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

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* Gal4-EED/luc cells IFC - stain with anti-luc overnight
* Gal4-EED/luc cells IFC - stain with anti-luc overnight
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* PcTF transfection of U2OS
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* Use 1:200 dilution of rabbit (poly) anti-luc
* Use 1:200 dilution of rabbit (poly) anti-luc
* Incubate under parafilm "covers" overnight at 4°C
* Incubate under parafilm "covers" overnight at 4°C
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 +
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----
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'''Lipo transfection'''<br>
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> Cells were 100% confluent<br>
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> Re-plate 1:2 dilution prior to transfection in 3.4 mL p/s-free medium<br>
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 +
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{| class="wikitable" border="0" cellspacing="5" <!-- Lipofectamine transfection table -->
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|-
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| <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>  ||
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|-
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| 1            || KAH126        || 2 μg      || 9.0 μL +11.0  || 570 μL          || 2.5 μL          || 7.5 μL
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|-
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| 2            || KAH126        || 1 μg      || 4.5 μL +15.5  || "            || "          || "
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|-
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| 3            || KAH126        || 0.5 μg    || 2.3 μL +17.7  || "            || "          || "
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|-
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| 4            || KAH126        || 0.2 μg    || 0.9 μL +19.1  || "            || "          || "
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|-
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| 5            || KAH126        || 0.1 μg    || 0.5 μL +19.5  || "            || "          || "
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|-
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| 6            || none          || none      || ---          || none          || none          || none
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|}
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> Add 570 μL Opti-MEM to each 20 μL DNA sample <br>
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> Add 2.5 μL PLUS reagent to each DNA+Opti-MEM sample. 5 min/room temp<br>
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> Add 7.5 μL Lipofectamine LTX to each DNA+Opti-MEM+PLUS reagent sample. 30 min/ room temp <br>
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> Add 600 μL complexes to each well (final volume of medium = 4 ml/well)<br>
 +
> Add 1 μg/μL dox<br>
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> Grow cells at 37&deg;C overnight<br>
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 +

Revision as of 22:52, 12 June 2013

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 two 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
  • Incubate under parafilm "covers" overnight at 4°C



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





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