User:Karmella Haynes/Notebook/PcTF Genomics/2013/06/30: Difference between revisions
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* | * 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 ELISA set-up | |||
---- | ---- | ||
''' | '''Gal4-EED/luc cells IFC'''<br> | ||
> | * Previous trial, saw no difference in repression for 0.1, 0.2, 0.5, 1.0, 2.0 μg/mL dox. Try lower range. | ||
# 1.0 μg/mL dox | |||
# 10<sup>-1</sup> | |||
# 10<sup>-2</sup> | |||
# 10<sup>-3</sup> | |||
# 10<sup>-4</sup> | |||
# 0 | |||
{| class="wikitable" border="0" cellspacing=" | ---- | ||
'''Lipo transfection'''<br> | |||
> Samples are for RT-PCR | |||
> 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-TetO promoter (KAH126/MV2); ### ng/μL<br>; Note: no TetR in SK-N-SH, so expression will be constitutive | |||
{| 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 || 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 || " || " || " | |||
|} | |} | ||
--> | > 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 14:01, 30 June 2013
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6/30/13/dd/yy
Gal4-EED/luc cells IFC
Lipo transfection
> Add 570 μL Opti-MEM to each 20 μL DNA sample
|