User:Karmella Haynes/Notebook/Polycomb project/2011/08/06

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08/06/11

  • Transfections: Test new activation domains on K. Hansen's HEK293 luc reporter system



Transfections: H3me test reporters (Gal4)
> Lipofectamine LTX
> Follow Jason's protocol, 12-well plates
> Use 1000 ng total plasmid (transcription factor plus empty vector)
> Plate 1 = repressed HEK 23;4;9 cells (5-day dox treatment, ~4 day wash-out)
> Plate 2 = non-repressed HEK 23;4;9 cells

Plate 1, 2

Wells TF plasmid (RFP) Description 200 ng DNA = Empty pcDNA3.1+ (MV1), 800 ng DNA =
1 KAH170/MV1 VP64-no DB 0.5 μL 0.8 μL
2 KAH60/pcVN VP64-Gal4DB 0.4 μL "
3 KAH160/MV1 VP64-hPCD 0.6 μL "
4 KAH220/MV1 SP1A-no DB 0.5 μL "
5 KAH214/MV1 SP1A-Gal4DB 0.6 μL "
6 KAH223/MV1 SP1A-hPCD 0.5 μL "
7 KAH221/MV1 SP1B-no DB 0.6 μL "
8 KAH215/MV1 SP1B-Gal4DB 1.0 μL "
9 KAH224/MV1 SP1B-hPCD 0.6 μL "
10 KAH221/MV1 p65-no DB 1.3 μL "
11 KAH215/MV1 p65-Gal4DB 0.7 μL "
12 KAH224/MV1 p65-hPCD 0.9 μL "

Lipo LTX, Note: double each reaction
> Dilute 1 μg DNA in dH2O (10 μL final)
> Add 190 μL Opti-MEM to 10 μL DNA
> Add 1 μL PLUS reagent --> R.T/ 5 min.
> Add 3 μL Lipo LTX --> R.T/ 30 min.
> Add ~200 μL complexes (drop-wise) to each well (1 ml med. each); Grow cells at 37°C

> Express for two days; Luciferase assay early Monday morning


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