User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2013/10/10: Difference between revisions

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* Run 5 μL of each PCR reaction on 10% agarose gel.
* Run 5 μL of each PCR reaction on 10% agarose gel.


[[Image:Image:BD002-SD-SAPI.png]]
[[Image:BD002-SD-SAPI.png]]

Revision as of 09:48, 13 October 2013

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10/10/12

Site Directed Mutagenesis-Removing the BSMBI cut site from BD002 reporter gene

  1. Template strands are about 7kb for BD002.
  2. Forward primer: 0.15 mg and 32.6 nm, add 326μL dH2O to make 100μM and then diluted to 10μM concentration.
  3. Reverse primer: 0.16mg and 34.1 nm, add 341μL dH2O to make 100μM and then diluted to 10μM concentration.
  4. DNA template (BD002) miniprep concentration: 116ng/μL. Accuracy of the BD002 where checked with SapI and NotI cut.
  5. Serial dilution of template to: 1. 100 ng/μL 2. 80 ng/μL 3. 10 ng/μL 4. 1 ng/μL 5. 200 pg/μL
Reagents 1 2 3 4 5
Plasmid DNA 0.2 μL 0.5 μL 0.5 μL 0.5 μL 1 μL
primer 1 (10 μM, 125 ng) 1 μL 1 μL 1 μL 1 μL 1 μL
primer 2 (10 μM, 125 ng) 1 μL 1 μL 1 μL 1 μL 1 μL
2X GOTAG (PCR Master Mix) 25μL 25μL 25μL 25μL 25μL
dH2O 22.8 μL 22.5 μL 22.5 μL 22.5 μL 22 μL
Total 50 μL 50 μL 50 μL 50 μL 50 μL
  • No control reaction, I will test the accuracy of the mutagenesis with BSMBI restriction enzyme.
  • Thermal Cycling
    • 95°C/ 30 sec
    • [95°C/ 30 sec; 55°C/ 1 min; 72°C/ 6 min (1 min/kb plasmid length)]x30 cycle
    • 4°C, ∞
  • Run 5 μL of each PCR reaction on 10% agarose gel.
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