User:Karmella Haynes/Notebook/Polycomb project/2009/11/19

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11/19/09

  • ✓ Transfections: test Gal4/mCh/VP64 ATF's (trial 2)

Transfections
> HEK293 cells + Gal4/mCh/VP64 constructs
> Use Fugene, 24-well format
> Dilute each plasmid to 100 ng/ul in st dH2O

Wells Construct DNA Volumes Fugene Opti-MEM
1-6 KAH59/VNpc 25-800 ng 0.3, 0.5, 1.0, 2.0, 4.0, 8.0 ul 1.8 ul 18.2 ul
7-12 KAH60/VNpc " " " "
13-18 KAH54/VNpc " " " "
19-24 KAH66/MV5 " " " "
25-30 KAH65/MV5 " " " "
31-36 KAH59/MV5 " " " "
37-42 KAH36 " " " "
43-48 none " " " "

> Fugene + Opti-MEM --> R.T/ 5 min.
> Add DNA --> R.T./ 20 min.
> Add complexes to each well (1 ml med. each); 37°C
> Assay RFP/ Luc after 18-24 hours



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