User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2013/06/06

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06/06/2013

  • ✓ Transfections: H3K27me3 reporters into cell lines BD002 (KAH201+KAH182+V0200)
  • ✓ Polycomb-ATF negative control lines: colony plates



Transfections
> U2OS Flp-in T-REx lines (puro/zeo/blast) + FRT-marked mammalian transfection vector (hygro)
--> Note: co-transfect with FlpE (1:3); hygro resistance replaces zeo resistance after "Flp" in
> Use Lipofectamine, 6-well format
> Plates:

  1. BD002 (3:1) Flp-in T-REx
  2. BD002 (4:1) Flp-in T-REx

BD002 (3:1) = 339 ng/μl BD002 (4:1) = 317 ng/μl PlpE = 192 ng/μl

Plate-Well Plasmid DNA Volume dH2O Lipo Opti-MEM (total)
1-1 BD002 (3:1) + FlpE 1.5 + 0.5 μg 4.42 + 2.60 μL 12.98μL 4 μL 500 μL
1-2 BD002 (3:1) + FlpE " 4.42 + 2.60 μL 12.98μL " "
1-3 BD002 (4:1) + FlpE " 4.73 + 2.60 μL 12.67μL " "
1-4 BD002 (4:1)+ FlpE " 4.73 + 2.60 μL 12.67μL " "
1-5 BD002 (3:1) + --- 4.42 + --- 15.58μL
1-6 BD002 (4:1) + --- 4.73 + --- 15.58μL


> Add 16 μL (4x) Lipo to 1000 μL (4x) Opti-MEM --> R.T/ 5 min.
> Add DNA to 250 μL Opti-MEM
> Add 250 Lipo mix into each 250 DNA mix --> R.T./ 20 min.
> Add 500 μL complexes to each 3.5 mL well (4 ml med. total each); Grow cells at 37°C
> Refresh medium after 5 hours (ab-free)
> Grow for one/two days, then plate dilution cultures in selection medium (+hygro)



Polycomb-ATF negative control lines: colony plates
> For each line make 2 plates: ~1x105 and 5x104 cells
> 25 ml 1 μg/mL puro, 15 μg/mL blast, 200 μg/mL zeo
> Also make 6-well back-up plate for all transfections (~3x105 cells in selection medium)



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