User:Karmella Haynes/Notebook/Polycomb project/2010/07/24

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07/24/10

  • ✓ Transfections: Test Gal4-ATF's on KAH142 H3me reporter clones



Fugene transfection
> 12-well plate
1-4. KAH142-1
5-8. KAH142-3
9-12. KAH142-5

Wells Plasmid DNA Volume Fugene Opti-MEM
1,5,9 no plasmid --- 0 μL 1.8 μL 18.2 μL
2,6,10 KAH54/pcVN " 0.4 μL " "
3,7,11 KAH59/pcVN " 0.6 μL " "
4,8,12 KAH60/pcVN " 0.4 μL " "

> Master mixes (3x mix, 4 mixes): 5.4 μL Fugene + 54.6 μL Opti-MEM --> R.T/ 5 min.
> Add 3x vol DNA --> R.T./ 20 min.
> Add 20 μL complexes to each well (1 ml med. each); Grow cells at 37°C overnight


7/25/10
Results:

  • Background YFP expression: two or three cells for KAH142-5, none for KAH142-3, KAH142-1 control well was contaminated
  • KAH54/pcVN (Gal4DB-VP64): YFP+ cells in KAH142-3
  • KAH59/pcVN (Gal4DB-mCh-mCh-VP64): RFP+/YFP+ cells in KAH142-3 and KAH142-5
  • KAH60/pcVN (Gal4DB-mCh-VP64): strange result; RFP+/YFP- or RFP-/YFP+ cells

Conclusions:

  • Keep KAH142-3 and 142-5 for frozen stocks; use 142-3 for further experimentation
  • Use KAH59/pcVN to screen other H3me Reporter clones



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