User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2013/08/08: Difference between revisions
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'''08/08/2013''' | |||
''' | = Gal4-VP64, Cyan Transactivation = | ||
# | |||
'''1 day before transfection:'''<br> | |||
# Seed cultures in 6-well plates at ~2.5x10<suP>5</sup> so that cells will be ~5.0x10<sup>5</sup> 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'''<br> | |||
> U2OS Flp-in T-REx lines (puro/zeo/blast) + FRT-marked mammalian transfection vector (hygro)<br> | |||
--> Note: co-transfect with FlpE (1:3); hygro resistance replaces zeo resistance after "Flp" in<br> | |||
> Use Lipofectamine, 6-well format<br> | |||
> Plates: | |||
#BD002 (4:1) Flp-in T-REx | |||
BD002 (3:1) = 183.42 ng/μl | |||
BD002 (3:1) = 317.00 ng/μl | |||
BD002 (4:1) = 346.61 ng/μl | |||
KAH187 = 441.5 ng/μl | |||
FlpE = 192 ng/μl | |||
{| {{table}} cellspacing="7" width=700px | |||
|- | |||
| Plate-Well || Plasmid || DNA || Volume|| dH2O|| Lipo || PLUS reagent || <u>Opti-MEM (total)</u> | |||
|- | |||
| 1-1 || BD002 (3:1) + FlpE || 1.5 + 0.5 μg || 8.187 + 2.60 μL || 9.22μL|| 2.5μL|| 7.5 μL || 570 μL | |||
|- | |||
| 1-2 || BD002 (4:1) + FlpE || " || 4.32 + 2.60 μL || 13.08μL|| " || " || " | |||
|- | |||
| 1-3 || BD002 (4:1) + FlpE || 1.5 + 0.5 μg || 4.73 + 2.6 μL || 12.67μL || " || " || " | |||
|- | |||
| 1-4 (-)Ctrl || BD002 (3:1) + --- || 2 μg || 8.18 + --- μL || 11.82μL|| " || " || " | |||
|- | |||
|1-5 (-) Ctrl || --- || --- || --- || 20μL|| " || "||" | |||
|- | |||
|1-6 || KAH187 || 2 μg || 4.53 || 15.46μL|| " || "||" | |||
|} | |||
# Prepare DNA-Lipofectamine complexes (600 μl per sample) as follows: | |||
## Label sterile microfuge (1.5 ml) tubes. | |||
## Add '''570 μL Opti-MEM''' to each 20 μL DNA sample. | |||
## Add '''2.5 μL PLUS reagent''' to each DNA+Opti-MEM sample. Incubate for 5 minutes at room temperature. | |||
## Add '''7.5 μL Lipofectamine LTX''' to each DNA+Opti-MEM+PLUS reagent sample. Incubate for 30 minutes at room temperature. | |||
# 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 | |||
# (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. | |||
---- | |||
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__NOTOC__ |
Revision as of 22:37, 8 August 2013
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08/08/2013 Gal4-VP64, Cyan Transactivation1 day before transfection:
Transfections > U2OS Flp-in T-REx lines (puro/zeo/blast) + FRT-marked mammalian transfection vector (hygro)
BD002 (3:1) = 183.42 ng/μl BD002 (3:1) = 317.00 ng/μl BD002 (4:1) = 346.61 ng/μl KAH187 = 441.5 ng/μl FlpE = 192 ng/μl
Transgene expression should be detectable after 18 hours.
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