User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2013/08/08

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(Gal4-VP64, Cyan Transactivation)
(Gal4-VP64, Cyan Transactivation)
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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.
# Add the total 600 μL of DNA+Opti-MEM+PLUS reagent+Lipofectamine mixture '''drop-wise''' to each appropriate well of cells.
# Incubate cells at 37°C in a CO<sub>2</sub> incubator  
# Incubate cells at 37°C in a CO<sub>2</sub> incubator  
-
# (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.  
+
# To reduce toxicity, after 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.
Transgene expression should be detectable after 18 hours.

Revision as of 15:53, 9 August 2013

PcTF Subcloning in E. coli Main project page
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08/08/2013

Gal4-VP64, Cyan Transactivation

1 day before transfection:

  1. Seed cultures in 6-well plates at ~2.5x105 so that cells will be ~5.0x105 at the time of transfection.
  • 22.5mL U2OS media (p/s) + 2.5mL trypsinized cells from T-75 flask with 100% confluent cells. Incubate 6-well plate overnight.

Transfections

> U2OS Flp-in T-REx lines (puro/zeo/blast) + FRT-marked mammalian transfection vector (hygro)
--> Note: BD002 co-transfect with Gal4-VP64 (10:1); hygro resistance replaces zeo resistance after transfection
> Use Lipofectamine, 6-well format
> Plasmids:

  1. BD002 = 346.61 ng/μl
  2. Gal4-VP64 transactivation plasmids
    1. KAH59 (MV5) = 277.719
    2. KAH54 (PCVN) = 217.267
    3. KAH59 (PCVN) = 203.300
    4. KAH60 (PCVN) = 169.931
    5. KAH66 (MV5) = 174.819


Plate-Well Plasmid DNA Volume dH2O Lipo PLUS reagent Opti-MEM (total)
1-1 BD002 + KAH59(PcVN) 1.81 + 0.19 μg 5.23 + 0.89 μL 13.88μL 2.5μL 7.5 μL 570 μL
1-2 BD002 + KAH60(PcVN) " 5.23 + 1.07 μL 13.7μL " " "
1-3 BD002 + KAH54(PcVN) " 5.23 + 0.83 μL 13.94μL " " "
1-4 (-)Ctrl BD002 + KAH59(MV5) " 5.23 + 0.65 μL 14.12μL " " "
1-5 (-) Ctrl BD002 + KAH66(MV5) " 5.23+1.04 13.73μL " ""
  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. To reduce toxicity, after 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.




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