User:Karmella Haynes/Notebook/PcTF Genomics/2013/06/30

From OpenWetWare

Jump to: navigation, search
Pc-TF Genomics Main project page
Previous entry      Next entry


6/30/13

  • Gal4-EED/luc cells - split 6-well culture plate (shipment-2 cells) and treat 1 plate with dox
  • PcTF transfection of SK-N-SH



Gal4-EED/luc cells IFC

  • Previous trial, saw no difference in repression for 0.1, 0.2, 0.5, 1.0, 2.0 μg/mL dox. Try lower range.
  1. 1.0 μg/mL dox
  2. 10-1
  3. 10-2
  4. 10-3
  5. 10-4
  6. 0

Addition of serial dilutions of dox to cells

  • Start with 3.5 mL growth medium in cell culture wells. Remove some growth medium from cells where necessary.
  • Set up the following serial dilutions (using appropriate growth medium) in sterile 1 mL tubes...
  tube 1 2 3 4 5 6
1 mg/mL dox (μL) 8.0 --- --- --- --- ---
mixture from prior tube (μL) --- 100 100 100 100 ---
+growth med. (μL) 1000 900 900 900 900 ---
Final [dox] (μg/mL) in 4 mL med. 1.0 10-1 10-2 10-3 10-4 0



Lipo transfection
> Samples are for RT-PCR > 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-TetO promoter (KAH126/MV2); 226 ng/μL; Note: no TetR in SK-N-SH, so expression will be constitutive


Wells Plasmid DNA Volume + dH2O Opti-MEM PLUS Lipo
1 KAH126/MV2 2 μg 8.8 μL +11.2 570 μL 2.5 μL 7.5 μL
2 KAH126/MV2 2 μg 8.8 μL +11.2 " " "
3 KAH126/MV2* 2 μg 8.8 μL +11.2 " " "
4 mock none 0 μL + 20.0 " " "
5 mock none 0 μL + 20.0 " " "
6 mock none 0 μL + 20.0 " " "

(*Ran out of plasmid DNA)

> 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




Personal tools