User:Karmella Haynes/Notebook/Polycomb project/2011/07/21

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07/21/11

  • ✓ Transfections: HEK Flp-in T-REx cells, H3me test reporters



Transfections: H3me test reporters (Gal4)
> Lipofectamine LTX
> Follow Jason's protocol, 12-well plates
> Use ~1:10 ratio of transactivator plasmid to reporter plasmid
> Dilute KAH60/pcVN and empty pcVN to 100 ng/μL

Plate 2

Wells Reporter plasmid (CFP) 900 ng DNA = TA plasmid (RFP) 100 ng DNA =
1 207/V0200 5.5 μL empty pcVN 1 μL
2 207/V0200 5.5 μL KAH60/pcVN 1 μL
3 208/V0200 6.2 μL empty pcVN 1 μL
4 208/V0200 6.2 μL KAH60/pcVN 1 μL
5 209/V0200 5.0 μL empty pcVN 1 μL
6 209/V0200 5.0 μL KSH60/pcVN 1 μL

Lipo LTX
> 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

> Flow cytometry tomorrow (7/22/11)

--> Results: Induction with the transactivator (KAH60) works! KAH208/V0200 shows optimal induction, so the spacer appears to help. 209 not much different. No spacer (207) appears to be repressed by KAH60.


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