User:Karmella Haynes/Notebook/PcTF Genomics/2013/07/05

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07/05/13

  • HEK293 Gal4-EED transfection - for PcTF expression and flow cytometry



Lipo transfection
> Samples are for flow cytometry > Seed new 6-well plates w/ ~5x105 cells/well in 3.4 mL plain medium (don't have any p.s.-free)
> Transfect human PcTF under control of constitutive CMV-TetO promoter (KAH160/MV1); 347 ng/μL; Note: this cell line has TetR, so constitutive CMV is needed.


Wells Plasmid DNA Volume + dH2O Opti-MEM PLUS Lipo
1 KAH160/MV1 2 μg 5.8 μL +14.2 570 μL 2.5 μL 7.5 μL
2 KAH160/MV1 2 μg 5.8 μL +14.2 " " "
3 mock none 0 μL + 20.0 " " "
4 none none --- --- --- ---

> Add 570 μL Opti-MEM to each 20 μL DNA sample
> Add 2.5 μL PLUS reagent to each DNA+Opti-MEM sample. 5 min/room temp
> Add 7.5 μL Lipofectamine LTX to each DNA+Opti-MEM+PLUS reagent sample. 30 min/ room temp
> Add 600 μL complexes to each well (final volume of medium = 4 ml/well)
> Grow cells at 37°C overnight

Results (7/06/13)

  • Success! Very high levels of RFP expression in wells 1 & 2 (microscopy), zero signal in mock and untreated (3 & 4)
  • Harvest cells for flow cytometry



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