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

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06/06/2013

  • ✓ Transfections: H3me reporters into cell lines KAH126-1, 130-2, 132-8
  • ✓ Polycomb-ATF negative control lines: colony plates



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

  1. KAH126-1 Flp-in T-REx
  2. KAH130-2 Flp-in T-REx
  3. KAH132-8 Flp-in T-REx


Plate-Well Plasmid DNA Volume Lipo Opti-MEM (total)
1-1 KAH96/V0201 + FlpE 1.5 + 0.5 μg 2.4 + 1.4 μL 4 μL 500 μL
1-2 KAH142/V0201 + FlpE " 2.4 + 1.4 μL " "
1-3 KAH146/V0201 + FlpE " 6.4 + 1.4 μL " "
1-4 KAH147/V0201 + FlpE " 9.6 + 1.4 μL " "
---
2-1 KAH96/V0201 + FlpE 1.5 + 0.5 μg 2.4 + 1.4 μL 4 μL 500 μL
2-2 KAH142/V0201 + FlpE " 2.4 + 1.4 μL " "
2-3 KAH146/V0201 + FlpE " 6.4 + 1.4 μL " "
2-4 KAH147/V0201 + FlpE " 9.6 + 1.4 μL " "
---
3-1 KAH96/V0201 + FlpE 1.5 + 0.5 μg 2.4 + 1.4 μL 4 μL 500 μL
3-2 KAH142/V0201 + FlpE " 2.4 + 1.4 μL " "
3-3 KAH146/V0201 + FlpE " 6.4 + 1.4 μL " "
3-4 KAH147/V0201 + FlpE " 9.6 + 1.4 μL " "

> 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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