Lipofection of 293GPG cells



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Lipofection
1. Plate cells onto 10 -cm dishes and grow to near- confluency in “293 GPG Medium.”

2. Mix in eppendorf tube:


 * 750 ul OPTIMEM
 * 5 ug DNA

3. Allow to sit 15 minutes, room temp.

4. Mix in second eppendorf tube:


 * 750 ul OPTIMEM
 * 10 ul lipofectamine

5. Add contents of 2nd tube to 1st, mix, and allow to sit 15 minutes at room temp.

6. While step 5 is going on,  wash plate(s) in PBS 1 X.

7. Add 1.5 ml OPTIMEM to each plate.

8. Add DNA mix to plate(s). (Total volume ~3 ml)

9. Incubate 4 to 6 hours at 37 degrees, 5% CO2.

10. Aspirate transfection mix.

11. Add 5 ml complete media to each plate.

12. Change media daily.

13. Harvest media after 72 h, 96 h, 120 h, 144 h.

14. Store harvested medai at -80 degrees.

15. Frozen supernatants can be used for later infections.

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