Plasmid/short hairpin RNA construction and virus infection

Lentivirus Packaging and Infection Protocol

Prepare plasmids: core plasmids, two packaging plasmids (pMD2.G and pSPAX2), and the plasmid concentration>100ng/uL can be used.

Prepare Reagents: opti-MEM, PEI transfection reagent, Polybrene.

Part I. Packaging

Day1:

Plate 293T cells in 6-well plates. Cells should be ~80% confluent next day.

Day2：

Transfection plasmid into 293T cells, prepare the following mastermix:

100ul opti-MEM;

0.6ug pMD2.G;

1.2ug pSPAX2;

**2ug specific lentivector;

Incubate each individual mix for 5 min. at room temperature.

Then 8ul PEI is added, mixed gently and incubate at room temperature for 30min.

After incubation, add mixture on the SIDE of one well. Place plate in incubator for 36-48h.

Part II: Infection

Day3:

**Day before infection: Plate cells targeted for infection in 6-well plate. Cells should be ~30% confluent next day.

Day4：

Harvest virus particles by collecting medium, centrifuge at 1500 rpm for 5 min, and filter with 0.22um filter membrane.

Add 1ml media, then 0.5 ml virus, to 6-well plate.

Add 1.5 ul of 8 mg/ml polybrene  (want final concentration of 8 ug/ml) to each well.

**Virus can now be stored at 4°C for a few weeks. For long-term storage, aliquot and store in -80°C.

Day5：

Change media. 

Day6:

Add selection marker/puromycin (2 ug/ml) to each well containing virus, as well as to one of the control wells. **Cells in the control wells treated with antibiotics should display 100% death.

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