User:Karmella Haynes/Notebook/Polycomb project/2010/06/25

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06/25/10

  • ✓ Minipreps: KAH96, 142, 146, 147/V0201 DNAme-
  • ✓ Transfection: KAH96, 142, 146, 147/V0201 DNAme-
  • ✓ ChIP: cross-link FTRx and 154-2 cells



Minipreps
> DNA methylation-free plasmid preps for transfection (grown in C2925)
> Check KAH96, 142, 146, 147/V0201 with XbaI digest (add 641 to expected insert size)

Reagent Volume Expected:
V0201 band = 4516
Insert band = BB size + 641 bp
1-3. KAH96/V0201 = 3466
4,5. KAH142/V0201 = 5212
6-8. KAH146/V0201 = 7027
9-12. KAH147/V0201 = 8773
Miniprep digests 6/25/10
15 μL/lane; 1% agarose
DNA(plasmid) 2.0 μL
10X buffer 1.5
XbaI 1.0
dH2O 10.5
  15 μL --> 37°C/ ~15 min.




Lipo transfection
> KAH96 [5xGal4 : HSVtkTATA : K : YFP : YFP : NLSstop2 : polyA : HS4 : HS4]
> KAH142 [DPRE : 5xGal4 : HSVtkTATA : K : YFP : YFP : NLSstop2 : polyA : HS4 : HS4]
> KAH146 [[KAH96] + EED expression cassette]
> KAH147 [[KAH142] + EED expression cassette]

Wells Plasmid DNA Volume Lipo Opti-MEM (total)
1 KAH96/ FlpE 1.5, 0.5 μg 1.5, 1.0 μL 4 μL 500 μL
2 KAH142/ FlpE 1.5, 0.5 μg 1.8, 1.0 μL " "
3 KAH146/ FlpE 1.5, 0.5 μg 2.0, 1.0 μL " "
4 KAH147/ FlpE 1.5, 0.5 μg 2.5, 1.0 μL " "
5 Lipo only --- --- " "

> Add Lipo to 250 μL Opti-MEM --> R.T/ 5 min.
> Add DNA to 250 μL Opti-MEM
> Add DNA mix to Lipo mix --> R.T./ 20 min.
> Add 500 μL complexes to each well (4 ml med. total each); Grow cells at 37°C
> Refresh medium after 5 hours (ab-free)



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