Difference between revisions of "Transfecting Cells"

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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