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

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=Date=
'''08/08/2013'''


'''List title'''
= Gal4-VP64, Cyan Transactivation =
# List items
 
 
'''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 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: co-transfect with FlpE (1:3); hygro resistance replaces zeo resistance after "Flp" in
> Use Lipofectamine, 6-well format
> Plates:

  1. 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

Plate-Well Plasmid DNA Volume dH2O Lipo PLUS reagent Opti-MEM (total)
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 " " "
  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.



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