Lipofection of 293GPG cells: Difference between revisions
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=='''Lipofection'''== | |||
1. Plate cells onto 10 -cm dishes and grow to near- confluency in “293 GPG Medium.” | 1. Plate cells onto 10 -cm dishes and grow to near- confluency in “293 GPG Medium.” | ||
Revision as of 13:17, 4 August 2006
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.
