User:Karmella Haynes/Notebook/PcTF Genomics/2013/06/12: Difference between revisions

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# 2.0
# 2.0


* Use 1:200 dilution of rabbit (poly) anti-luc
* Use 1:200 dilution of rabbit (poly) anti-luc (in 15 μL Gentex horse serum blocking solution per well)
* Incubate under parafilm "covers" overnight at 4°C
* Incubate under parafilm "covers" overnight at 4°C in box with wet paper towels




Revision as of 21:29, 12 June 2013

Pc-TF Genomics <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
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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 row 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 (in 15 μL Gentex horse serum blocking solution per well)
  • Incubate under parafilm "covers" overnight at 4°C in box with wet paper towels


Lipo transfection
> Cells were 100% confluent
> Re-plate 1:2 dilution prior to transfection in 3.4 mL p/s-free medium
> Transfect human PcTF under control of constitutive CMV promoter (KAH160/MV1); 346.6 ng/μL
> Samples are for RT-PCR


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 KAH160/MV1 2 μg 5.8 μL +14.2 " " "
4 mock none 0 μL + 20.0 " " "
5 mock none 0 μL + 20.0 " " "
6 mock none 0 μL + 20.0 " " "

> 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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