User:Karmella Haynes/Notebook/Polycomb project/2009/12/18

From OpenWetWare
Jump to: navigation, search
Owwnotebook icon.png Project name Report.pngMain project page
Resultset previous.pngPrevious entry      Next entryResultset next.png

12/18/09

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

Transfection
> Test of Neo-inducible translation in U2OS (plain) cells

Wells Plasmid DNA Volume Fugene Opti-MEM
1-6 KAH105/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 overnight

Day 2
> Check RFP expression
--> Much less RFP in K-stop samples than K controls; some leaky K-stop expression (recorded images) > Refresh medium and add varying amounts of neomycin (G418)
--> Make neo dilutions in 50 μL Opti-MEM (see table)

Wells Transfection Neomycin (final conc. in well) 50 mg/mL neo Vol. Opti-MEM
1-6 KAH5105/MV2 0, 50, 100, 200, 400, 500 μg/mL 0, 1, 2, 4, 8, 10 μL 50 μL
7-12 KAH106/MV2 " " "
13-18 KAH107/MV2 " " "
19-24 KAH108/MV2 " " "

> Assay RFP after 18-24 hours
--> Checked after 48 hours on 12/21/09(snowstorm on Sunday); indication of induction in some cells
--> Move forward with neo-inducible Gal4-EED; quantify by Western blot