User:Karmella Haynes/Notebook/Polycomb project/2010/04/22

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04/22/10

  • ✓ Transfections: Polycomb-ATF negative controls for stable lines



Transfections
> U2OS Flp-in T-REx cells + Polycomb-ATF plasmids (puro)
> Use Lipofectamine, 6-well format
> Make duplicate plates (one will be induced with dox); 12 wells total

Wells Plasmid DNA Volume Lipo Opti-MEM (total)
1 KAH154/MV2 2 μg 19.0 μL 4 μL 500 μL
2 KAH155/MV2 " 12.0 μL " "
3 KAH156/MV2 " 19.0 μL " "
4 KAH157/MV2 " 6.0 μL " "
5 KAH158/MV2 " 13.0 μL " "
6 KAH159/MV2 " 9.0 μL " "

> Add 8 μL (2x) Lipo to 500 μL (2x) Opti-MEM --> R.T/ 5 min.
> Add 2x DNA to 500 μL (2x) Opti-MEM
> Add DNA mix to Lipo mix --> R.T./ 20 min.
> Add 500 μL complexes to each 3.5 mL well (4 ml med. total each); Grow cells at 37°C
> Refresh medium after 5 hours (ab-free)
--> Add 1μg/mL dox to one plate, check for RFP expression tomorrow
> Grow for one/two days, then plate dilution cultures in selection medium (puro)


> 4/23/10
--> Dox+ plate results: ~50% of the cells are RFP+ (high expression and efficiency). For colony plates, use ~50x105 (1/20) and ~25x105 (1/40) cells in a large (~20 cm) gridded plate



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