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

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Line 24: Line 24:
 
# 0.5
 
# 0.5
 
# 1.0
 
# 1.0
# 2.0
+
# 2.0 (induced on 6/02/13, 10 days)
  
* 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
  
  
Line 48: Line 48:
 
| 3            || KAH160/MV1        || 2 μg    || 5.8 μL +14.2  || "            || "          || "
 
| 3            || KAH160/MV1        || 2 μg    || 5.8 μL +14.2  || "            || "          || "
 
|-
 
|-
| 4            || mock        || ---     || 0 μL + 20.0  || "            || "          || "
+
| 4            || mock        || none     || 0 μL + 20.0  || "            || "          || "
 
|-
 
|-
| 5            || mock        || ---     || 0 μL + 20.0  || "            || "          || "
+
| 5            || mock        || none     || 0 μL + 20.0  || "            || "          || "
 
|-  
 
|-  
 
| 6            || mock          || none      || 0 μL + 20.0          || "          || "          || "
 
| 6            || mock          || none      || 0 μL + 20.0          || "          || "          || "
Line 59: Line 59:
 
> Add 7.5 μL Lipofectamine LTX to each DNA+Opti-MEM+PLUS reagent sample. 30 min/ room temp <br>
 
> Add 7.5 μL Lipofectamine LTX to each DNA+Opti-MEM+PLUS reagent sample. 30 min/ room temp <br>
 
> Add 600 μL complexes to each well (final volume of medium = 4 ml/well)<br>
 
> Add 600 μL complexes to each well (final volume of medium = 4 ml/well)<br>
> Add 1 μg/μL dox<br>
 
 
> Grow cells at 37&deg;C overnight<br>
 
> Grow cells at 37&deg;C overnight<br>
  

Revision as of 13:02, 30 June 2013

Owwnotebook icon.pngPc-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 (induced on 6/02/13, 10 days)
  • 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)
> Grow cells at 37°C overnight