User:Karmella Haynes/Notebook/Polycomb project/2011/07/31

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07/31/11

  • ✓ Transfection: for H3me reporter stable lines



Transfections: H3me reporters stables
> Lipofectamine LTX
> Follow Jason's protocol, 6-well plates
> Use ~1:3 ratio of FlpE plasmid to reporter plasmid
> Two different types of Flp-in T-REx: HEK293, U2OS

Plate 1: HEK293 FTRx

Wells Reporter plasmid (CFP) 1.5 μg DNA = Recombinase 0.5 μg DNA =
1 208/V0200 10.4 μL FlpE 1.0 μL
2 DPRE-208/V0200 6.2 μL FlpE 1.0 μL

Plate 2: U2OS FTRx

Wells Reporter plasmid (CFP) 1.5 μg DNA = Recombinase 0.5 μg DNA =
1 208/V0200 10.4 μL FlpE 1.0 μL
2 DPRE-208/V0200 6.2 μL FlpE 1.0 μL

Lipo LTX
> Dilute 2 μg DNA in dH2O (20 μL final)
> Add 580 μL Opti-MEM to 20 μL DNA
> Add 2.5 μL PLUS reagent --> R.T/ 5 min.
> Add 7.5 μL Lipo LTX --> R.T/ 30 min.
> Add ~600 μL complexes (drop-wise) to each well (1 ml med. each); Grow cells at 37°C


8/01/11
> Trypsinize cells w/ 0.5 mL medium; bring volume to 2 mL with selective medium

  1. U20S: McCoy's 5A complete (tet screened), 200μg/mL hygromycin, 15 μg/mL blasticidin
  2. HEK: DMEM complete (tet screened), 200μg/mL hygromycin, 15 μg/mL blasticidin

> Passage transfected and untransfected control cells to new plates...

  1. 100 μL cells, 10 mL, 10 cm dish
  2. 500 μL cells, 10 mL, 10 cm dish
  3. 1 mL cells, 4mL, 6-well plate (single well)

> Look for colony growth over time