User:Karmella Haynes/Notebook/PcTF Genomics/2013/06/12: Difference between revisions
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# 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 | ||
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> 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 | |||
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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> || | | <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 || | | 1 || KAH160/MV1 || 2 μg || 5.8 μL +14.2 || 570 μL || 2.5 μL || 7.5 μL | ||
|- | |- | ||
| 2 || | | 2 || KAH160/MV1 || 2 μg || 5.8 μL +14.2 || " || " || " | ||
|- | |- | ||
| 3 || | | 3 || KAH160/MV1 || 2 μg || 5.8 μL +14.2 || " || " || " | ||
|- | |- | ||
| 4 || | | 4 || mock || none || 0 μL + 20.0 || " || " || " | ||
|- | |- | ||
| 5 || | | 5 || mock || none || 0 μL + 20.0 || " || " || " | ||
|- | |- | ||
| 6 || | | 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> | ||
> Grow cells at 37°C overnight<br> | > Grow cells at 37°C overnight<br> | ||
Revision as of 14:02, 30 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
|