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126 lines
4.7 KiB
YAML
126 lines
4.7 KiB
YAML
---
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# Protocol metadata
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id: PROT-0020
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name: 24-well Plate siRNA Transfection (RNAiMAX)
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version: 1.0
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description: Protocol for transfecting 4 siRNAs (including siNC) into cells in a 24-well plate using Lipofectamine RNAiMAX
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author: Lab Agent
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created: 2024-06-08
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last_updated: 2025-05-07
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category: cell-culture
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# Materials required
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materials:
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- name: siRNA#1
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concentration: 10 nM final
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storage: -20°C or -80°C
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- name: siRNA#2
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concentration: 10 nM final
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storage: -20°C or -80°C
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- name: siRNA#3
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concentration: 10 nM final
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storage: -20°C or -80°C
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- name: siNC (negative control)
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concentration: 10 nM final
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storage: -20°C or -80°C
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- name: Lipofectamine RNAiMAX
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amount: 1.5 µL per well
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storage: 4°C
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- name: Opti-MEM Reduced Serum Medium
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storage: 4°C
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- name: Complete growth medium
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notes: Appropriate for your cell line, without antibiotics during transfection
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# Equipment required
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equipment:
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- name: 24-well tissue culture plate
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type: Cell culture grade
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- name: Pipettes and sterile tips
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range: 0.5-1000 µL
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- name: CO2 incubator
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settings: 37°C, 5% CO2, humidified
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- name: Biosafety cabinet
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certification: Class II
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- name: Centrifuge
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type: Benchtop (if needed for cell preparation)
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# Protocol steps
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steps:
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- step: 1
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action: "Seed cells in 24-well plate"
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details: "Seed 5x10^4 cells/well 24 hours prior to transfection to achieve 60-80% confluence at time of transfection"
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- step: 2
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action: "Prepare siRNA dilutions"
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details: "For each siRNA, dilute 10 pmol siRNA in 50 µL Opti-MEM per well"
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- step: 3
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action: "Prepare RNAiMAX dilution"
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details: "In a separate tube, dilute 1.5 µL RNAiMAX in 50 µL Opti-MEM per well"
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- step: 4
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action: "Create transfection complexes"
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details: "Combine diluted siRNA and RNAiMAX solutions, mix gently, and incubate for 10-20 min at room temperature"
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- step: 5
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action: "Add complexes to cells"
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details: "Add 100 µL siRNA-RNAiMAX complex dropwise to each well containing cells in 400 µL growth medium"
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- step: 6
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action: "Incubate cells"
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details: "Incubate cells at 37°C, 5% CO2 for 24-72 hours before assessing knockdown efficiency"
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- step: 7
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action: "Change medium (optional)"
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details: "After 4-6 hours, replace transfection medium with fresh complete medium if needed for sensitive cells"
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# Experimental design
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experimental_design:
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- layout: |
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Typical 24-well plate layout:
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| 1-siNC | 2-siNC | 3-siNC | 4-empty |
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| 1-siRNA1| 2-siRNA1| 3-siRNA1| 4-empty |
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| 1-siRNA2| 2-siRNA2| 3-siRNA2| 4-empty |
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| 1-siRNA3| 2-siRNA3| 3-siRNA3| 4-empty |
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| 5-empty | 6-empty |etc.
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# Critical parameters
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critical_parameters:
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- parameter: "Cell density"
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details: "60-80% confluence at time of transfection is optimal"
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- parameter: "siRNA concentration"
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details: "10 nM is standard but may need optimization (5-50 nM range)"
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- parameter: "RNAiMAX:siRNA ratio"
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details: "May need adjustment for different cell lines"
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- parameter: "Incubation time"
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details: "10-20 minutes for complex formation; overly long incubation may reduce efficiency"
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# Troubleshooting
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troubleshooting:
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- problem: "Low transfection efficiency"
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solution: "Optimize cell density, increase RNAiMAX amount, extend transfection time"
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- problem: "High cytotoxicity"
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solution: "Reduce siRNA/RNAiMAX concentrations, change medium 4-6h post-transfection"
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- problem: "Poor knockdown"
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solution: "Verify siRNA design, extend incubation time post-transfection, consider alternative delivery method"
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# Safety considerations
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safety:
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ppe: "Lab coat, gloves, and eye protection required"
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hazards: "RNAiMAX may cause irritation; handle with care"
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# Quality control
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quality_control:
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- check: "Include siNC (non-targeting control) in every experiment"
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criteria: "Should show no target gene knockdown"
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- check: "Verify knockdown efficiency"
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criteria: "Typically by qPCR or Western blot 24-72h post-transfection"
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# References
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references:
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- "Lipofectamine RNAiMAX manufacturer's protocol"
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- "Dorsett Y, Tuschl T. (2004) siRNAs: applications in functional genomics and potential as therapeutics. Nat Rev Drug Discov. 3(4):318-329"
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# Notes
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notes: |
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- siNC is a non-targeting negative control siRNA
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- Adjust cell number and reagent volumes as needed for specific cell lines
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- For sensitive cells, transfect in antibiotic-free medium
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- Assay for knockdown 24-72 hours post-transfection depending on target protein half-life
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- Consider optimizing siRNA concentration (5-50 nM range) for your specific cell line
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- Perform reverse transfection as an alternative by adding complexes to wells before adding cells
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--- |