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

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# Prepare DNA-Lipofectamine complexes (600 μl per sample) as follows:
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## Label sterile microfuge (1.5 ml) tubes.
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## Add '''570 μL Opti-MEM''' to each 20 μL DNA sample.
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## Add '''2.5 μL PLUS reagent''' to each DNA+Opti-MEM sample. Incubate for 5 minutes at room temperature.
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## Add '''7.5 μL Lipofectamine LTX''' to each DNA+Opti-MEM+PLUS reagent sample. Incubate for 30 minutes at room temperature.
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# Add the total 600 μL of DNA+Opti-MEM+PLUS reagent+Lipofectamine mixture '''drop-wise''' to each appropriate well of cells.
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# Incubate cells at 37°C in a CO<sub>2</sub> incubator
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# (Optional) To reduce toxicity, after 5-6 hours wash cells once with warm 1xPBS to remove excess DNA-Lipo complexes. Add fresh antibiotic-free growth medium.
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Transgene expression should be detectable after 18 hours.
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> Add 16 μL (4x) Lipo to 1000 μL (4x) Opti-MEM --> R.T/ 5 min. <br>
 
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> Add DNA to 250 μL Opti-MEM <br>
 
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> Add 250 Lipo mix into each 250 DNA mix --> R.T./ 20 min. <br>
 
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> Add 500 μL complexes to each 3.5 mL well (4 ml med. total each); Grow cells at 37&deg;C<br>
 
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> Refresh medium after 5 hours (ab-free)<br>
 
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> Grow for one/two days, then plate dilution cultures in selection medium (+hygro)
 

Revision as of 15:07, 6 June 2013

PcTF Subcloning in E. coli Main project page
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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 PLUS reagent Opti-MEM (total)
1-1 BD002 (3:1) + FlpE 1.5 + 0.5 μg 4.42 + 2.60 μL 12.98μL 2.5μL 7.5 μL 570 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 " ""
  1. Prepare DNA-Lipofectamine complexes (600 μl per sample) as follows:
    1. Label sterile microfuge (1.5 ml) tubes.
    2. Add 570 μL Opti-MEM to each 20 μL DNA sample.
    3. Add 2.5 μL PLUS reagent to each DNA+Opti-MEM sample. Incubate for 5 minutes at room temperature.
    4. Add 7.5 μL Lipofectamine LTX to each DNA+Opti-MEM+PLUS reagent sample. Incubate for 30 minutes at room temperature.
  2. Add the total 600 μL of DNA+Opti-MEM+PLUS reagent+Lipofectamine mixture drop-wise to each appropriate well of cells.
  3. Incubate cells at 37°C in a CO2 incubator
  4. (Optional) To reduce toxicity, after 5-6 hours wash cells once with warm 1xPBS to remove excess DNA-Lipo complexes. Add fresh antibiotic-free growth medium.

Transgene expression should be detectable after 18 hours.



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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