Sack: Cell Transfection (Lipofectamine)

Sack and Yarov-Yarovoy Labs

Department of Physiology and Membrane Biology
University of California, Davis

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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