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

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(06/12/13)
Current revision (16:02, 30 June 2013) (view source)
(06/12/13)
 
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Line 16: Line 16:
----
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'''Gal4-EED/luc cells IFC'''<br>
'''Gal4-EED/luc cells IFC'''<br>
-
* Samples: first two of wells in the 24-well plate.
+
* Samples: first row of wells in the 24-well plate.
* Keep all others under PBS
* Keep all others under PBS
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 34: Line 34:
> Cells were 100% confluent<br>
> Cells were 100% confluent<br>
> Re-plate 1:2 dilution prior to transfection in 3.4 mL p/s-free medium<br>
> Re-plate 1:2 dilution prior to transfection in 3.4 mL p/s-free medium<br>
 +
> Transfect human PcTF under control of constitutive CMV promoter (KAH160/MV1); 346.6 ng/μL<br>
 +
> Samples are for RT-PCR
Line 40: Line 42:
| <u>Wells</u> || <u>Plasmid</u> || <u>DNA</u> || <u>Volume + dH<sub>2</sub>O</u> || <u>Opti-MEM</u> || <u>PLUS</u> || <u>Lipo</u>  ||  
| <u>Wells</u> || <u>Plasmid</u> || <u>DNA</u> || <u>Volume + dH<sub>2</sub>O</u> || <u>Opti-MEM</u> || <u>PLUS</u> || <u>Lipo</u>  ||  
|-
|-
-
| 1            || KAH126         || 2 μg      || 9.0 μL +11.0 || 570 μL          || 2.5 μL          || 7.5 μL
+
| 1            || KAH160/MV1         || 2 μg      || 5.8 μL +14.2 || 570 μL          || 2.5 μL          || 7.5 μL
|-
|-
-
| 2            || KAH126         || 1 μg      || 4.5 μL +15.5 || "            || "          || "
+
| 2            || KAH160/MV1         || 2 μg      || 5.8 μL +14.2 || "            || "          || "
|-
|-
-
| 3            || KAH126         || 0.5 μg    || 2.3 μL +17.7 || "            || "          || "
+
| 3            || KAH160/MV1         || 2 μg    || 5.8 μL +14.2 || "            || "          || "
|-
|-
-
| 4            || KAH126         || 0.2 μg     || 0.9 μL +19.1 || "            || "          || "
+
| 4            || mock         || none     || 0 μL + 20.0 || "            || "          || "
|-
|-
-
| 5            || KAH126         || 0.1 μg     || 0.5 μL +19.5 || "            || "          || "
+
| 5            || mock         || none     || 0 μL + 20.0 || "            || "          || "
|-  
|-  
-
| 6            || none           || none      || ---           || none         || none         || none
+
| 6            || mock           || none      || 0 μL + 20.0           || "         || "         || "
|}
|}
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> 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>

Current revision

Pc-TF Genomics 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





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