VIRUSES PACKING AND CELL INFECTION
Reagents
OPTIMEM.
HEK 293 T cells.
Polybrene (8mg/ml) 1000X
Syringes.
0,45um filtres.
Plasmid DNA: ENV Plasmid(VSV-G), pMDLg/pRRE plasmid, REV plasmid and
transfer vector plasmid.
VIRUSES PACKING
Initially we packed virus with transfer vector plasmid using a calcium phosphate
protocol but lipofectamine 2000 transfection protocol works better.
All the volumen and quantities are for a p100 plate.
Plate 4x106cells 24h before transfection.
One hour before transfection remove medium and add 10ml of fresh medium.
Eppendorf A: 360ul OPTIMEM + 14ul lipofectamine 2000.
Eppendorf B: 360ul OPTIMEM, 3ug ENV plasmid, 5ug pMDL plasmid, 2,5ug REV
plasmid and 10-15ug transfer vector plasmid(never more than 40ug).
Incubate 5 minutes RT.
Mix eppendorf A and eppendorf b and incubate 20minutes.
Remove medium from plate and add 2,5ml of OPTIMEM.
After 20 minutes add transfection mixture drop-wise.
After 4-5hours remove medium and add 8ml of fresh medium.
After 48hours harvest medium and centrifuge 2000g 2-3minutes.
Filter supernatant using a 0,45um filter.
Make aliquots in 1.5ml eppendorf(0,5ml each) and store at -80ºC.
LENTIVIRUS INFECTION
Plate 50000-100000 cells/well in a 6-multiwell a day before transduction.
Aspirate the media from the cells and add 0,5ml fresh medium-polybrene(2X) to the
cells.
Add 0,5ml of the virus supernatant to the cells.
After 16h add 1ml or fresh medium to the cells.
After 48-72h cells can be trypsinized, passaged like regular cells and selected with
puromycin or another antibiotic.