User:Karmella Haynes/Notebook/Polycomb project/2010/11/27

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11/27/10

  • ✓ HEK293 Gal4-EED: luc repression assay
  • ✓ luc reactivation: Fugene transfection
  • ✓ HEK293 Gal4-EED stocks: change to puro medium



Transfections
> HEK293 luc-repressed cells + Gal4/mCh/VP64 constructs

> Do luc assay to confirm silencing in 8, 6, 4, 2 and 0 day silenced cells
--> 4, 6, and 8-day treatments show full luc repression

> Set up 24-well plates (5 ml from 10 ml plate; added to ; aliquot 1 ml cells per well):

  1. 8-day silenced (10 cm plate #1)
  2. 4-day silenced (#3)
  3. no silencing (#5)
  • Note: dox wash-out; cells were washed 1x w/ PBS before resuspending in complete medium

Set-up per plate
> Use Fugene, 24-well format
> Dilute each plasmid to 100 ng/ul in 1x PBS

Wells Construct DNA Volumes Fugene Opti-MEM
1-6 KAH60/VNpc 0, 50-800 ng 0.0, 0.5, 1.0, 2.0, 4.0, 8.0 ul 1.8 ul 18.2 ul
7-12 KAH54/VNpc " " " "
13-18 KAH36(VNpc) " " " "
19-24 KAH59/Npc " " " "

> 25x master mix: 45 ul Fugene + 455 ul Opti-MEM --> R.T/ 5 min.
> Add 8.0, 7.5, 7.0, 6.0, 4.0, 0.0 ul PBS to each row of tubes
> Add 0.0, 0.5, 1.0, 2.0, 4.0, 8.0 ul 100 ng/ul DNA to each row
> Add 20 ul Fugene master mix to each sample --> R.T./ 20 min.
> Add complexes to each well (1 ml med. each); 37°C
> Assay RFP & luc after 48 hours


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