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Lipofectamine 2000 method (Invitrogen) (for 1 well in 6-well cluster 10cm2, small dish of 35 mm).
- The day before transfection, pass confluent cell 1:3 in 10%FBS without Penicillin/Straptomyosin.
- Check the cell condition, if it is in 60-80% confluency, proceed, otherwise, start a new experiment.
- 1 ug DNA gently mixed into 100 ul Optimum only, gently tap the tip of vial to mix.
- 2 ul lipofectamine mixed into 100 ul Optimum only, gently tap the tip of vial to mix.
- Wait for 5 min, gently apply lipofectamine-Optimum to DNA-Optimum. Tilt and rotate the DNA tube, while adding lipo-optimum drop by drop on its wall.
- Tap or reverse the tube gently to mix.
- Incubate in RT for 20 min for the complex formation between DNA and lipofectamine
- Apply the DNA-Lipofectamine complex Optimum to cells. Gently swirl the dish to mix.
- Incubate 5.5 hr in incubator. check cell toxicity, if severe, stop the incubation and apply fresh 0.5% FBS-DMEM, if not, continue incubation until next day and change to 0.5%FBS DMEM.
- The optimal time interval between passing cells and transfection is 15-18 hours; and that between transfection and imaging is 36-54 hours.