User:Karmella Haynes/Notebook/Polycomb project/2011/02/28

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02/28/11

  • ✓ Transfection: Pc-TF's into luc-silenced HEK cells (Fugene)



Fugene transfection

  1. KAH160: human Pc-TF
  2. KAH170: deleted PCD
Wells Plasmid DNA Volume Fugene Opti-MEM
1-6 KAH160/MV1 0, 50, 100, 200, 400, 800 ng 2.5 μL = 800 ng 1.8 μL 18.2 μL
7-12 " " " " "
13-18 KAH170/MV1 0, 50, 100, 200, 400, 800 ng 2.1 μL = 800 ng " "
19-24 " " " " "

> Add DNA to PBS for a total volume of 10 μL --> 2x master mix = 20 μL (start w/ 1600 ng/ 20 μL; do a serial dilution to get lower concentrations) > Master mixes (x2, 12 total): 3.6 μL Fugene + 36.4 μL Opti-MEM --> R.T/ 5 min.
> Add 20 μL DNA mix to each Fugene mix --> R.T./ 20 min.
> Add 30 μL complexes to each well (1 ml med. each); Grow cells at 37°C
> Assay luc activity after 2 days


> Note: when plating cells, use total cells from two dox+ 10 cm plates (~40% confluent); also use ~10% of the cells (2 mL of 10 mL resuspension) to seed eachnew 10 cm plate (2 total) for continued dox treatment


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