Plate Lenti-X cells 2.5 x 10^6/10 cm 24h 0m 0s before transfection.

Warm OptiMEM, X-tremeGENE HP DNA Transfection Reagent, and DNA to warm to Room temperature.

Add 1mL OptiMEM to a tube.

Add plasmid DNA to the OptiMEM, gently pipetting to mix.

A	B	C
psPAX2	0.65 pmol	4600 ng
pMD2.G	0.36 pmol	1400 ng
Transfer vector	0.82 pmol

Add X-tremeGENE HP DNA Transfection Reagent to OptiMEM/DNA (4µL per 1µg of DNA).

Incubate for 0h 15m 0s at Room temperature.

Add OptiMEM/DNA/X-tremeGENE to the plate of cells dropwise, then gently swirl the plate.

The day after transfection, replace the medium with

72h 0m 0s after transfection, collect the medium in a 15 ml conical.

Centrifuge 500x g,0h 0m 0s x 0h 5m 0s, then pass the supernatant through a .22 µm filter.

Add 3mL of LentiX concentrator to the supernatant, invert 6 times to mix, then incubate 0h 45m 0s at 4°C.

Centrifuge at 1500x g,0h 0m 0s for 0h 45m 0s at 4°C.

Remove the supernatant and resuspend the pellet in 120µL ice cold PBS.

Make aliquots in multiples of 14µL and freeze at -70°C.