User:Karmella Haynes/Notebook/Polycomb project/2010/02/02

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02/02/10

  • ✓ Minipreps: KAH139-141/V0201
  • ✓ Transfection: Gal4-EED expression constructs
  • ✓ Thaw cells: HEK293 Luc #4 (to test Gal4-EED function)



Minipreps
> Check with XbaI digest

Reagent Volume Expected:Vector = 4500
1,2. KAH139/V0201 = 3655
1,2. KAH140/V0201 = 3599
1,2. KAH141/V0201 = 3187
File:KAH 020210 gel11.tif
15 μL/lane; 1% agarose
DNA(plasmid) 2.0 μL
10X buffer 1.5
XbaI 1.0
dH2O 10.5
  15 μL --> 37°C/ ~15 min.

--> Combine duplicate samples, measure conc., and use for Lipo transfection



Lipo transfection
> Details

Wells Plasmid DNA Volume Lipo Opti-MEM (total)
1 KAH139/V0201 2 μg 4.4 μL 4 μL 500 μL
2 " (+50 μg/mL neo) " 4.4 μL " "
3 " (+100 μg/mL neo) " 4.4 μL " "
4 " (+200 μg/mL neo) " 4.4 μL " "
5 " (+400 μg/mL neo) " 4.4 μL " "
6 " (+500 μg/mL neo) " 4.4 μL " "
7 KAH140/V0201 " 5.8 μL " "
8 KAH141/V0201 " 5.1 μL " "
9 no DNA " --- " "

> Add Lipo to 250 μL Opti-MEM --> R.T/ 5 min.
> Add DNA to 250 μL Opti-MEM
> Add DNA mix to Lipo mix --> R.T./ 20 min.
> Starting vol. is 3.5 mL, no need to remove 500 μL; Add 500 μL complexes to each well (4 ml med. each); Grow cells at 37°C
> Refresh medium after 5 hours (ab-free)
> Add neomycin to samples 2-6


Day 2
> Do protein preps on all samples (for EED Western blot)