User:Karmella Haynes/Notebook/Polycomb project/2010/02/09

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02/09/10

  • ✓ U2OS Flp-in T-REx: add dox to Dox+ wells (6-well plate from 2/08)
  • ✓ Transfection: Gal4-EED constructs in HEK293 luc reporter line
  • ✓ Polycomb-ATF lines: frozen stocks for 90% confluent plates: 127-4, 128-8, 132-8; 1x 1:10 plate (per line)

Transfection
> HEK293 cells + Gal4-EED constructs (KAH139, 140, 141/V0201)
> Use Fugene, 24-well format
> Serial dilutions of plasmids: 800 - 25 ng in 5 μL
--> Start with 1600 ng in 10 μL OptiMEM

Plasmid ng/μL Add (μL)... to OptiMEM (μL)
1. KAH139/V0201 454.2 3.5 7.5
2. KAH140/V0201 342.3 4.7 5.3
3. KAH141/V0201 388.8 4.1 5.9

--> Add 5 μL plasmid to 5 μL OptiMEM for each dilution step

Wells Plasmid DNA Volume Fugene Opti-MEM
1-6 KAH139/V0201 25, 50, 100, 200, 400, 800 ng 5.0 μL 1.8 μL 18.2 μL
7-12 KAH140/V0201 " " " "
13-18 KAH141/V0201 " " " "
19-24 no DNA " " " "

> Master mix: 90 μL Fugene + 910 μL Opti-MEM --> R.T/ 5 min.
> Add 20 μL Fugene mix to DNA --> R.T./ 20 min.
> Add 25 μL complexes to each well (1 ml med. each); Grow cells at 37°C
> Assay Luc after 18-24 hours


> Day 2
--> Refresh medium. Do luc assay next day (2/11).