Difference between revisions of "Transfecting Cells"

Revision as of 21:23, 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

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