User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2013/06/06: Difference between revisions
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==06/06/2013== | ==06/06/2013== | ||
==05/06/10== | |||
<!-- 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> | |||
Revision as of 13:34, 6 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> | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||
06/06/201305/06/10
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 PlpE = 192 ng/μl
Transgene expression should be detectable after 18 hours.
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