in specific IP vs. IgG control IP"
|
||||
- step: 2
|
||||
action: "Calculate percent input"
|
||||
details: "Compare RNA abundance in IP vs. a defined percentage of input material"
|
||||
- step: 3
|
||||
action: "Statistical analysis"
|
||||
details: "Perform appropriate statistical tests on biological replicates"
|
||||
|
||||
# Best practices
|
||||
best_practices:
|
||||
- "Run pilot experiments to optimize crosslinking conditions for specificity vs. yield"
|
||||
- "Test different crosslinking strengths and confirm with a known positive RNA target"
|
||||
- "Include IgG control or nonspecific antibody to measure background binding"
|
||||
- "Use input RNA to normalize or calculate percentage of input in qPCR"
|
||||
- "If possible, use a known RNA-protein interaction as positive control"
|
||||
- "Incorporate RNase inhibitors in all buffers and keep samples cold"
|
||||
- "For many RIP-qPCR experiments, mild UV crosslinking at 254 nm is preferred"
|
||||
|
||||
# References
|
||||
references:
|
||||
- "Peritz T, et al. (2006) Immunoprecipitation of mRNA-protein complexes. Nat Protoc. 1(2):577-580"
|
||||
- "Keene JD, et al. (2006) RIP-Chip: the isolation and identification of mRNAs, microRNAs and protein components of ribonucleoprotein complexes from cell extracts. Nat Protoc. 1(1):302-307"
|
||||
- "Niranjanakumari S, et al. (2002) Reversible cross-linking combined with immunoprecipitation to study RNA-protein interactions in vivo. Methods. 26(2):182-190"
|
||||
|
||||
# Notes
|
||||
notes: |
|
||||
- Two crosslinking methods are described: UV (254 nm) and formaldehyde
|
||||
- UV crosslinking is more specific but requires specialized equipment
|
||||
- Formaldehyde crosslinking is simpler but may have higher background
|
||||
- Always include appropriate controls (IgG, input RNA)
|
||||
- Consider CLIP-based methods for more precise mapping of binding sites
|
||||
- For studying RNA binding proteins that interact with many targets, consider combining with RNA-seq (RIP-seq)
|
||||
---
|
||||
144
protocols/seeding_96well_plate_v1.yaml
Normal file
144
protocols/seeding_96well_plate_v1.yaml
Normal file
@ -0,0 +1,144 @@
|
||||
---
|
||||
# Protocol metadata
|
||||
id: PROT-0033
|
||||
name: Seeding 96-Well Plate Protocol
|
||||
version: 1.0
|
||||
description: Protocol for seeding cells in a 96-well plate format
|
||||
author: Lab Staff
|
||||
created: 2025-05-07
|
||||
last_updated: 2025-05-07
|
||||
category: cell-culture
|
||||
|
||||
# Materials required
|
||||
materials:
|
||||
- name: 96-well tissue culture plate
|
||||
type: Clear for brightfield / black for fluorescence
|
||||
preparation: Sterile
|
||||
- name: Cell suspension
|
||||
preparation: Freshly harvested and counted
|
||||
concentration: Adjusted for desired cells/well
|
||||
- name: Complete media
|
||||
temperature: Pre-warmed to 37°C
|
||||
storage: 4°C
|
||||
- name: Sterile 25-mL reagent reservoir
|
||||
type: Sterile, disposable
|
||||
- name: 70% ethanol
|
||||
use: Surface disinfection
|
||||
|
||||
# Equipment required
|
||||
equipment:
|
||||
- name: Multi-channel pipette
|
||||
type: 8 or 12 channel, 50-200 μL
|
||||
- name: Biosafety cabinet
|
||||
certification: Class II
|
||||
- name: Pipette aid
|
||||
range: For handling cell suspension volumes
|
||||
- name: Hemocytometer or cell counter
|
||||
use: For determining cell concentration
|
||||
- name: CO2 incubator
|
||||
settings: 37°C, 5% CO2, humidified
|
||||
|
||||
# Protocol steps
|
||||
steps:
|
||||
- step: 1
|
||||
action: "Prepare workspace"
|
||||
details: "Sterilize all materials and working area with 70% ethanol in biosafety cabinet"
|
||||
- step: 2
|
||||
action: "Prepare experimental plan"
|
||||
details: "Complete plate layout and cell number calculations before beginning seeding procedure"
|
||||
- step: 3
|
||||
action: "Open sterile plate"
|
||||
details: "Unwrap 96-well tissue culture plate and place in biosafety cabinet"
|
||||
- step: 4
|
||||
action: "Calculate required volumes"
|
||||
details: "For a full 96-well plate with 100 μL per well, prepare at least 10 mL of cell suspension"
|
||||
- step: 5
|
||||
action: "Prepare diluted cell suspension"
|
||||
details: "Dilute 9 mL of cell suspension with 14 mL complete media to achieve desired cell concentration"
|
||||
- step: 6
|
||||
action: "Transfer to reservoir"
|
||||
details: "Gently swirl to mix and pour into a sterile 25-mL reagent reservoir"
|
||||
- step: 7
|
||||
action: "Seed plate"
|
||||
details: "Using a multi-channel pipette, seed 100 μL of cell suspension per well"
|
||||
- step: 8
|
||||
action: "Mix during seeding"
|
||||
details: "Mix cell suspension periodically while seeding to ensure even cell distribution"
|
||||
- step: 9
|
||||
action: "Complete plate"
|
||||
details: "Continue until all required wells are seeded according to the plate layout"
|
||||
- step: 10
|
||||
action: "Clean up"
|
||||
details: "Aspirate leftover cell suspension and discard disposables appropriately"
|
||||
- step: 11
|
||||
action: "Incubate plate"
|
||||
details: "Place the plate in the incubator at 37°C, 5% CO2 for the required attachment period"
|
||||
- step: 12
|
||||
action: "Document"
|
||||
details: "Record plate layout, cell density, and seeding time in lab notebook"
|
||||
|
||||
# Cell density guidelines
|
||||
cell_density_guidelines:
|
||||
- cell_type: "HEK293"
|
||||
density: "10,000-15,000 cells/well"
|
||||
notes: "Fast-growing, adhere quickly"
|
||||
- cell_type: "HepG2"
|
||||
density: "15,000-20,000 cells/well"
|
||||
notes: "Form clumps, mix well before seeding"
|
||||
- cell_type: "Primary hepatocytes"
|
||||
density: "20,000-40,000 cells/well"
|
||||
notes: "Use collagen-coated plates"
|
||||
- cell_type: "Fibroblasts"
|
||||
density: "5,000-10,000 cells/well"
|
||||
notes: "Spread widely, use lower density"
|
||||
- cell_type: "MCF7"
|
||||
density: "10,000-15,000 cells/well"
|
||||
notes: "Medium growth rate"
|
||||
|
||||
# Critical parameters
|
||||
critical_parameters:
|
||||
- parameter: "Cell suspension homogeneity"
|
||||
details: "Mix suspension frequently to prevent cell settling and ensure even distribution"
|
||||
- parameter: "Seeding technique"
|
||||
details: "Maintain consistent angle and speed with multi-channel pipette to ensure uniform volumes"
|
||||
- parameter: "Edge effects"
|
||||
details: "Consider filling outer wells with PBS for long-term experiments to minimize evaporation"
|
||||
|
||||
# Troubleshooting
|
||||
troubleshooting:
|
||||
- problem: "Uneven cell distribution"
|
||||
solution: "Mix cell suspension more frequently; check pipette calibration; avoid bubbles when dispensing"
|
||||
- problem: "Edge wells drying out"
|
||||
solution: "Fill outer wells with sterile PBS or water; use humidified chamber in incubator"
|
||||
- problem: "Cell clumping"
|
||||
solution: "Triturate cell suspension gently before seeding; ensure complete dissociation during harvesting"
|
||||
|
||||
# Safety considerations
|
||||
safety:
|
||||
ppe: "Lab coat, gloves, and eye protection required"
|
||||
hazards: "Follow appropriate biosafety procedures for cell line being used"
|
||||
|
||||
# Quality control
|
||||
quality_control:
|
||||
- check: "Even seeding"
|
||||
criteria: "Visually inspect plate under microscope for uniform cell distribution"
|
||||
- check: "Cell density"
|
||||
criteria: "Count cells in representative wells to verify target density was achieved"
|
||||
- check: "Sterility"
|
||||
criteria: "Include at least one well with media only to check for contamination"
|
||||
|
||||
# References
|
||||
references:
|
||||
- "Promega Corporation. (2018). Cell Culture Basics: Passage Number Effects in Cell Lines. Cell Notes, 15:2-5."
|
||||
- "Freshney RI. (2016) Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, 7th Edition."
|
||||
|
||||
# Notes
|
||||
notes: |
|
||||
- Plate type should be selected based on the planned assay (clear for brightfield, black for fluorescence)
|
||||
- Always mix suspension frequently to ensure even cell distribution
|
||||
- Consistent seeding technique is important for reproducible results
|
||||
- For cell attachment assays, check attachment at 2-4 hours post-seeding
|
||||
- For proliferation assays, cells typically need 24 hours to recover before treatment
|
||||
- Consider using a repeater pipette for very large numbers of plates
|
||||
- For high-throughput screening, automated cell dispensers can improve consistency
|
||||
---
|
||||
149
protocols/seeding_based_on_confluence_v1.yaml
Normal file
149
protocols/seeding_based_on_confluence_v1.yaml
Normal file
@ -0,0 +1,149 @@
|
||||
---
|
||||
# Protocol metadata
|
||||
id: PROT-0028
|
||||
name: Seeding Based on Confluence Protocol
|
||||
version: 1.0
|
||||
description: Protocol for seeding cells based on confluence rather than direct cell counting
|
||||
author: Lab Staff
|
||||
created: 2025-05-07
|
||||
last_updated: 2025-05-07
|
||||
category: cell-culture
|
||||
|
||||
# Materials required
|
||||
materials:
|
||||
- name: Cell culture flask
|
||||
type: T-25 (25 cm²)
|
||||
condition: 90-100% confluent cells
|
||||
- name: Sterile PBS
|
||||
temperature: Room temperature
|
||||
storage: Room temperature
|
||||
- name: Trypsin-EDTA
|
||||
concentration: 0.25% or 0.05% (cell line dependent)
|
||||
temperature: 37°C (pre-warmed)
|
||||
storage: -20°C (stock), 4°C (working solution)
|
||||
- name: Complete media
|
||||
composition: Base media with 10% FBS + 1% Penicillin/Streptomycin
|
||||
temperature: 37°C (pre-warmed)
|
||||
storage: 4°C
|
||||
- name: Culture plates
|
||||
type: 6-well or 24-well
|
||||
preparation: Sterile
|
||||
|
||||
# Equipment required
|
||||
equipment:
|
||||
- name: Biosafety cabinet
|
||||
certification: Class II
|
||||
- name: CO2 incubator
|
||||
settings: 37°C, 5% CO2, humidified
|
||||
- name: Microscope
|
||||
type: Inverted, phase contrast
|
||||
- name: Pipettes and sterile tips
|
||||
range: Various sizes for cell culture
|
||||
- name: Aspiration system
|
||||
type: Vacuum or manual
|
||||
|
||||
# Protocol steps
|
||||
steps:
|
||||
- step: 1
|
||||
action: "Prepare materials"
|
||||
details: "Pre-warm media and trypsin; gather plates and other supplies in biosafety cabinet"
|
||||
- step: 2
|
||||
action: "Examine source flask"
|
||||
details: "Confirm flask is 90–100% confluent using inverted microscope"
|
||||
- step: 3
|
||||
action: "Wash cells"
|
||||
details: "Aspirate media from flask and wash with 10 mL sterile PBS to remove serum"
|
||||
- step: 4
|
||||
action: "Add trypsin"
|
||||
details: "Add 1 mL trypsin, ensure all cells are covered, and incubate for ~5 minutes at 37°C"
|
||||
- step: 5
|
||||
action: "Check cell detachment"
|
||||
details: "Observe under microscope to confirm cells have rounded up and detached"
|
||||
- step: 6
|
||||
action: "Neutralize trypsin"
|
||||
details: "Add 9 mL complete media (10% FBS + 1% PS) to neutralize trypsin"
|
||||
- step: 7
|
||||
action: "Mix cell suspension"
|
||||
details: "Gently pipette up and down to create uniform cell suspension"
|
||||
- step: 8
|
||||
action: "Seed 6-well plates"
|
||||
details: "Pipet 350 μL cell suspension into each well of a 6-well plate"
|
||||
- step: 9
|
||||
action: "Add media to 6-well plates"
|
||||
details: "Add 1600 μL complete media to each well (total volume 1950 μL per well)"
|
||||
- step: 10
|
||||
action: "Seed 24-well plates"
|
||||
details: "For 24-well plates, pipet 200 μL cell suspension into each well"
|
||||
- step: 11
|
||||
action: "Add media to 24-well plates"
|
||||
details: "Add 800 μL complete media to each well (total volume 1000 μL per well)"
|
||||
- step: 12
|
||||
action: "Distribute cells"
|
||||
details: "Gently rock/swirl plate to ensure even distribution of cells"
|
||||
- step: 13
|
||||
action: "Incubate plates"
|
||||
details: "Place in incubator at 37°C, 5% CO2 for 24–48 hours before further manipulation"
|
||||
- step: 14
|
||||
action: "Monitor confluence"
|
||||
details: "Check confluence after 24 hours to determine if enough time has passed for experiments"
|
||||
|
||||
# Common dilution ratios
|
||||
dilution_ratios:
|
||||
- plate_format: "6-well"
|
||||
ratio: "1:29 (350 μL cells : 1600 μL media)"
|
||||
expected_confluence: "30-40% after 24h; 60-70% after 48h"
|
||||
- plate_format: "24-well"
|
||||
ratio: "1:5 (200 μL cells : 800 μL media)"
|
||||
expected_confluence: "30-40% after 24h; 60-70% after 48h"
|
||||
- plate_format: "12-well"
|
||||
ratio: "1:10 (250 μL cells : 1000 μL media)"
|
||||
expected_confluence: "Similar to 24-well"
|
||||
- plate_format: "96-well"
|
||||
ratio: "1:20 (10 μL cells : 190 μL media)"
|
||||
expected_confluence: "May need optimization for specific cell lines"
|
||||
|
||||
# Critical parameters
|
||||
critical_parameters:
|
||||
- parameter: "Source flask confluence"
|
||||
details: "Starting flask should be 90-100% confluent; lower confluence may yield inconsistent results"
|
||||
- parameter: "Cell suspension homogeneity"
|
||||
details: "Ensure thorough but gentle mixing to achieve uniform cell suspension"
|
||||
- parameter: "Incubation timing"
|
||||
details: "Fast-growing cells may need less than 24h before treatment/transfection"
|
||||
|
||||
# Troubleshooting
|
||||
troubleshooting:
|
||||
- problem: "Uneven cell distribution"
|
||||
solution: "Ensure thorough mixing of cell suspension; gently rock plates after seeding"
|
||||
- problem: "Low attachment"
|
||||
solution: "Check trypsin activity; ensure adequate neutralization; verify plate surface is suitable for cell type"
|
||||
- problem: "Inconsistent confluence between wells"
|
||||
solution: "Mix cell suspension more frequently while dispensing; consider cell counter for future experiments"
|
||||
|
||||
# Safety considerations
|
||||
safety:
|
||||
ppe: "Lab coat, gloves, and eye protection required"
|
||||
hazards: "Follow appropriate biosafety procedures for cell line being used"
|
||||
|
||||
# Quality control
|
||||
quality_control:
|
||||
- check: "Confluence check at 24h"
|
||||
criteria: "Wells should show consistent cell density across all wells"
|
||||
- check: "Cell morphology"
|
||||
criteria: "Cells should display normal morphology for the cell type"
|
||||
|
||||
# References
|
||||
references:
|
||||
- "Freshney RI. (2016) Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, 7th Edition."
|
||||
- "Davis JM. (2011) Animal Cell Culture: Essential Methods. Wiley-Blackwell."
|
||||
|
||||
# Notes
|
||||
notes: |
|
||||
- This protocol uses a fixed dilution from a confluent flask rather than direct cell counting
|
||||
- Adjust volumes proportionally for different plate formats
|
||||
- Incubation time may vary depending on cell type and growth rate
|
||||
- For fast-growing cells like HEK293, check confluence after 18-20 hours
|
||||
- For slow-growing primary cells, 48-72 hours may be required
|
||||
- The protocol can be adapted for different flask sizes (T-75, T-175) by scaling the cell suspension volume
|
||||
- While less precise than direct counting, this method is suitable for routine passaging and experiments where exact cell number is not critical
|
||||
---
|
||||
126
protocols/siRNA_transfection_24well_rnaimax_v1.yaml
Normal file
126
protocols/siRNA_transfection_24well_rnaimax_v1.yaml
Normal file
@ -0,0 +1,126 @@
|
||||
---
|
||||
# Protocol metadata
|
||||
id: PROT-0020
|
||||
name: 24-well Plate siRNA Transfection (RNAiMAX)
|
||||
version: 1.0
|
||||
description: Protocol for transfecting 4 siRNAs (including siNC) into cells in a 24-well plate using Lipofectamine RNAiMAX
|
||||
author: Lab Agent
|
||||
created: 2024-06-08
|
||||
last_updated: 2025-05-07
|
||||
category: cell-culture
|
||||
|
||||
# Materials required
|
||||
materials:
|
||||
- name: siRNA#1
|
||||
concentration: 10 nM final
|
||||
storage: -20°C or -80°C
|
||||
- name: siRNA#2
|
||||
concentration: 10 nM final
|
||||
storage: -20°C or -80°C
|
||||
- name: siRNA#3
|
||||
concentration: 10 nM final
|
||||
storage: -20°C or -80°C
|
||||
- name: siNC (negative control)
|
||||
concentration: 10 nM final
|
||||
storage: -20°C or -80°C
|
||||
- name: Lipofectamine RNAiMAX
|
||||
amount: 1.5 µL per well
|
||||
storage: 4°C
|
||||
- name: Opti-MEM Reduced Serum Medium
|
||||
storage: 4°C
|
||||
- name: Complete growth medium
|
||||
notes: Appropriate for your cell line, without antibiotics during transfection
|
||||
|
||||
# Equipment required
|
||||
equipment:
|
||||
- name: 24-well tissue culture plate
|
||||
type: Cell culture grade
|
||||
- name: Pipettes and sterile tips
|
||||
range: 0.5-1000 µL
|
||||
- name: CO2 incubator
|
||||
settings: 37°C, 5% CO2, humidified
|
||||
- name: Biosafety cabinet
|
||||
certification: Class II
|
||||
- name: Centrifuge
|
||||
type: Benchtop (if needed for cell preparation)
|
||||
|
||||
# Protocol steps
|
||||
steps:
|
||||
- step: 1
|
||||
action: "Seed cells in 24-well plate"
|
||||
details: "Seed 5x10^4 cells/well 24 hours prior to transfection to achieve 60-80% confluence at time of transfection"
|
||||
- step: 2
|
||||
action: "Prepare siRNA dilutions"
|
||||
details: "For each siRNA, dilute 10 pmol siRNA in 50 µL Opti-MEM per well"
|
||||
- step: 3
|
||||
action: "Prepare RNAiMAX dilution"
|
||||
details: "In a separate tube, dilute 1.5 µL RNAiMAX in 50 µL Opti-MEM per well"
|
||||
- step: 4
|
||||
action: "Create transfection complexes"
|
||||
details: "Combine diluted siRNA and RNAiMAX solutions, mix gently, and incubate for 10-20 min at room temperature"
|
||||
- step: 5
|
||||
action: "Add complexes to cells"
|
||||
details: "Add 100 µL siRNA-RNAiMAX complex dropwise to each well containing cells in 400 µL growth medium"
|
||||
- step: 6
|
||||
action: "Incubate cells"
|
||||
details: "Incubate cells at 37°C, 5% CO2 for 24-72 hours before assessing knockdown efficiency"
|
||||
- step: 7
|
||||
action: "Change medium (optional)"
|
||||
details: "After 4-6 hours, replace transfection medium with fresh complete medium if needed for sensitive cells"
|
||||
|
||||
# Experimental design
|
||||
experimental_design:
|
||||
- layout: |
|
||||
Typical 24-well plate layout:
|
||||
| 1-siNC | 2-siNC | 3-siNC | 4-empty |
|
||||
| 1-siRNA1| 2-siRNA1| 3-siRNA1| 4-empty |
|
||||
| 1-siRNA2| 2-siRNA2| 3-siRNA2| 4-empty |
|
||||
| 1-siRNA3| 2-siRNA3| 3-siRNA3| 4-empty |
|
||||
| 5-empty | 6-empty |etc.
|
||||
|
||||
# Critical parameters
|
||||
critical_parameters:
|
||||
- parameter: "Cell density"
|
||||
details: "60-80% confluence at time of transfection is optimal"
|
||||
- parameter: "siRNA concentration"
|
||||
details: "10 nM is standard but may need optimization (5-50 nM range)"
|
||||
- parameter: "RNAiMAX:siRNA ratio"
|
||||
details: "May need adjustment for different cell lines"
|
||||
- parameter: "Incubation time"
|
||||
details: "10-20 minutes for complex formation; overly long incubation may reduce efficiency"
|
||||
|
||||
# Troubleshooting
|
||||
troubleshooting:
|
||||
- problem: "Low transfection efficiency"
|
||||
solution: "Optimize cell density, increase RNAiMAX amount, extend transfection time"
|
||||
- problem: "High cytotoxicity"
|
||||
solution: "Reduce siRNA/RNAiMAX concentrations, change medium 4-6h post-transfection"
|
||||
- problem: "Poor knockdown"
|
||||
solution: "Verify siRNA design, extend incubation time post-transfection, consider alternative delivery method"
|
||||
|
||||
# Safety considerations
|
||||
safety:
|
||||
ppe: "Lab coat, gloves, and eye protection required"
|
||||
hazards: "RNAiMAX may cause irritation; handle with care"
|
||||
|
||||
# Quality control
|
||||
quality_control:
|
||||
- check: "Include siNC (non-targeting control) in every experiment"
|
||||
criteria: "Should show no target gene knockdown"
|
||||
- check: "Verify knockdown efficiency"
|
||||
criteria: "Typically by qPCR or Western blot 24-72h post-transfection"
|
||||
|
||||
# References
|
||||
references:
|
||||
- "Lipofectamine RNAiMAX manufacturer's protocol"
|
||||
- "Dorsett Y, Tuschl T. (2004) siRNAs: applications in functional genomics and potential as therapeutics. Nat Rev Drug Discov. 3(4):318-329"
|
||||
|
||||
# Notes
|
||||
notes: |
|
||||
- siNC is a non-targeting negative control siRNA
|
||||
- Adjust cell number and reagent volumes as needed for specific cell lines
|
||||
- For sensitive cells, transfect in antibiotic-free medium
|
||||
- Assay for knockdown 24-72 hours post-transfection depending on target protein half-life
|
||||
- Consider optimizing siRNA concentration (5-50 nM range) for your specific cell line
|
||||
- Perform reverse transfection as an alternative by adding complexes to wells before adding cells
|
||||
---
|
||||
143
protocols/spectramax_plate_reader_v1.yaml
Normal file
143
protocols/spectramax_plate_reader_v1.yaml
Normal file
@ -0,0 +1,143 @@
|
||||
---
|
||||
# Protocol metadata
|
||||
id: PROT-0035
|
||||
name: SpectraMax Plate Reader Protocol
|
||||
version: 1.0
|
||||
description: Protocol for using the SpectraMax plate reader for absorbance measurements
|
||||
author: JJ
|
||||
created: 2025-03-22
|
||||
last_updated: 2025-05-07
|
||||
category: equipment-usage
|
||||
|
||||
# Materials required
|
||||
materials:
|
||||
- name: 96-well plate with samples
|
||||
type: Clear bottom preferred
|
||||
preparation: Bubble-free samples with adequate volume
|
||||
- name: SpectraMax plate reader
|
||||
model: Specify model (e.g., M5, i3x)
|
||||
location: Core facility
|
||||
- name: USB drive
|
||||
use: For data export
|
||||
size: Any capacity (files are small)
|
||||
|
||||
# Equipment required
|
||||
equipment:
|
||||
- name: Computer with SpectraMax software
|
||||
version: Latest SoftMax Pro software
|
||||
location: Connected to the plate reader
|
||||
- name: Plate reader cover
|
||||
use: To prevent light interference during reading
|
||||
|
||||
# Protocol steps
|
||||
steps:
|
||||
- step: 1
|
||||
action: "Start the software"
|
||||
details: "Open the SpectraMax software on the computer"
|
||||
- step: 2
|
||||
action: "Create a new experiment"
|
||||
details: "Click Protocol > New Experiment"
|
||||
- step: 3
|
||||
action: "Configure settings"
|
||||
details: "Click the gear icon above the plate grid to adjust settings"
|
||||
- step: 4
|
||||
action: "Set reading parameters"
|
||||
details: "For BCA assay, select:
|
||||
- Absorbance mode
|
||||
- Wavelength = 562 nm
|
||||
- 96-well, clear bottom plate type
|
||||
- No shaking
|
||||
- Fast Read option is fine"
|
||||
- step: 5
|
||||
action: "Select wells to read"
|
||||
details: "Paint (select) the wells you want to measure in the grid"
|
||||
- step: 6
|
||||
action: "Open the plate drawer"
|
||||
details: "Click the open/close plate reader button in the software"
|
||||
- step: 7
|
||||
action: "Insert plate"
|
||||
details: "Insert your plate with well H1 positioned in the bottom-left corner"
|
||||
- step: 8
|
||||
action: "Start measurement"
|
||||
details: "Press Read button in the software"
|
||||
- step: 9
|
||||
action: "Export data"
|
||||
details: "Copy paste the results grid to Excel or export directly to file"
|
||||
- step: 10
|
||||
action: "Save data"
|
||||
details: "Save Excel file to a USB drive or to the hard drive and transfer it off the core facility computer"
|
||||
- step: 11
|
||||
action: "Clean up"
|
||||
details: "Close the software, remove your plate, and log out of the system if required"
|
||||
|
||||
# Common assay settings
|
||||
common_assays:
|
||||
- assay: "BCA protein assay"
|
||||
wavelength: 562 nm
|
||||
plate_type: "96-well, clear bottom"
|
||||
mode: "Absorbance"
|
||||
- assay: "Bradford assay"
|
||||
wavelength: 595 nm
|
||||
plate_type: "96-well, clear bottom"
|
||||
mode: "Absorbance"
|
||||
- assay: "ELISA (TMB substrate)"
|
||||
wavelength: 450 nm
|
||||
reference: 630 nm
|
||||
plate_type: "96-well, clear bottom"
|
||||
mode: "Absorbance"
|
||||
- assay: "Fluorescence assay"
|
||||
excitation: "Depends on fluorophore"
|
||||
emission: "Depends on fluorophore"
|
||||
plate_type: "96-well, black or clear bottom"
|
||||
mode: "Fluorescence"
|
||||
|
||||
# Critical parameters
|
||||
critical_parameters:
|
||||
- parameter: "Plate orientation"
|
||||
details: "Always insert plate with well H1 in bottom-left corner to match software layout"
|
||||
- parameter: "Bubbles in wells"
|
||||
details: "Remove bubbles as they can interfere with readings"
|
||||
- parameter: "Blank wells"
|
||||
details: "Include blank wells for background subtraction"
|
||||
- parameter: "Wavelength selection"
|
||||
details: "Match wavelength to assay requirements"
|
||||
|
||||
# Troubleshooting
|
||||
troubleshooting:
|
||||
- problem: "Abnormally high readings"
|
||||
solution: "Check for bubbles; ensure proper blank subtraction; verify wavelength setting"
|
||||
- problem: "Low or no signal"
|
||||
solution: "Verify wavelength; check sample preparation; ensure plate orientation is correct"
|
||||
- problem: "Inconsistent readings"
|
||||
solution: "Mix wells before reading; check for condensation on plate lid; avoid edge effects"
|
||||
- problem: "Software errors"
|
||||
solution: "Restart software; contact core facility manager if issues persist"
|
||||
|
||||
# Safety considerations
|
||||
safety:
|
||||
ppe: "Follow standard laboratory safety procedures"
|
||||
hazards: "Low risk; handle plates containing potentially hazardous materials appropriately"
|
||||
|
||||
# Data analysis tips
|
||||
data_analysis:
|
||||
- tip: "Always subtract blank well readings"
|
||||
details: "Average multiple blank wells for more accurate background correction"
|
||||
- tip: "Standard curves"
|
||||
details: "Use appropriate curve fitting (linear, 4-parameter, etc.) based on assay type"
|
||||
- tip: "Technical replicates"
|
||||
details: "Calculate mean and standard deviation across replicate wells"
|
||||
|
||||
# References
|
||||
references:
|
||||
- "SpectraMax user manual (available at core facility)"
|
||||
- "Assay-specific references based on experiment type"
|
||||
|
||||
# Notes
|
||||
notes: |
|
||||
- Settings may need to be adjusted depending on the specific assay
|
||||
- Always ensure the plate is properly oriented when loading (H1 position at bottom-left)
|
||||
- Remember to transfer your data before leaving the facility
|
||||
- For specialized readings (kinetic, spectral scans, etc.), consult the core facility manager
|
||||
- Some assays may benefit from plate shaking prior to reading
|
||||
- If using a plate with a lid, remove it before measurement unless protocol specifically indicates otherwise
|
||||
---
|
||||
122
protocols/subculturing_cells_v1.yaml
Normal file
122
protocols/subculturing_cells_v1.yaml
Normal file
@ -0,0 +1,122 @@
|
||||
---
|
||||
# Protocol metadata
|
||||
id: PROT-0036
|
||||
name: Subculturing Cells Protocol
|
||||
version: 1.0
|
||||
description: Protocol for passaging and subculturing adherent cell lines
|
||||
author: Lab Staff
|
||||
created: 2025-05-07
|
||||
last_updated: 2025-05-07
|
||||
category: cell-culture
|
||||
|
||||
# Materials required
|
||||
materials:
|
||||
- name: Cell culture flask with cells
|
||||
notes: 70-90% confluent
|
||||
- name: PBS (Phosphate Buffered Saline)
|
||||
concentration: 1X, sterile
|
||||
temperature: Room temperature
|
||||
- name: Tryp-LE or trypsin-EDTA
|
||||
temperature: Pre-warmed to 37°C
|
||||
storage: -20°C (stock), 4°C (working)
|
||||
- name: Complete media
|
||||
temperature: Pre-warmed to 37°C
|
||||
- name: New culture flask
|
||||
type: T25, T75, or T175 as needed
|
||||
- name: 70% ethanol
|
||||
preparation: Freshly prepared or commercially available
|
||||
use: Surface disinfection
|
||||
|
||||
# Equipment required
|
||||
equipment:
|
||||
- name: Biosafety cabinet
|
||||
certification: Class II
|
||||
- name: CO2 incubator
|
||||
settings: 37°C, 5% CO2, humidified
|
||||
- name: Centrifuge
|
||||
settings: 200-300g for 5 minutes
|
||||
- name: Aspiration system
|
||||
type: Vacuum or manual
|
||||
- name: Hemocytometer or cell counter (optional)
|
||||
use: Cell counting if precise seeding is required
|
||||
|
||||
# Protocol steps
|
||||
steps:
|
||||
- step: 1
|
||||
action: "Prepare workstation"
|
||||
details: "Turn on biosafety cabinet, sterilize surfaces and materials with 70% ethanol"
|
||||
- step: 2
|
||||
action: "Maintain sterile technique"
|
||||
details: "Spray gloved hands with 70% ethanol after touching anything outside the hood"
|
||||
- step: 3
|
||||
action: "Aspirate old media"
|
||||
details: "Carefully remove all media without disturbing cell layer"
|
||||
- step: 4
|
||||
action: "Wash cells with PBS"
|
||||
details: "Add sufficient PBS to cover the cell layer, gently rock, then aspirate"
|
||||
- step: 5
|
||||
action: "Add dissociation reagent"
|
||||
details: "Add 1 ml Tryp-LE per T25 flask (scale accordingly for larger flasks)"
|
||||
- step: 6
|
||||
action: "Incubate cells"
|
||||
details: "Place in incubator for 5-10 minutes until cells detach"
|
||||
- step: 7
|
||||
action: "Check cell detachment"
|
||||
details: "Observe under microscope to confirm cells are rounded and detached"
|
||||
- step: 8
|
||||
action: "Neutralize and collect cells"
|
||||
details: "Add 9 ml complete media, pipette gently to dislodge and disperse cells"
|
||||
- step: 9
|
||||
action: "Seed new flask"
|
||||
details: "Transfer 1 ml cell suspension to new flask containing 9 ml complete media (1:10 split ratio)"
|
||||
- step: 10
|
||||
action: "Label flask"
|
||||
details: "Note cell line, passage number, date, and your initials"
|
||||
- step: 11
|
||||
action: "Incubate cells"
|
||||
details: "Place in incubator at 37°C with 5% CO₂"
|
||||
|
||||
# Critical parameters
|
||||
critical_parameters:
|
||||
- parameter: "Confluence level"
|
||||
details: "Ideal passage at 70-90% confluence; overconfluent cells may exhibit altered properties"
|
||||
- parameter: "Dissociation time"
|
||||
details: "Excessive trypsinization can damage cells; insufficient time leads to poor detachment"
|
||||
- parameter: "Split ratio"
|
||||
details: "Adjust based on growth rate and experimental timeline (1:3 to 1:20 depending on cell line)"
|
||||
|
||||
# Troubleshooting
|
||||
troubleshooting:
|
||||
- problem: "Cells not detaching"
|
||||
solution: "Extend incubation time with dissociation reagent, tap flask gently, or try fresh trypsin"
|
||||
- problem: "Cell clumping"
|
||||
solution: "Pipette gently up and down to create single-cell suspension; use cell strainer if needed"
|
||||
- problem: "Poor cell recovery/growth"
|
||||
solution: "Check media quality, incubator conditions, and avoid over-trypsinization"
|
||||
|
||||
# Safety considerations
|
||||
safety:
|
||||
ppe: "Lab coat, gloves, and closed-toe shoes required"
|
||||
hazards: "Handle biological materials according to biosafety level requirements"
|
||||
|
||||
# Quality control
|
||||
quality_control:
|
||||
- check: "Cell morphology"
|
||||
criteria: "Cells should maintain typical morphology post-passage"
|
||||
- check: "Growth rate"
|
||||
criteria: "Consistent doubling time compared to historical data for the cell line"
|
||||
|
||||
# References
|
||||
references:
|
||||
- "Freshney RI. (2016) Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, 7th Edition"
|
||||
|
||||
# Notes
|
||||
notes: |
|
||||
- This protocol describes a 1:10 split ratio
|
||||
- Incubation time with Tryp-LE may vary depending on cell line
|
||||
- Maintain sterile technique throughout the procedure
|
||||
- Consider counting cells for experiments requiring precise cell numbers
|
||||
- Record passage number with each subculture
|
||||
- Some cell lines may require specialized coating on flasks (collagen, poly-L-lysine, etc.)
|
||||
- Check specific requirements for your cell line, as some may need different media or culture conditions
|
||||
---
|
||||
133
protocols/treating_cells_v1.yaml
Normal file
133
protocols/treating_cells_v1.yaml
Normal file
@ -0,0 +1,133 @@
|
||||
---
|
||||
# Protocol metadata
|
||||
id: PROT-0034
|
||||
name: Treating Cells Protocol
|
||||
version: 1.0
|
||||
description: Protocol for treating cultured cells with control or treatment media
|
||||
author: Lab Staff
|
||||
created: 2025-05-07
|
||||
last_updated: 2025-05-07
|
||||
category: cell-culture
|
||||
|
||||
# Materials required
|
||||
materials:
|
||||
- name: Culture plate
|
||||
type: 96-well, 24-well, or 6-well
|
||||
preparation: Cells at appropriate confluence for treatment
|
||||
- name: Control media
|
||||
temperature: Pre-warmed to 37°C
|
||||
storage: 4°C
|
||||
- name: Treatment media
|
||||
temperature: Pre-warmed to 37°C
|
||||
storage: As appropriate for treatment compound
|
||||
- name: PBS
|
||||
temperature: Room temperature or 37°C
|
||||
use: Optional for washing (compound-dependent)
|
||||
|
||||
# Equipment required
|
||||
equipment:
|
||||
- name: Thermal bead bath
|
||||
temperature: 37°C
|
||||
use: For warming media
|
||||
- name: Biosafety cabinet
|
||||
certification: Class II
|
||||
- name: Aspirator
|
||||
type: Vacuum or manual
|
||||
- name: Pipettes and sterile tips
|
||||
range: Appropriate for plate format
|
||||
- name: CO2 incubator
|
||||
settings: 37°C, 5% CO2, humidified
|
||||
|
||||
# Protocol steps
|
||||
steps:
|
||||
- step: 1
|
||||
action: "Prepare treatment media"
|
||||
details: "Prepare fresh treatment media with the compound of interest at desired concentration"
|
||||
- step: 2
|
||||
action: "Pre-warm media"
|
||||
details: "Place control and treatment media in thermal bead bath at 37°C for at least 15 minutes"
|
||||
- step: 3
|
||||
action: "Remove plate from incubator"
|
||||
details: "Transfer cell culture plate to biosafety cabinet"
|
||||
- step: 4
|
||||
action: "Aspirate old media"
|
||||
details: "Carefully remove all existing media from wells without disrupting cell layer"
|
||||
- step: 5
|
||||
action: "Optional wash step"
|
||||
details: "If needed, gently wash cells with PBS to remove residual media/serum"
|
||||
- step: 6
|
||||
action: "Add appropriate volume of media"
|
||||
details: "Add pre-warmed control or treatment media according to plate format:
|
||||
- 150 μL for 96-well plates
|
||||
- 1 mL for 24-well plates
|
||||
- 2 mL for 6-well plates"
|
||||
- step: 7
|
||||
action: "Return to incubator"
|
||||
details: "Place plate back in incubator for the duration of the treatment period"
|
||||
- step: 8
|
||||
action: "Document treatment"
|
||||
details: "Record treatment start time, concentration, and other relevant details in lab notebook"
|
||||
|
||||
# Volume reference
|
||||
volume_reference:
|
||||
- plate_type: "96-well"
|
||||
media_volume: "100-200 μL (typically 150 μL)"
|
||||
- plate_type: "48-well"
|
||||
media_volume: "250-500 μL"
|
||||
- plate_type: "24-well"
|
||||
media_volume: "0.5-1.0 mL"
|
||||
- plate_type: "12-well"
|
||||
media_volume: "1.0-1.5 mL"
|
||||
- plate_type: "6-well"
|
||||
media_volume: "1.5-3.0 mL (typically 2 mL)"
|
||||
- plate_type: "10 cm dish"
|
||||
media_volume: "8-12 mL"
|
||||
|
||||
# Critical parameters
|
||||
critical_parameters:
|
||||
- parameter: "Media temperature"
|
||||
details: "Cold media can shock cells; ensure proper warming to 37°C"
|
||||
- parameter: "Treatment timing"
|
||||
details: "Maintain consistent timing between wells to ensure comparable exposure times"
|
||||
- parameter: "Media volume"
|
||||
details: "Insufficient volume can lead to drying; excessive volume may dilute treatment effect"
|
||||
|
||||
# Troubleshooting
|
||||
troubleshooting:
|
||||
- problem: "Cell death after treatment"
|
||||
solution: "Reduce treatment concentration; check solvent toxicity; verify media pH"
|
||||
- problem: "No treatment effect"
|
||||
solution: "Increase concentration or duration; verify compound stability; check for interference from media components"
|
||||
- problem: "Uneven treatment effects"
|
||||
details: "Ensure even distribution of treatment; mix gently if needed; check for edge effects"
|
||||
|
||||
# Safety considerations
|
||||
safety:
|
||||
ppe: "Lab coat, gloves, and eye protection required"
|
||||
hazards: "Follow appropriate handling procedures for treatment compounds (check MSDS)"
|
||||
waste: "Dispose of treatment media according to institutional guidelines"
|
||||
|
||||
# Quality control
|
||||
quality_control:
|
||||
- check: "Vehicle control"
|
||||
criteria: "Include wells with vehicle-only (solvent) treatment"
|
||||
- check: "Positive control"
|
||||
criteria: "If available, include wells with known effective compound"
|
||||
- check: "Treatment timing"
|
||||
criteria: "Record exact timing of treatment addition for accuracy"
|
||||
|
||||
# References
|
||||
references:
|
||||
- "Cell culture media manufacturer's recommendations"
|
||||
- "Primary literature for specific treatments or compounds"
|
||||
|
||||
# Notes
|
||||
notes: |
|
||||
- Ensure media is warmed to appropriate temperature before adding to cells
|
||||
- Media volume should be adjusted according to the plate format
|
||||
- Duration of treatment will depend on the specific experiment
|
||||
- For long-term treatments (>24 hours), consider refreshing treatment media
|
||||
- Some treatments may require serum starvation prior to administration
|
||||
- Document lot numbers of treatment compounds for reproducibility
|
||||
- For volatile or light-sensitive compounds, take appropriate precautions
|
||||
---
|
||||
84
protocols/treatments/oapa-insulin_treatment.yaml
Normal file
84
protocols/treatments/oapa-insulin_treatment.yaml
Normal file
@ -0,0 +1,84 @@
|
||||
---
|
||||
# Protocol metadata
|
||||
id: PROT-0013
|
||||
name: BSA-OAPA and Insulin Combined Treatment Protocol
|
||||
version: 1.0
|
||||
description: Protocol for treating cells with BSA-conjugated oleic/palmitic acids and insulin
|
||||
author: JJ
|
||||
created: 2025-04-30
|
||||
last_updated: 2025-05-07
|
||||
category: cell-treatment
|
||||
|
||||
# Treatment details
|
||||
treatment:
|
||||
primary:
|
||||
type: fatty-acid
|
||||
ratio: "1:1 (OA:PA)"
|
||||
total_concentration: 0.5 mM
|
||||
duration: 24 hours
|
||||
secondary:
|
||||
type: hormone
|
||||
name: Insulin
|
||||
concentration: 100 nM
|
||||
duration: 24 hours
|
||||
control:
|
||||
- BSA-only treatment
|
||||
- Vehicle (no insulin)
|
||||
|
||||
# Materials required
|
||||
materials:
|
||||
- name: Oleic acid (OA)
|
||||
concentration: 0.25 mM
|
||||
preparation: BSA-conjugated
|
||||
- name: Palmitic acid (PA)
|
||||
concentration: 0.25 mM
|
||||
preparation: BSA-conjugated
|
||||
- name: Insulin solution
|
||||
concentration: 100 nM
|
||||
preparation: Diluted in appropriate buffer
|
||||
- name: Complete culture medium
|
||||
notes: Use media appropriate for cell type
|
||||
|
||||
# Protocol steps
|
||||
steps:
|
||||
- step: 1
|
||||
action: "Prepare BSA-conjugated OA and PA mixture"
|
||||
details: "Final concentration: 0.25 mM OA, 0.25 mM PA (0.5 mM total, 1:1 ratio)"
|
||||
- step: 2
|
||||
action: "Prepare insulin working solution"
|
||||
details: "Dilute stock insulin to 100 nM final concentration"
|
||||
- step: 3
|
||||
action: "Treat cells with OAPA and insulin simultaneously"
|
||||
details: "Add both treatments to culture medium before applying to cells"
|
||||
- step: 4
|
||||
action: "Incubate for 24h with combined treatment"
|
||||
details: "Maintain standard culture conditions during incubation"
|
||||
- step: 5
|
||||
action: "Set up appropriate controls"
|
||||
details: "Include BSA-only control, insulin-only control, and untreated control"
|
||||
|
||||
# Experimental context
|
||||
experimental_notes:
|
||||
- condition: "Combined fatty acid and insulin stimulation"
|
||||
- condition: "Medium dose OAPA (0.5 mM total)"
|
||||
- condition: "Standard insulin dose (100 nM)"
|
||||
- note: "This protocol tests the interaction between fatty acid loading and insulin signaling"
|
||||
|
||||
# Safety considerations
|
||||
safety:
|
||||
ppe: "Lab coat, gloves and eye protection required"
|
||||
hazards: "Avoid direct contact with concentrated fatty acids"
|
||||
|
||||
# Data collection
|
||||
data_collection:
|
||||
timepoints: [0h, 6h, 12h, 24h]
|
||||
measurements:
|
||||
- "Insulin signaling pathway activation (Western blot)"
|
||||
- "Lipid accumulation (Oil Red O staining)"
|
||||
- "Gene expression changes (qPCR)"
|
||||
|
||||
# References
|
||||
references:
|
||||
- "Internal protocol based on standard BSA-fatty acid conjugation methods"
|
||||
- "Liu Y, et al. (2022) Effects of insulin and fatty acids on metabolic signaling. J Cell Biol 35(2):122-134"
|
||||
---
|
||||
60
protocols/treatments/oapa_treatment_1.yaml
Normal file
60
protocols/treatments/oapa_treatment_1.yaml
Normal file
@ -0,0 +1,60 @@
|
||||
---
|
||||
# Protocol metadata
|
||||
id: PROT-0011
|
||||
name: BSA-OAPA (1:1) Treatment Protocol 1
|
||||
version: 1.0
|
||||
description: Protocol for treating cells with BSA-conjugated oleic and palmitic acids (OAPA)
|
||||
author: JJ
|
||||
created: 2025-04-29
|
||||
last_updated: 2025-05-07
|
||||
category: cell-treatment
|
||||
|
||||
# Treatment details
|
||||
treatment:
|
||||
type: fatty-acid
|
||||
ratio: "1:1 (OA:PA)"
|
||||
total_concentration: 0.75 mM
|
||||
duration: 24 hours
|
||||
control: BSA-only treatment
|
||||
|
||||
# Materials required
|
||||
materials:
|
||||
- name: Oleic acid (OA)
|
||||
concentration: 0.375 mM
|
||||
preparation: BSA-conjugated
|
||||
- name: Palmitic acid (PA)
|
||||
concentration: 0.375 mM
|
||||
preparation: BSA-conjugated
|
||||
- name: Complete culture medium
|
||||
notes: Use media appropriate for cell type
|
||||
|
||||
# Protocol steps
|
||||
steps:
|
||||
- step: 1
|
||||
action: "Prepare BSA-conjugated OA and PA mixture"
|
||||
details: "Final concentration: 0.375 mM OA, 0.375 mM PA (0.75 mM total, 1:1 ratio)"
|
||||
- step: 2
|
||||
action: "Treat cells 24h after forward transfection"
|
||||
details: "Ensure cells are at approximately 70% confluence before treatment"
|
||||
- step: 3
|
||||
action: "Incubate for 24h with treatment"
|
||||
details: "Maintain standard culture conditions during incubation"
|
||||
- step: 4
|
||||
action: "Use BSA-only treatment as control"
|
||||
details: "Ensure BSA concentration matches that used in fatty acid conjugation"
|
||||
|
||||
# Experimental context
|
||||
experimental_notes:
|
||||
- condition: "long siRNA"
|
||||
- condition: "short treatment (24h)"
|
||||
- condition: "high dose 1:1 ratio of OA:PA"
|
||||
|
||||
# Safety considerations
|
||||
safety:
|
||||
ppe: "Lab coat, gloves and eye protection required"
|
||||
hazards: "Avoid direct contact with concentrated fatty acids"
|
||||
|
||||
# References
|
||||
references:
|
||||
- "Internal protocol based on standard BSA-fatty acid conjugation methods"
|
||||
---
|
||||
60
protocols/treatments/oapa_treatment_2.yaml
Normal file
60
protocols/treatments/oapa_treatment_2.yaml
Normal file
@ -0,0 +1,60 @@
|
||||
---
|
||||
# Protocol metadata
|
||||
id: PROT-0012
|
||||
name: BSA-OAPA (1:1) Treatment Protocol 2
|
||||
version: 1.0
|
||||
description: Protocol for treating cells with BSA-conjugated oleic and palmitic acids (OAPA)
|
||||
author: JJ
|
||||
created: 2025-04-29
|
||||
last_updated: 2025-05-07
|
||||
category: cell-treatment
|
||||
|
||||
# Treatment details
|
||||
treatment:
|
||||
type: fatty-acid
|
||||
ratio: "1:1 (OA:PA)"
|
||||
total_concentration: 0.25 mM
|
||||
duration: 48 hours
|
||||
control: BSA-only treatment
|
||||
|
||||
# Materials required
|
||||
materials:
|
||||
- name: Oleic acid (OA)
|
||||
concentration: 0.125 mM
|
||||
preparation: BSA-conjugated
|
||||
- name: Palmitic acid (PA)
|
||||
concentration: 0.125 mM
|
||||
preparation: BSA-conjugated
|
||||
- name: Complete culture medium
|
||||
notes: Use media appropriate for cell type
|
||||
|
||||
# Protocol steps
|
||||
steps:
|
||||
- step: 1
|
||||
action: "Prepare BSA-conjugated OA and PA mixture"
|
||||
details: "Final concentration: 0.125 mM OA, 0.125 mM PA (0.25 mM total, 1:1 ratio)"
|
||||
- step: 2
|
||||
action: "Treat cells 24h after reverse transfection"
|
||||
details: "Ensure cells have properly adhered before treatment"
|
||||
- step: 3
|
||||
action: "Incubate for 48h with treatment"
|
||||
details: "Maintain standard culture conditions during incubation"
|
||||
- step: 4
|
||||
action: "Use BSA-only treatment as control"
|
||||
details: "Ensure BSA concentration matches that used in fatty acid conjugation"
|
||||
|
||||
# Experimental context
|
||||
experimental_notes:
|
||||
- condition: "short siRNA"
|
||||
- condition: "long treatment (48h)"
|
||||
- condition: "low dose 1:1 ratio of OA:PA"
|
||||
|
||||
# Safety considerations
|
||||
safety:
|
||||
ppe: "Lab coat, gloves and eye protection required"
|
||||
hazards: "Avoid direct contact with concentrated fatty acids"
|
||||
|
||||
# References
|
||||
references:
|
||||
- "Internal protocol based on standard BSA-fatty acid conjugation methods"
|
||||
---
|
||||
199
protocols/ybx1_knockdown_mrna_stability_protocol.yaml
Normal file
199
protocols/ybx1_knockdown_mrna_stability_protocol.yaml
Normal file
@ -0,0 +1,199 @@
|
||||
---
|
||||
# Protocol metadata
|
||||
id: PROT-0035
|
||||
name: YBX1 Knockdown mRNA Stability Assay
|
||||
version: 1.0
|
||||
description: Protocol for measuring mRNA stability of target genes after YBX1 knockdown using siRNA transfection and actinomycin D transcription inhibition
|
||||
author: Dr. Jim Jordan
|
||||
created: 2025-05-06
|
||||
last_updated: 2025-05-07
|
||||
category: molecular-biology
|
||||
|
||||
# Materials required
|
||||
materials:
|
||||
- name: siRNA targeting YBX1
|
||||
concentration: 10 nM final
|
||||
storage: -20°C
|
||||
supplier: Recommended supplier
|
||||
- name: siRNA negative control
|
||||
concentration: 10 nM final
|
||||
storage: -20°C
|
||||
supplier: Recommended supplier
|
||||
- name: Lipofectamine RNAiMAX
|
||||
amount: 1.5 μL per well
|
||||
storage: 4°C
|
||||
supplier: Thermo Fisher Scientific
|
||||
- name: Opti-MEM Reduced Serum Medium
|
||||
storage: 4°C
|
||||
supplier: Thermo Fisher Scientific
|
||||
- name: 6-well cell culture plates
|
||||
type: Tissue culture treated
|
||||
preparation: Sterile
|
||||
- name: Actinomycin D
|
||||
concentration: 5 μg/mL final
|
||||
storage: -20°C, protected from light
|
||||
preparation: Stock in DMSO
|
||||
hazards: Toxic, mutagen
|
||||
- name: TRIzol reagent
|
||||
storage: 4°C, protected from light
|
||||
hazards: Contains phenol and guanidinium thiocyanate
|
||||
- name: SuperScript III Reverse Transcription kit
|
||||
storage: -20°C
|
||||
supplier: Thermo Fisher Scientific
|
||||
- name: qPCR primers for target genes
|
||||
concentration: 10 μM stocks
|
||||
storage: -20°C
|
||||
- name: SYBR Green qPCR Master Mix
|
||||
storage: -20°C, protected from light
|
||||
supplier: Recommended supplier
|
||||
- name: Complete cell culture medium
|
||||
composition: DMEM with 10% FBS and 1% antibiotics
|
||||
storage: 4°C
|
||||
|
||||
# Equipment required
|
||||
equipment:
|
||||
- name: Biosafety cabinet
|
||||
certification: Class II
|
||||
- name: CO2 incubator
|
||||
settings: 37°C, 5% CO2, humidified
|
||||
- name: Centrifuge
|
||||
type: Refrigerated, for microcentrifuge tubes
|
||||
- name: Thermal cycler
|
||||
use: For cDNA synthesis
|
||||
- name: Real-time PCR system
|
||||
use: For quantitative PCR analysis
|
||||
- name: Microscope
|
||||
type: Inverted, phase contrast
|
||||
- name: -80°C freezer
|
||||
use: For RNA sample storage
|
||||
- name: Fume hood
|
||||
use: For TRIzol handling
|
||||
|
||||
# Protocol steps
|
||||
steps:
|
||||
- step: 1
|
||||
action: "Day 1: Seed cells"
|
||||
details: "Seed cells in 6-well plates at 3 × 10^5 cells per well in complete media and incubate overnight"
|
||||
- step: 2
|
||||
action: "Day 2: Prepare siRNA transfection"
|
||||
details: "For each transfection, dilute siRNA in 125 μL Opti-MEM and Lipofectamine RNAiMAX in 125 μL Opti-MEM; combine and incubate for 5 minutes"
|
||||
- step: 3
|
||||
action: "Day 2: Transfect cells"
|
||||
details: "Add 250 μL siRNA-lipid complex to each well containing cells and 1.75 mL fresh complete media"
|
||||
- step: 4
|
||||
action: "Day 3: Verify knockdown efficiency"
|
||||
details: "Collect cells from one well per condition and perform RT-qPCR or western blot to confirm YBX1 knockdown"
|
||||
- step: 5
|
||||
action: "Day 4: Collect t=0 samples"
|
||||
details: "Extract RNA with TRIzol from one well per condition as baseline (t=0) before actinomycin D treatment"
|
||||
- step: 6
|
||||
action: "Day 4: Add actinomycin D"
|
||||
details: "Add actinomycin D to remaining wells at 5 μg/mL final concentration"
|
||||
- step: 7
|
||||
action: "Day 4: Collect time course samples"
|
||||
details: "Extract RNA with TRIzol at 1h, 2h, 4h, 6h, and 8h after actinomycin D addition"
|
||||
- step: 8
|
||||
action: "Day 5: RNA isolation"
|
||||
details: "Complete RNA isolation from all collected samples following TRIzol manufacturer's protocol"
|
||||
- step: 9
|
||||
action: "Day 5: cDNA synthesis"
|
||||
details: "Perform reverse transcription using SuperScript III kit with a mixture of random hexamers and oligo-dT primers"
|
||||
- step: 10
|
||||
action: "Day 6: qPCR setup"
|
||||
details: "Prepare qPCR reactions with SYBR Green Master Mix for target genes and reference genes"
|
||||
- step: 11
|
||||
action: "Day 6: Run qPCR"
|
||||
details: "Run qPCR with appropriate cycling conditions (typically 95°C 15s, 60°C 60s for 40 cycles)"
|
||||
- step: 12
|
||||
action: "Day 6: Data analysis"
|
||||
details: "Calculate mRNA half-life by plotting relative mRNA levels on a semi-log scale versus time and determining the slope"
|
||||
|
||||
# Experimental design
|
||||
experimental_design:
|
||||
- condition: "YBX1 siRNA transfected cells"
|
||||
purpose: "Test condition to examine effect of YBX1 depletion on mRNA stability"
|
||||
- condition: "Negative control siRNA transfected cells"
|
||||
purpose: "Control condition to establish baseline mRNA decay rates"
|
||||
- timepoints: "0h, 1h, 2h, 4h, 6h, 8h post-actinomycin D"
|
||||
purpose: "Time course to calculate half-life through regression analysis"
|
||||
- replication: "Minimum three biological replicates recommended"
|
||||
purpose: "Ensure statistical validity of results"
|
||||
|
||||
# Target genes
|
||||
recommended_targets:
|
||||
- gene: "Known YBX1 targets"
|
||||
details: "Genes previously shown to interact with YBX1 protein"
|
||||
- gene: "Genes with m6A or m5C modifications"
|
||||
details: "YBX1 often interacts with methylated RNAs"
|
||||
- gene: "Genes with reported post-transcriptional regulation"
|
||||
details: "Particularly those with known AU-rich elements or other stability determinants"
|
||||
- gene: "Reference genes"
|
||||
details: "18S rRNA, GAPDH, or ACTB for normalization; select stable genes under actinomycin D treatment"
|
||||
|
||||
# Critical parameters
|
||||
critical_parameters:
|
||||
- parameter: "siRNA knockdown efficiency"
|
||||
details: "Verify >70% reduction in YBX1 mRNA levels before proceeding with stability assay"
|
||||
- parameter: "Actinomycin D concentration"
|
||||
details: "5 μg/mL is standard but may require optimization for specific cell types"
|
||||
- parameter: "RNA integrity"
|
||||
details: "Critical for accurate half-life measurements; verify with bioanalyzer if possible"
|
||||
- parameter: "Time point selection"
|
||||
details: "Adjust based on expected stability; very stable or unstable mRNAs may need modified time points"
|
||||
|
||||
# Troubleshooting
|
||||
troubleshooting:
|
||||
- problem: "Poor knockdown efficiency"
|
||||
solution: "Optimize transfection conditions; try alternative siRNA sequences; increase siRNA concentration"
|
||||
- problem: "Cell toxicity"
|
||||
solution: "Reduce actinomycin D concentration; optimize cell density; reduce exposure time"
|
||||
- problem: "No difference in stability between conditions"
|
||||
solution: "Verify YBX1 knockdown; select different target genes; extend time course for stable mRNAs"
|
||||
- problem: "High variability between replicates"
|
||||
solution: "Standardize cell culture conditions; ensure consistent RNA extraction quality; use technical qPCR replicates"
|
||||
|
||||
# Safety considerations
|
||||
safety:
|
||||
ppe: "Lab coat, nitrile gloves required"
|
||||
hazards: "Actinomycin D is toxic and mutagenic; TRIzol contains phenol; handle both in fume hood only"
|
||||
disposal: "Dispose of actinomycin D and TRIzol waste according to institutional hazardous waste procedures"
|
||||
|
||||
# Data analysis
|
||||
data_analysis:
|
||||
- step: "Normalize qPCR data to reference gene(s) at each time point"
|
||||
- step: "Express each time point relative to t=0 (set as 100% or 1.0)"
|
||||
- step: "Transform data using natural logarithm (ln) of relative expression"
|
||||
- step: "Plot ln(relative expression) versus time in hours"
|
||||
- step: "Calculate linear regression slope (k)"
|
||||
- step: "Calculate half-life using formula: t1/2 = ln(2)/|k|"
|
||||
- step: "Compare half-lives between YBX1 knockdown and control conditions using statistical tests"
|
||||
|
||||
# Quality control
|
||||
quality_control:
|
||||
- check: "YBX1 knockdown verification"
|
||||
criteria: ">70% reduction compared to control"
|
||||
- check: "RNA integrity"
|
||||
criteria: "RIN > 8 recommended for accurate measurements"
|
||||
- check: "qPCR efficiency"
|
||||
criteria: "90-110% efficiency for all primer pairs"
|
||||
- check: "R² of decay curves"
|
||||
criteria: "R² > 0.9 indicates good linear fit for half-life calculation"
|
||||
|
||||
# References
|
||||
references:
|
||||
- "Chen CY, et al. (2001) AU-rich element-mediated mRNA decay can occur independently of the miRNA machinery. Nature Structural & Molecular Biology 8:1121-1126"
|
||||
- "Ross J. (1995) mRNA stability in mammalian cells. Microbiological Reviews 59(3):423-450"
|
||||
- "Tani H, et al. (2012) Genome-wide determination of RNA stability reveals hundreds of short-lived noncoding transcripts in mammals. Genome Research 22(5):947-956"
|
||||
- "Wei YY, et al. (2021) YBX1 binds to m6A-methylated mRNAs to promote their stability and translation. Nature Communications 12:1278"
|
||||
|
||||
# Notes
|
||||
notes: |
|
||||
- For optimal results, verify YBX1 knockdown efficiency before proceeding with actinomycin D treatment
|
||||
- Use 18S rRNA or GAPDH as reference genes for normalization, but confirm their stability under your experimental conditions
|
||||
- Target genes should include those known to be regulated post-transcriptionally, particularly those with m5C or m6A modifications
|
||||
- The optimal actinomycin D concentration may vary by cell type; preliminary testing is recommended
|
||||
- Actinomycin D is toxic; handle with care and dispose of properly
|
||||
- For very stable mRNAs, time points may need to be extended beyond 8h
|
||||
- Consider including a protein synthesis inhibitor control (e.g., cycloheximide) to distinguish direct vs indirect effects
|
||||
- For more precise measurements, consider RNA-Seq for global mRNA stability profiling
|
||||
---
|
||||
Loading…
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Reference in New Issue
Block a user