User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2013/06/06: Difference between revisions
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<!-- Precede each finished task with a checkmark ✓ --> | |||
* ✓ Minipreps: FlpE (4) | |||
---- | |||
'''Minipreps'''<br> | |||
> Check with E, P, and E/P digests | |||
{| {{table}} cellspacing="7" width=700px | |||
|-valign="top" | |||
| <u>Reagent</u> || <u>1,4,7,10</u> || <u>2,5,8,11</u> || <u>3,6,9,12</u> | |||
| rowspan="7" | <u>Expected:</u><br>1. FlpE, E = 7160, 546<br>2. FlpE, P = 7706<br>3.FlpE, E/P=5649, 1511, 546 | |||
| rowspan="7" | [[Image:Flp-E.jpg|300px|E/P digests 05/06/10]]<br>15 μL/lane; 1% agarose | |||
|- | |||
| DNA(plasmid) || 2.0 || 2.0 || 2.0 | |||
|- | |||
| 10X buffer || 1.5 || 1.5 || 1.5 | |||
|- | |||
| EcoRI || 1.0 || --- || 1.0 | |||
|- | |||
| PstI || --- || 1.0 || 1.0 | |||
|- | |||
| dH<sub>2</sub>O || 10.5 || 10.5 || 9.5 | |||
|- | |||
| <br> <br> <br> || | |||
|} | |||
15 μL --> 37°C/ ~15 min. | |||
--> Conclusion: Success! Combine 4 preps (300μL total) for DNA clean-up (tomorrow)<br> | |||
--> Streak clone (saved toothpick) onto 100 μg/mL Amp plate.<br> | |||
<!-- Precede each finished task with a checkmark ✓ --> | <!-- Precede each finished task with a checkmark ✓ --> | ||
* ✓ Transfections: | * ✓ Transfections: H3K27me3 reporters into cell lines BD002 (KAH201+KAH182+V0200) | ||
* ✓ Polycomb-ATF negative control lines: colony plates | * ✓ Polycomb-ATF negative control lines: colony plates | ||
---- | ---- | ||
'''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. | |||
'''Transfections'''<br> | '''Transfections'''<br> | ||
> U2OS Flp-in T-REx lines (puro/zeo/blast) + | |||
> 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> | --> Note: co-transfect with FlpE (1:3); hygro resistance replaces zeo resistance after "Flp" in<br> | ||
> Use Lipofectamine, 6-well format<br> | > Use Lipofectamine, 6-well format<br> | ||
> Plates: | > Plates: | ||
# | # BD002 (3:1) Flp-in T-REx | ||
# | #BD002 (4:1) Flp-in T-REx | ||
BD002 (3:1) = 339 ng/μl | |||
BD002 (4:1) = 317 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 || 4.42 + 2.60 μL || 12.98μL|| 2.5μL|| 7.5 μL || 570 μL | ||
|- | |- | ||
| | | 1-2 || BD002 (4:1) + FlpE || " || 4.73 + 2.60 μL || 12.67μL|| " || " || " | ||
|- | |- | ||
| | | 1-3 (-) Ctrl || BD002 (3:1) + --- || 2 μg || 4.42 + --- μL || 15.58μL || " || " || " | ||
|- | |- | ||
| | | 1-4 (-)Ctrl || --- + --- || 0 μg || --- + --- μL || 20.0μL|| " || " || " | ||
|- | |- | ||
| - | |1-5 || KAH187 || 20 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. | |||
---- | |||
<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### --> | <!-- ##### DO NOT edit below this line unless you know what you are doing. ##### --> | ||
__NOTOC__ | __NOTOC__ |
Revision as of 13:39, 19 June 2013
PcTF Subcloning in E. coli | <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page <html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> | ||||||||||||||||||||||||||||||||||||||||||||||||
06/06/2013
Minipreps 15 μL --> 37°C/ ~15 min.
1 day before transfection:
Transfections > U2OS Flp-in T-REx lines (puro/zeo/blast) + FRT-marked mammalian transfection vector (hygro)
BD002 (3:1) = 339 ng/μl BD002 (4:1) = 317 ng/μl KAH187 = 441.5 ng/μl FlpE = 192 ng/μl
Transgene expression should be detectable after 18 hours.
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