User:Karmella Haynes/Notebook/PcTF Genomics/2015/06/27

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06/27/15

  • Cas-tone - cell transfections (Gal4EED/luc)



Cas-tone - transfections

  • Final H3#_MV11 builds verified on 6/24/15
  • Co-transfect H3#_MV11 with pSP-guide RNA plasmid (built by Ben Nyer)
  • Use HEK293 Gal4EED/luc cells, no dox (from Rene)
  1. H340_MV11 + g034_pSPgRNA
  2. H340_MV11 (no gRNA)
  3. H360_MV11 + g034_pSPgRNA
  4. H360_MV11 (no gRNA)
  5. H380_MV11 + g034_pSPgRNA
  6. H380_MV11 (no gRNA)
Well H3#_MV11 (1.0 μg) g034_pSPgRNA (1.0 μg) dH2O OptiMEM PLUS rgt. Lipo LTX
1 5.0 1.6 13.4 570.0 2.5 7.5
2 5.0 --- 15.0 570.0 2.5 7.5
3 5.0 1.6 13.4 570.0 2.5 7.5
4 5.0 --- 15.0 570.0 2.5 7.5
5 5.6 1.6 12.8 570.0 2.5 7.5
6 5.6 --- 14.4 570.0 2.5 7.5
  • Pre-warm ps-free DMEM
  • Make 20 μL plasmid/dH2O mixes in sterile 1.5 ml tubes
  • Add OptiMEM
  • Add PLUS --> 5 min./ RT
  • Add Lipo LTX --> 30 min./ RT
  • Cells - refresh growth medium w/ ps-free DMEM
  • Add 600 μL complexes, drop-wise
  • Incubate overnight



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