User:Karmella Haynes/Notebook/PcTF Genomics/2014/09/23

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09/23/14

  • Transductions: U2OS, SK-N-SH, K562

Transductions
Note: cells are in standard complete medium

  1. Plate 1: U2OS
  2. Plate 2: SK-N-SH
  3. Plate 4: K562 b (1 mL) - no one was around to consult on whether I should pellet the 2mL and replate

Wells (for all plates) - added 400 μL virus

  1. well 1 - GFP
  2. well 2 - KAH160/015 (2010)
  3. well 3 - KAH160/015 (2014)
  4. well 4 - KAH165/015 (2010)
  5. well 6 - KAH165/015 (2014)
  6. well 7 - KAH170/015 (2014)


Day 1: Abbreviated protocol (from Laura Gonzalez)

  • Retrieve frozen aliquots of harvested virus from -80C freezer (just outside TC room)
  • Thaw and quick-spin to get virus off of the inside of lid
  • Biohazard set-up: 10 mL Wescodyne waste conical (50 mL), 10 mL Wescodyne rinse
  • Remove 1mL of media from each well (except for K562)
  • Add 5ul of 2.2mg/ml Polybrene (thawed) to each well. Note: final concentration of Polybrene in well will be 8μg/ml after 400 μL viral aliquot is added
  • Spin at 2250 rpm for 25 minutes
  • Place the plates in the incubator (37°C) overnight.



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