User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2013/09/05: Difference between revisions
From OpenWetWare
Line 43: | Line 43: | ||
* Add 1 μL DpnI enzyme to the reaction. | * Add 1 μL DpnI enzyme to the reaction. | ||
* Gently and thoroughly mix the reaction, spin down in a microcentrifuge for 1 minute, and immediately incubate at 37°C for 1 hour to digest the parental supercoiled dsDNA | * Gently and thoroughly mix the reaction, spin down in a microcentrifuge for 1 minute, and immediately incubate at 37°C for 1 hour to digest the parental supercoiled dsDNA | ||
* Transform | |||
'''Transformation''' | |||
* Transform 10μL of Dpn I treated DNA ftom the reaction tube to the 50μL aliquot of XL1-Blue supercompetent cells (Comes with the QuikChange Kit) '''No colony grow''' | |||
* Transform 40μL of Dpn I-treated DNA from the reaction tube to the 50μL aliquot of DH5α-T. '''One colony grow''' | |||
*Transform 1μL of control plasmid from the kit to 50μL of XL1-Blue supercompetent cells. '''100s colonies grow''' | |||
*''Do the traditional transformation process: 10minutes on ice, 45 seconds at 42°C, 1 minute on ice, add SOC and incubate 45 minutes at 37°C, Spin down the cells and resuspend the pallet with 100μL LB AMP broth, spread on AMP agar plate and incubate for 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 11:38, 9 September 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> | ||||||||||||||||
September 5, 2013
Transformation
|