User:Karmella Haynes/Notebook/Polycomb project/2010/07/20

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07/20/10

  • ✓ Transfection: test H3me reporter clones
  • ✓ Transformation: KAH60/VNpc and FuW-Oct4 for minipreps
  • ✓ Cytology: fix cells for microscopy tomorrow (mCh vs. DAPI)



Transfections
> Screen for successful YFP-H3me3 reporter integration by inducing expression from UAS-TATA promoter using Gal4-RFP-VP64 (KAH60/VNpc)
> Use Fugene, 12-well format
> Samples (plated in duplicate, transfect just one):
--> KAH96-1, 2, 3, 5, 6, 7, 8, 9, 10, 11, 12 (11 total)
--> KAH142-1, 3, 5, 6, 7, 8, 10, 11, 12 (9 total)
--> KAH146-1, 2, 5, 6, 10, 11, 12 (7 total)
--> KAH147-3, 7, 9, 10, 11, 12 (6 total)
--> Note: Not enough plasmid DNA for all samples; do KAH96, 142 today, others tomorrow


Wells Plasmid DNA Volume Fugene Opti-MEM
18 total KAH60/VNpc 500 ng 1.2 μL 1.5 μL 48.5 μL

> Cells: change medium to ab-free for transfection samples > Master mix (18x): 27 μL Fugene + 873 μL Opti-MEM --> R.T./ 5 min.
> Add 21.6 μL DNA --> R.T./ 20 min.
> Add 51.2 μL DNA/Fugene mix to each well (2 ml medium/ well); Grow cells at 37°C
> Check RFP/ YFP after 18-24 hours


7/21/10
> Results: For KAH96 and 142 clones, some clones showed two or three dim YFP+ cells. Odd...cells with very little or no RFP showed YFP expression. Try transfecting some of these clones with other Gal4-ATF's to find one that can activate the YFP reporter.