Twice purified by CsCl. MSCV backbone, encoding oncogene and marker of choice (Neo, GFP, etc.)
EcoPak
also called pMCV-Ecopac: ecotropic packaging plasmid, encoding gag-pol-env
2x HBS
For 500 ml: 8.0 g NaCl + 0.37 g KCl + 106.5 mg Na2HPO4 (anhydrous; 201.1 mg if 7xH2O) + 1.0 g dextrose (D-glucose) + 5.0 g HEPES powder. Dissolve in 450 ml dH2O (milli-Q), adjust pH to 7.05 with NaOH, then complete to 500 ml with dH2O. Sterile filter thru 0.45 µ filter. Store at room temperature, with the cap on tight.
2M CaCl2
Sigma
miliQ H2O
sterile-filtered
Chloroquine
1000x stock is 25 mM in PBS- (w/o Ca2+/Mg2+), stored at -20C. Add fresh to the medium when needed.
6 cm plates
for tissue culture
Incubator
for tissue culture. Set at 37C, 10% CO2.
10 cc sterile syringes
Sterile. One per virus type.
18G needles
single use, one per virus type.
45 um syringe filters
5 ml plastic tubes
Sterile, to mix transfection solution, for up to 2 plates at a time (2 ml). We use Falcon tubes.
50 ml conical tubes
cryovials
2 and 4 ml, for virus aliquots.
All reagents used in the transfection must be sterile-filtered (CaCl2, 2x HBS, miliQ H2O) and kept sterile.
Gavrilescu, L. C., Van Etten, R. A. Production of Replication-Defective Retrovirus by Transient Transfection of 293T cells. J. Vis. Exp. (10), e550, doi:10.3791/550 (2007).