User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2013/10/10: Difference between revisions
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''' | '''Site Directed Mutagenesis-Removing the BSMBI cut site from BD002 reporter gene''' | ||
# | |||
# 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 10μM concentration. | |||
# Reverse primer: 0.16mg and 34.1 nm, add 341μL dH2O to make 100μM and then diluted to 10μM concentration. | |||
# DNA template (BD002) miniprep concentration: 116ng/μL. Accuracy of the BD002 where checked with SapI and NotI cut. | |||
# 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 | |||
{| class="wikitable" border=1 cellpadding="5" cellspacing="0" | |||
|- | |||
| 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 | |||
|- | |||
| dH<sub>2</sub>O || 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, ∞ |
Revision as of 09:25, 13 October 2013
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10/10/12Site Directed Mutagenesis-Removing the BSMBI cut site from BD002 reporter gene
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