Basic LentiVirus Prep

Please note that once cells are transfected they MUST be grown in the virus room and all manipulations must be done in the virus hood. Anything that touches the media should be considered infectious, so all pipets, filters, syringes tubes etc. should be bleached for a minimum of 20 minutes before disposal (preferably in the red bin for extra safety). Use double gloves, wear a lab coat and use plastic aspirating pipets instead of glass for safety.

~1 week before transfection

Thaw 293T cells and grow them in DMEM + 10% FBS or CS, 1% PSF (293C)

Split them 1-2x before use for virus prep so they are growing in log phase

note: do not let them overgrow and form tight clusters of cells, this should be at or less than 10 x106 per 10 cm dish, if overgrown, split 1-2 more times at lower density so the cells are not clumped.

Split and plate cells so that there will be enough cells to plate 5-6 x 106 cells per 10 cm dish to be transfected on the day of transfection

Coat 10 cm plates with poly-lysine (dilute stock 1:10 in PBS and incubate 10 minutes on plate, wash with PBS). This helps prevent cell loss during virus production

Trypsinize and count 293T cells

Remove enough cells for 5-6 x 106 cells per 10 cm dish for the required number of plates.  Spin down and resuspend in DMEM + 10% FBS without antibiotics. Plate 10 ml of this mixture per plate prior to setting up transfection mix.

Transfection mix:

For each 10 cm dish-

Mix 600 ml serum-free antibiotic-free DMEM + 20 ml FuGENE6  (Can do 600 – volume of fugene + DNA)

Incubate RT 5’

Add DNAs:

3 mg of viral vector

2 mg of pCMV DR 8.2 Dvpr

1 mg of pCMV-VSVG

Incubate 20-30’ RT

Add mix dropwise to plate, incubate overnight (transfect in the late afternoon/evening)