User:Karmella Haynes/Notebook/Polycomb project/2009/12/07

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12/07/09

  • ✓ Transfection: transfection and neo-induction of K-stop constructs

Transfection
> Test of Neo-inducible translation

Wells Plasmid DNA Volume Fugene Opti-MEM
1-6 KAH5105/MV2 200 ng 0.5 μL 1.8 μL 18.2 μL
7-12 KAH106/MV2 " 0.4 μL " "
13-18 KAH107/MV2 " 0.4 μL " "
19-24 KAH108/MV2 " 0.4 μL " "

> Master mixes (4, x6): 10.8 μL Fugene + 109.2 μL Opti-MEM --> R.T/ 5 min.
> Add 6x vol DNA --> R.T./ 20 min.
> Add 20 μL complexes to each well (1 ml med. each); Grow cells at 37°C
> After 4-5 hours, refresh medium and add varying amounts of neomycin (G418); make serial dilutions (1:2)
--> Initial dilution = 400 μg/ 100 μL (8 μL 50 mg/mL stock in 92 μL Opti-MEM)
--> 4x (= 400 μL) = 32 μL 50 mg/mL stock in 368 μL Opti-MEM
--> Final vol. of each dilution is 50 μL (4x = 200 μL)

Wells Plasmid Neomycin (final conc.) Diluted Neo Vol.
1-6 KAH5105/MV2 0, 25, 50, 100, 200, 400 μg/mL 50 μL
7-12 KAH106/MV2 " "
13-18 KAH107/MV2 " "
19-24 KAH108/MV2 " "

> Assay RFP after 18-24 hours