22 Expression in Eukaryotic Cells: Gene Transfer Using Retroviral Vectors
345
Materials
• \lf2 cells
• cDMEM
• Calf serum
• PBS
• 75 cm 2 tissue culture flasks
• Penicillin/Streptomycin
• Trypsin/EDT A
• LB medium
• Ampicillin
• Geneticin (G418)
• Polybrene (Abbott Labs., Sigma))
• DMSO
• Sterile 0.2 and 0.45 Jlm filters
• Ultrapure water
Procedure
Transfeetion
Pick a positive clone which has been analyzed for the presence of the insert
from the master plate (see above). Grow the bacteria in 5 ml LB media supplemented with 20 J.lg/ml ampicillin overnight at 37°C in an orbitalshakerat
250 rpm.
1. Prepare the plasmid DNA using the "miniprep" protocol (see Subprotocol 4). Phenol/chloroform extract the DNA at least four times and
ethanol precipitate the DNA. Wash the DNA overnight with 70% ethanol.
2. Grow 'lf2 cells in 75 cm 2 tissue culture flasks in cDMEM (complete Dulbecco's modified Eagle's medium) supplemented with penicillin, Streptomycin and 10% bovine calf serum (available from Gibco or Sigma). Be
345
Materials
• \lf2 cells
• cDMEM
• Calf serum
• PBS
• 75 cm 2 tissue culture flasks
• Penicillin/Streptomycin
• Trypsin/EDT A
• LB medium
• Ampicillin
• Geneticin (G418)
• Polybrene (Abbott Labs., Sigma))
• DMSO
• Sterile 0.2 and 0.45 Jlm filters
• Ultrapure water
Procedure
Transfeetion
Pick a positive clone which has been analyzed for the presence of the insert
from the master plate (see above). Grow the bacteria in 5 ml LB media supplemented with 20 J.lg/ml ampicillin overnight at 37°C in an orbitalshakerat
250 rpm.
1. Prepare the plasmid DNA using the "miniprep" protocol (see Subprotocol 4). Phenol/chloroform extract the DNA at least four times and
ethanol precipitate the DNA. Wash the DNA overnight with 70% ethanol.
2. Grow 'lf2 cells in 75 cm 2 tissue culture flasks in cDMEM (complete Dulbecco's modified Eagle's medium) supplemented with penicillin, Streptomycin and 10% bovine calf serum (available from Gibco or Sigma). Be