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May 31, 1990
Rosalie Veile
Purpose:
Time required:
Procedure:
Thawing cells for transformation:
Solutions:
To 500 ml of sterile RPMI 1640 with 2 mM L-glutamine, add: 90.0 ml FBS 6.0 ml 200 mM (100X) L-glutamine 0.6 ml 50 mg/ml gentamicin reagent Note: Filter sterilize through a 0.22 µm filter and store up to 2 weeks at 4 degrees C.
To 1 liter of sterile RPMI 1640 with 2 mM L-glutamine, add: 10.0 ml 2.5 M (100X) hepes buffer 1.2 ml 50 mg/ml gentamicin reagent Note: Filter sterilize through a 0.22 µm filter and store up to 2 weeks at 4 degrees C.
To 100 ml of growth media add 2 ml of 100X Cyclosporin A.
Dissolve 1 mg CSA in 0.1 ml ethanol, add 0.02 ml Tween 80 and mix well. While continually stirring, add 1 ml RPMI, drop by drop. Quanitate to a final volume of 100 ml with RPMI. Filter sterilize and store at 4 degrees C for up to 4 months.
References:
Chenevix-Trench, G., et al, Am. J. Hum. Genet. 46:635-636, 1990.