User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2013/09/05: Difference between revisions
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= | ==September 5, 2013== | ||
* '''QuickChange Site Directed Mutagenesis of V0200 to remove BSMBI cut site in the Hygro resistance sequence''' | |||
# Use 50 ng of dsDNA template [http://openwetware.org/wiki/User:Behzad_Damadzadeh/Notebook/PcTF_Subcloning_in_E-coli/2013/05/06 (BD002)] and 125 ng of each primer. | |||
# Template strands are about 7kb for BD002. | |||
#Forward primer: 0.15 mg and 32.6 nm, add 326μL dH2O to make 100μM and then diluted to 10μL concentration. | |||
#Reverse primer: 0.16mg and 34.1 nm, add 341μL dH2O to make 100μM and then diluted to 10μL concentration. | |||
#DNA template (BD002) miniprep concentration: 88ng/μL. | |||
''' | {| class="wikitable" border=1 cellpadding="5" cellspacing="0" | ||
# | |- | ||
| Reagents || BD002 | |||
|- | |||
| Plasmid DNA (50 ng) || 0.6 μL | |||
|- | |||
| primer 1 (10 μM, 125 ng) || 2.7 | |||
|- | |||
| primer 2 (10 μM, 125 ng) || 2.66 | |||
|- | |||
| 10x reaction buffer|| 5.0 | |||
|- | |||
| dNTP mix || 1.0 | |||
|- | |||
| dH<sub>2</sub>O || 38.04 | |||
|- | |||
| Total || 50.0 | |||
|} | |||
*No control reaction, I will test the accuracy of the mutagenesis with BSMBI restriction enzyme. | |||
* Add 1 μl of PfuTurbo DNA polymerase (2.5 U/μl) to the sample reaction | |||
* Thermal cycling | |||
** 95°C/ 30 sec | |||
** [95°C/ 30 sec; 55°C/ 1 min; 68°C/ '''7''' min (1 min/kb plasmid length)]x18 | |||
** 4°C, ∞ | |||
* DpnI Digest (gets rid of methylated template DNA) | |||
* 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 | |||
'''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. '' | |||
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Revision as of 08:56, 13 October 2013
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September 5, 2013
Transformation
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