User:Karmella Haynes/Notebook/PcTF Genomics/2015/10/20

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10/20/15

  • Luc/CFP swap - Colony growth

Luc/CFP swap - Colony growth

  • 10/19/15 - Ben Nyer - Gal4EED/luc HEK293 cells were co-transfected with lipofectamine LTX and (1) HPK-CFP gene flanked with homology arms that match luciferase site DBN007_pSB1A3, (2) Cas9/gRNA34 plasmid, and (3) Cas9/gRNA44 plasmid.
  • Today: plate cells at low concentration to try to get CFP-positive colonies


Images of transfected cells in 6-well dish:


Expansion procedure (to maintain back-up stocks):

  1. Wash with PBS and trypsinize in 0.5 mL trypsin medium
  2. Resuspend with 3.5 mL DMEM complete (+0.5 μg/mL puro); add to 6 mL DMEM complete (+0.5 μg/mL puro) in 10 cm dish


Dilution procedure:

  1. Wash with PBS and trypsinize in 0.5 mL trypsin medium
  2. Assume 1 million cells in 1 well...there are probably fewer) ≈ 1,000,000 cells
  3. Resuspend by adding 4.5 mL DMEM complete (+0.5 μg/mL puro) ≈ 200,000 cells/mL
  4. Add 0.5 mL resuspended cells to 10 mL medium in 10 cm plate ≈ 10,000 cells/mL
  5. Add 50 μL resuspended cells to 10 mL medium in 10 cm plate ≈ 1,000 cells/mL


Plates set up today:

  1. Luc/CFP Well 1 (expansion)
  2. Luc/CFP Well 2 1:100 (10,000 cells/mL)
  3. Luc/CFP Well 2 1:1000 (1,000 cells/mL)
  4. Luc/CFP Well 3 1:100 (10,000 cells/mL)
  5. Luc/CFP Well 3 1:1000 (1,000 cells/mL)
  6. Luc/CFP Well 4 (expansion)
  7. Luc/CFP Well 5 (expansion)




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