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Team:Warsaw/Calendar-Main/28 July 2009
From 2009.igem.org
(Difference between revisions)
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<p>Methods:</p> | <p>Methods:</p> | ||
<ul> | <ul> | ||
| - | <li>The plasmid digest mix was prepared as follows: 4μl purified plasmid | + | <li>The plasmid digest mix was prepared as follows: |
| - | <li>The mgtc promoter digest mix was prepared as follows: 10μl purified promoter | + | <pre>4μl purified plasmid |
| + | 2μl Tango buffer (Fermentas) | ||
| + | 1μl SpeI enzyme | ||
| + | 1μl XbaI enzyme</pre> | ||
| + | The solution was topped up with H2O to the final volume of 20μl. </li> | ||
| + | <li>The mgtc promoter digest mix was prepared as follows: | ||
| + | <pre>10μl purified promoter | ||
| + | 2μl Tango buffer (Fermentas) | ||
| + | 1μl SpeI enzyme | ||
| + | 1μl XbaI enzyme</pre> | ||
| + | The solution was topped up with H2O to the final volume of 20μl. </li> | ||
<li>The digest was carried out in 37°C for 3h. and then inactivated in 80°C for 20min.</li> | <li>The digest was carried out in 37°C for 3h. and then inactivated in 80°C for 20min.</li> | ||
<li>The digested plasmid was separated on 1% agarose gel.</li> | <li>The digested plasmid was separated on 1% agarose gel.</li> | ||
<li>The plasmid backbone was extracted from agarose gel using the GelOut kit (A&A Biotechnology) accrding to the manufacturers protocol (in the total volume of 50μl).</li> | <li>The plasmid backbone was extracted from agarose gel using the GelOut kit (A&A Biotechnology) accrding to the manufacturers protocol (in the total volume of 50μl).</li> | ||
| - | <li>The ligation mix was prepared as follows: 50μl purified backbone | + | <li>The ligation mix was prepared as follows: |
| + | <pre>50μl purified backbone | ||
| + | 20μl digested promoter | ||
| + | 8μl T4 ligase buffer (Fermentas) | ||
| + | 2μl T4 ligase (Fermentas)</pre></li> | ||
<li>The ligation was carried out in 18°C overnight.</li> | <li>The ligation was carried out in 18°C overnight.</li> | ||
Latest revision as of 16:26, 26 September 2009
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Cloning of the mgtc promoter into the pSB1A3 plasmid
Kamil
Tasks:
- Plasmid assembly
Methods:
- The plasmid digest mix was prepared as follows:
4μl purified plasmid 2μl Tango buffer (Fermentas) 1μl SpeI enzyme 1μl XbaI enzyme
The solution was topped up with H2O to the final volume of 20μl. - The mgtc promoter digest mix was prepared as follows:
10μl purified promoter 2μl Tango buffer (Fermentas) 1μl SpeI enzyme 1μl XbaI enzyme
The solution was topped up with H2O to the final volume of 20μl. - The digest was carried out in 37°C for 3h. and then inactivated in 80°C for 20min.
- The digested plasmid was separated on 1% agarose gel.
- The plasmid backbone was extracted from agarose gel using the GelOut kit (A&A Biotechnology) accrding to the manufacturers protocol (in the total volume of 50μl).
- The ligation mix was prepared as follows:
50μl purified backbone 20μl digested promoter 8μl T4 ligase buffer (Fermentas) 2μl T4 ligase (Fermentas)
- The ligation was carried out in 18°C overnight.
Construction of RBS.3-listeriolysin
Franek/Ania
Tasks:
- Transform chemocompetent cells with the ligation mixture
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