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130 lines
4.6 KiB
YAML
130 lines
4.6 KiB
YAML
---
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# Protocol metadata
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id: PROT-0030
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name: Preparation of Complete DME/F-12 Protocol
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version: 1.0
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description: Protocol for preparing complete DME/F-12 culture medium with FBS and antibiotics
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author: Lab Staff
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created: 2025-05-07
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last_updated: 2025-05-07
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category: reagent-preparation
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# Materials required
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materials:
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- name: DME/F-12 medium
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amount: 500 mL bottle
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storage: 4°C
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supplier: Recommended supplier
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- name: Fetal Bovine Serum (FBS)
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amount: 50 mL (10% final)
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storage: -20°C (stock), thawed before use
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supplier: Recommended supplier, heat-inactivated
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- name: Penicillin-Streptomycin (PS)
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amount: 5 mL (1% final)
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storage: -20°C
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supplier: Recommended supplier, 100X concentration
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- name: Sterile 50-mL tube
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type: Conical, polypropylene
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preparation: Sterile
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# Equipment required
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equipment:
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- name: Biosafety cabinet
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certification: Class II
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- name: Water bath or incubator
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temperature: 37°C
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use: For thawing FBS
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- name: Pipette aid
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range: For 5-50 mL volumes
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- name: Pipettes
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range: Serological, various sizes
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- name: Refrigerator
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temperature: 4°C
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use: For media storage
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# Protocol steps
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steps:
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- step: 1
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action: "Prepare materials"
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details: "Allow media to warm to room temperature; thaw FBS and PS at 37°C"
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- step: 2
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action: "Set up biosafety cabinet"
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details: "Turn on biosafety cabinet 15 minutes before use; clean with 70% ethanol"
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- step: 3
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action: "Aliquot plain media"
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details: "Transfer ~51 mL of DME/F-12 media into a sterile 50-mL tube and label as 'PLAIN DME/F-12'"
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- step: 4
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action: "Add FBS"
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details: "To the remaining 449 mL of media in the original bottle, add 50 mL Fetal Bovine Serum (FBS)"
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- step: 5
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action: "Add antibiotics"
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details: "Add 5 mL Penicillin-Streptomycin (PS) solution to the main bottle"
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- step: 6
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action: "Mix gently"
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details: "Invert bottle several times to mix thoroughly, avoiding bubble formation"
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- step: 7
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action: "Label media"
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details: "Label bottle with 'Complete DME/F-12', date of preparation, and expiration date (1 month from preparation)"
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- step: 8
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action: "Store media"
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details: "Store both complete and plain media at 4°C"
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# Final composition
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final_composition:
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- component: "DME/F-12 basal medium"
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percentage: "89%"
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function: "Provides essential nutrients and buffering"
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- component: "Fetal Bovine Serum"
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percentage: "10%"
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function: "Provides growth factors and proteins for cell growth"
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- component: "Penicillin-Streptomycin"
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percentage: "1%"
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function: "Prevents bacterial contamination"
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# Critical parameters
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critical_parameters:
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- parameter: "Sterility"
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details: "All components must be handled aseptically in a biosafety cabinet"
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- parameter: "FBS quality"
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details: "FBS should be heat-inactivated and from a validated lot known to support growth of target cells"
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- parameter: "Expiration"
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details: "Complete medium should be used within 1 month of preparation"
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# Troubleshooting
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troubleshooting:
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- problem: "Media color change"
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solution: "If phenol red indicator turns yellow, pH is too acidic; discard media"
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- problem: "Precipitation in media"
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solution: "Check for contamination; sometimes precipitates from FBS can be removed by filtration"
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- problem: "Cell growth issues"
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solution: "Test alternative FBS lots; verify proper storage conditions"
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# Safety considerations
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safety:
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ppe: "Lab coat, gloves required"
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hazards: "Minimal hazards; follow standard laboratory safety practices"
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# Quality control
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quality_control:
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- check: "pH indicator"
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criteria: "Phenol red should appear pink-red (not yellow or purple)"
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- check: "Sterility test"
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criteria: "Incubate a sample at 37°C for 72 hours to verify no contamination"
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- check: "Cell growth test"
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criteria: "Test with standard cell line to verify growth support"
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# References
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references:
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- "ATCC. Guidelines for Media Preparation and Methods. www.atcc.org"
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- "Freshney, R.I. Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, 7th Edition"
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# Notes
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notes: |
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- This protocol creates both complete DME/F-12 (with FBS and antibiotics) and plain DME/F-12
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- Complete medium contains 10% FBS and 1% Penicillin-Streptomycin
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- For specialty applications, additional supplements may be required
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- Some cell lines may require different FBS concentrations (5-20%)
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- If preparing larger or smaller volumes, adjust component volumes proportionally
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- For sensitive cell types, consider using reduced serum or serum-free formulations
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- For long-term storage, complete media can be aliquoted into working volumes to reduce freeze-thaw cycles
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--- |