User:Karmella Haynes/Notebook/Polycomb project/2010/08/30

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08/30/10

  • ✓ Transfection: Pc-ATF's into H3me3 reporter cells
  • ✓ Freeze-down: KAH142-3
  • ✓ ChIP/ Co-IP: x-link KAH130-4, 131-4 (formaldehyde x-link only, no DMA)



Fugene transfection
> Transfect the following cell lines (in 12-well plates, 6 wells per line):

  • KAH96-11
  • KAH142-3
  • KAH142-5
  • KAH146-1

with six different Pc-ATF constructs and neg. ctrls. (dox inducible). Test to see if Pc-ATF can activate YFP.
> Pc-ATF test constructs (human only for this trial):

  • KAH126: human PCD-ATF
  • KAH154: human PCD-RFP (no act. domain)
  • KAH130: human CBX8-ATF
  • KAH158: human CBX8-RFP (no act. domain)
  • KAH132: ATF (no Polycomb)
Wells Plasmid DNA Volume Fugene Opti-MEM
1 KAH126/MV2 200 ng 0.4 μL 1.8 μL 18.2 μL
2 KAH154/MV2 " 2.0 μL " "
3 KAH130/MV2 " 0.4 μL " "
4 KAH158/MV2 " 0.6 μL " "
5 KAH132/MV2 " 0.4 μL " "
6 none 0 --- " "

Use the same set-up (above) for:
1-6: KAH96-11 cells
7-12: KAH142-3 cells
13-18: KAH142-5 cells
19-24: KAH146-1 cells

> Master mixes (4, 4x mixes): 7.2 μL Fugene + 72.8 μL Opti-MEM --> R.T/ 5 min.
> Add 4x vol DNA --> R.T./ 20 min.
> Add 20 μL complexes to each well (2 ml med. each); Grow cells at 37°C overnight
> Tomorrow, photograph RFP/YFP without dox, then add dox and check for RFP/YFP in the evening


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