User:Karmella Haynes/Notebook/PcTF Genomics/2013/06/02: Difference between revisions

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'''Next day'''<br>
'''Next day'''<br>
> When harvesting cells for flow cytometry, use 1/4 of cells to seed new plate (for outgrowth)<br>
> Harvest cells for flow cytometry at Biodesign (contact Tong Xu)<br>
> Might run cells again to see when RFP reaches steady state




Revision as of 15:07, 11 June 2013

Pc-TF Genomics <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
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06/02/13

  • Transfection: KAH126 for flow cytometry on Monday
  • Gal4EED/luc test: induce repression with dox


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


Next day
> Harvest cells for flow cytometry at Biodesign (contact Tong Xu)


Gal4EED/luc test
> Dox dose response test
> 6-well plate (plated 5/29/13), shipment 1 cells

Wells dox μg/mL Volume dox (1 mg/mL) in 3.5 mL medium
1 0 ---
2 0.1 0.4 μL
3 0.2 0.8 μL
4 0.5 1.8 μL
5 1.0 3.5 μL
6 2.0 7.0 μL



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