User:Karmella Haynes/Notebook/PcTF Genomics/2013/06/12

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06/12/13

  • Gal4-EED/luc cells IFC - stain with anti-luc overnight
  • PcTF transfection of U2OS


Gal4-EED/luc cells IFC

  • Samples: first two of wells in the 24-well plate.
  • Keep all others under PBS
  1. 0 μg/mL dox
  2. 0.1
  3. 0.2
  4. 0.5
  5. 1.0
  6. 2.0
  • Use 1:200 dilution of rabbit (poly) anti-luc
  • Incubate under parafilm "covers" overnight at 4°C


Lipo transfection
> Cells were 100% confluent
> Re-plate 1:2 dilution prior to transfection in 3.4 mL p/s-free medium


Wells Plasmid DNA Volume + dH2O Opti-MEM PLUS Lipo
1 KAH126 2 μg 9.0 μL +11.0 570 μL 2.5 μL 7.5 μL
2 KAH126 1 μg 4.5 μL +15.5 " " "
3 KAH126 0.5 μg 2.3 μL +17.7 " " "
4 KAH126 0.2 μg 0.9 μL +19.1 " " "
5 KAH126 0.1 μg 0.5 μL +19.5 " " "
6 none none --- none 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)
> Add 1 μg/μL dox
> Grow cells at 37°C overnight





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