Lentivirus Packaging Mix QC

1. After MaxiPrep, measure OD260 and OD260/280 ratio.

2. Run agarose gel to check RNA contaimination. If you see a big RNA band, add 1 µl RNase and digest at 37 oC for 1 to 3 hours, purify DNA by phenol-chloroform extraction and then precipitate DNA using 3 M NaAc (or KAc) + 0.6 vol isopropanol. Wash DNA with 75% ethanol, dry under biosafety hood. Dissolve DNA in TE, measure DNA again.

3. Mix VSVG and Gag-Pol at 1:3 or 1:1 ratio, at 1 µg/µl concentration.

4. Transfect 293 cells with  1 µg of GFP or RFP lentivirus plasmid plus 1 µg V-G mix, use 6 µl transfection reagent for 12 well plate.

5. Collect 1 ml supernatant 2 days after transfection. Spin to remove cell debris.

6. Seed 293 cells to 12 well plate (50% confluence), add 2 µl 1000 X polybrene, add 1 ml supernatant containing lentivirus to one well.

7. Check cells everyday to see if there is any fluorescence. If cells become green or red, the VSVG/Gag-Pol  can be used to make lentivirus.

8. Check if cells have bacteria contamination. If you see bacteria, the V/G mix may be contaminated, and it should be QC again.

9. If you suspect V/G mix has been contaminated, use phenol-chloroform extraction method to kill bacteria.