Arman Sidhu (8/13/2012)
mod Jon Sack (8/14/2012)
- Prepare cells
- Plate cells in a 35mm dish at least a day before transfection
- Check and make sure to cells are 80% confluent
- ensure cells are in1 ml Ham’s F12 + 10%FBS solution with NO antibiotics and NO selection agents. NOTE it is OK to plate cells in 1 ml of this media in step 1a
- If there are antibiotics or selections media, rinse cells at least once with 2ml PBS and change media at least an hour before transfection
- Prepare a diluted solution of Lipofectamine
- In 1.5 ml tube, add 2ul of Lipofectamine 2000 and then add 100ul of Optimem
- Triturate mixture
- Prepare DNA and GFP solution
- In another 1.5 ml tube, add 0.5ug of ion channel DNA, add 0.5 ug of EGFP DNA, and then add 100ul of Optimem
- Triturate mixture
- 5 min after diluting Lipofectamine, mix the diluted Lipofectamine solution with the DNA mixture
- 20 minutes after mixing the Lipofectamine solution with the plasmid DNA solution, move cells to hood, apply the mixture to the cells, replace in incubator
- After 4-6 hours, replace media with 2 ml Ham’s F12 + 10% FBS + 1% pen/strep
- On the following day, record percent efficiency of transfection
- Patch cells in 2-3 days
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