Team:Warsaw/Calendar-Main/12 July 2009
From 2009.igem.org
(Difference between revisions)
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<h3>Insertion of the pho gene into the pKSII+ plasmid</h3> | <h3>Insertion of the pho gene into the pKSII+ plasmid</h3> | ||
<h4>Kama</h4> | <h4>Kama</h4> | ||
+ | <ul> | ||
+ | <li>Plasmid digest mix was prepared as follows: | ||
+ | <pre>2μl Tango buffer (Fermentas) | ||
+ | 1μl pKSII plasmid | ||
+ | 1μl XbaI enzyme | ||
+ | 1μl SmaI enzyme</pre> | ||
+ | the solution was topped up with H2O to the final volume of 20 μl.</li> | ||
- | <li> | + | <li>Pho gene digest mix was prepared as follows: |
+ | <pre>2μl Tango buffer (Fermentas) | ||
+ | 3μl purified gene | ||
+ | 1μl XbaI enzyme</pre> | ||
+ | the solution was topped up with H2O to the final volume of 20 μl.</li> | ||
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<li>The digest was kept for 3h at 37°C, and then the enzymes were inactivated for 20min. at 80°C.</li> | <li>The digest was kept for 3h at 37°C, and then the enzymes were inactivated for 20min. at 80°C.</li> | ||
- | <li><p>The ligation mix was prepared as follows: 20μl plasmid | + | <li><p>The ligation mix was prepared as follows: |
- | + | <pre>20μl plasmid | |
- | (Fermentas) | + | 20μl gene |
+ | 5μl ligation buffer G (Fermentas) | ||
+ | 2μl T4 DNA ligase (Fermentas)</pre> | ||
+ | the solution was topped up with H2O to the final volume of 50 μl. The ligation was carried out in 18°C overnight | ||
- | (~15h)</li> | + | (~15h)</li></ul> |
</html> | </html> | ||
Latest revision as of 21:21, 19 September 2009
Insertion of the pho gene into the pKSII+ plasmid
Kama
- Plasmid digest mix was prepared as follows:
2μl Tango buffer (Fermentas) 1μl pKSII plasmid 1μl XbaI enzyme 1μl SmaI enzyme
the solution was topped up with H2O to the final volume of 20 μl. - Pho gene digest mix was prepared as follows:
2μl Tango buffer (Fermentas) 3μl purified gene 1μl XbaI enzyme
the solution was topped up with H2O to the final volume of 20 μl. - The digest was kept for 3h at 37°C, and then the enzymes were inactivated for 20min. at 80°C.
The ligation mix was prepared as follows:
20μl plasmid 20μl gene 5μl ligation buffer G (Fermentas) 2μl T4 DNA ligase (Fermentas)
the solution was topped up with H2O to the final volume of 50 μl. The ligation was carried out in 18°C overnight (~15h)
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