User:Karmella Haynes/Notebook/PcTF Genomics/2013/06/12: Difference between revisions
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== | ==06/12/13== | ||
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* | * Gal4-EED/luc cells IFC - stain with anti-luc overnight | ||
* PcTF transfection of U2OS | |||
---- | |||
'''Gal4-EED/luc cells IFC'''<br> | |||
* Samples: first row of wells in the 24-well plate. | |||
* Keep all others under PBS | |||
# 0 μg/mL dox | |||
# 0.1 | |||
# 0.2 | |||
# 0.5 | |||
# 1.0 | |||
# 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'''<br> | ||
> Samples | > Cells were 100% confluent<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 | |||
{| class="wikitable" border="0" cellspacing=" | |||
{| class="wikitable" border="0" cellspacing="5" <!-- Lipofectamine transfection table --> | |||
|- | |- | ||
| | | <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 || 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 <br> | ||
> Add 2.5 μL PLUS reagent to each DNA+Opti-MEM sample. 5 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> | |||
> Grow cells at 37°C overnight<br> | |||
Revision as of 14:02, 30 June 2013
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06/12/13
Gal4-EED/luc cells IFC
Lipo transfection
> Add 570 μL Opti-MEM to each 20 μL DNA sample
|