User:Karmella Haynes/Notebook/PcTF Genomics/2013/06/12: Difference between revisions
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* Gal4-EED/luc cells IFC - stain with anti-luc overnight | * Gal4-EED/luc cells IFC - stain with anti-luc overnight | ||
* 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 | ||
---- | |||
'''Lipo transfection'''<br> | |||
> Cells were 100% confluent<br> | |||
> Re-plate 1:2 dilution prior to transfection in 3.4 mL p/s-free medium<br> | |||
{| 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 || 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 <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> | |||
> Add 1 μg/μL dox<br> | |||
> Grow cells at 37°C overnight<br> | |||
Revision as of 20:52, 12 June 2013
Pc-TF Genomics | <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page <html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> | ||||||||||||||||||||||||||||||||||||||||||||||||||
06/12/13
Gal4-EED/luc cells IFC
Lipo transfection
> Add 570 μL Opti-MEM to each 20 μL DNA sample
|