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Team:Warsaw/Calendar-Stage1/14 September 2010
From 2010.igem.org
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| + | <h2>Mutagenesis of MinC</h2> | ||
<h4>Kuba</h4> | <h4>Kuba</h4> | ||
<p align="justify">Directed mutagenesis was preformed on pSB-MinC in order to remove an additional PstI site. Two primers were used: MinCmutF (TAATTGCAGTCGCGCCGC) and MinCmutR (GTCGAAAACGCTTTGACCG). PCR was run with Yellow Pfu Polimerase (Eurex) in a standard reaction mix. | <p align="justify">Directed mutagenesis was preformed on pSB-MinC in order to remove an additional PstI site. Two primers were used: MinCmutF (TAATTGCAGTCGCGCCGC) and MinCmutR (GTCGAAAACGCTTTGACCG). PCR was run with Yellow Pfu Polimerase (Eurex) in a standard reaction mix. | ||
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<li>denaturation 95 <sup>o</sup>C; 30 s</li> | <li>denaturation 95 <sup>o</sup>C; 30 s</li> | ||
<li>annealing 51 <sup>o</sup>C; 30 s</li> | <li>annealing 51 <sup>o</sup>C; 30 s</li> | ||
| - | <li>elongation 72 <sup>o</sup>C<; 7'/li> | + | <li>elongation 72 <sup>o</sup>C<; 7'</li> |
<li>x 30 cycles</li> | <li>x 30 cycles</li> | ||
<li>72 <sup>o</sup>C; 10'</li> | <li>72 <sup>o</sup>C; 10'</li> | ||
</ul> | </ul> | ||
| - | <p align="justify">PCR product was purified by agarose electrophoresis | + | <p align="justify">PCR product was digested with DpnI amd purified by agarose electrophoresis. |
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Latest revision as of 20:24, 27 October 2010
Mutagenesis of MinC
Kuba
Directed mutagenesis was preformed on pSB-MinC in order to remove an additional PstI site. Two primers were used: MinCmutF (TAATTGCAGTCGCGCCGC) and MinCmutR (GTCGAAAACGCTTTGACCG). PCR was run with Yellow Pfu Polimerase (Eurex) in a standard reaction mix.
PCR conditions were as follows:
- initial denaturation 95 oC; 3'
- denaturation 95 oC; 30 s
- annealing 51 oC; 30 s
- elongation 72 oC<; 7'
- x 30 cycles
- 72 oC; 10'
PCR product was digested with DpnI amd purified by agarose electrophoresis.
