User:Karmella Haynes/Notebook/Polycomb project/2011/08/12

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08/12/11

  • ✓ Transfections: for H3me reporter (retry)



Transfections: H3me reporters stables
> Last attempt: cells did not survive hygromycin. May have selected too soon after transfection (24 hours). Try again with longer recovery after transfection to allow for hygromycin integration and expression. Use 12-well plates (transfection has worked very well with visible markers in this format)
> Lipofectamine LTX; follow Jason's protocol, 12-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
Plate 2: U2OS FTRx

Wells Reporter plasmid (CFP) 750 ng DNA = Recombinase 250 ng DNA =
1, 2. 208/V0200 3.6 μL FlpE 0.5 μL
3, 4. DPRE-208/V0200 3.1 μL FlpE 0.5 μL


Lipo LTX: Make 4x reactions (2 wells per plate for each transgene)
> Dilute 1 μg DNA in dH2O (10 μL final)
> Add 190 μL Opti-MEM to 10 μL DNA
> Add 1.0 μL PLUS reagent --> R.T/ 5 min.
> Add 3.0 μL Lipo LTX --> R.T/ 30 min.
> Add ~200 μL complexes (drop-wise) to each well (1 ml med. each); Grow cells at 37°C


8/14/11
> Harvest and plate 1 well of cells to 10 cm plate with either selective (100 μg/mL hygro, 15 μg/mL blast.) or non-selective (complete, no ab's) media.



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