Difference between revisions of "Transfecting Cells"
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* Work deep inside the hood | * Work deep inside the hood | ||
| + | ===Steps=== | ||
Transfection mix (for this example) | Transfection mix (for this example) | ||
Take sterile tubes for as many transfections you have, label | Take sterile tubes for as many transfections you have, label | ||
Revision as of 21:27, 20 July 2018
Summary
Cos 1 (African green monkey, from kidney cells). Cells are heterozygous, they all look different. We are transfecting Cos 1, 6 cm plates
- Make sure everything is clean and sterile
Materials
- DNA, transfection lipofectamine 2000
- Antibiotic free DMEM (no serum), kept in the fridge
- Sterile tips, tubes, pipettes (sprayed)
- Make sure cells are 50-70% confluent
- Work deep inside the hood
Steps
Transfection mix (for this example) Take sterile tubes for as many transfections you have, label In this example we needed six tubes Put 150 ul of media into tubes You can reuse the tip if you don’t touch anything Make sure you don’t go over any open tubes or bottles, close tubes and bottles right away Into the 150 ul of media add about 1-2 ug of DNA (optimize DNA concentration first) Tap gently to mix Add 6.5 ul of lipofectamine (transfection media) Pipette directly in the tube, flush, and tap re-cap / close Let it sit for 5-10 minutes (helps enhance transfection efficiency) After optional 5-10 minutes, grab cell plates from incubator (careful not to open lid yet) Make sure tubes and plates are in order (1-6), set pipette to 170 (ish) ul, take up media from tube and add to plate (pipette at an angle so nothing falls in) spread media in drops in a circle Give the dishes a gentle swirl, and place in incubator for a minimum of 24 hours and a maximum of three days. After 24 hours the DNa will be taken up, but after three days they will over grow and die
