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Team:Warsaw/Calendar-Main/5 May 2009
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| + | <h3>Cloning of hly gene into pKSII+ vector</h3> | ||
| + | <h4><font color="black">Kamil</font></h4> | ||
| + | <br /> | ||
| + | <p>Tasks:</p> | ||
| + | <ul> | ||
| + | <li>Verify the correctness of the pKS/hly constructs</li> | ||
| + | </ul> | ||
| + | <br /> | ||
| + | <p>Methods:</p> | ||
| + | <ul> | ||
| + | <li> The BanI enzyme was selected for the test because the hly insert bears an additional digest site.</li> | ||
| + | <li><p>Digest mixture's composition:</p> | ||
| + | <pre>2ul Orange buffer (Fermentas) | ||
| + | 1ul BanI enzyme | ||
| + | 5ul plasmid solution</pre> | ||
| + | The solution was topped up with H2O to 20ul. </li> | ||
| + | <li> The digest was kept at 37 °C overnight (~18h) and then inactivated at 65 °C for 10 min.</li> | ||
| + | <li>The electrophoretic separation was carried out on 1% agarose gel</li> | ||
| + | </ul> | ||
| + | <br /> | ||
| + | <p>Results:</p> | ||
| + | <aling="centre"><img src="https://static.igem.org/mediawiki/2009/0/0b/2009.05.05_-_trawienie_konstruktow_hly_opisany.jpg"/> | ||
| + | |||
| + | |||
| + | </aling> | ||
| + | <var> | ||
| + | <ul> | ||
| + | <li>Gel (from left)</li> | ||
| + | </ul> | ||
| + | <ol> | ||
| + | <li>GeneRuler DNA Ladder Mix #SM0333 (Fermentas) (3ul)</li> | ||
| + | <li>sample no. 14 (5ul)</li> | ||
| + | <li>sample no. 15 (5ul)</li> | ||
| + | <li>sample no. 16 (5ul)</li> | ||
| + | <li>sample no. 18 (5ul)</li> | ||
| + | <li>pKS plasmid (control) (5ul)</li> | ||
| + | |||
| + | |||
| + | </ol> | ||
| + | </var> | ||
| + | <br /> | ||
| + | <p>Discussion:</p> | ||
| + | <ul> | ||
| + | <li>The expected fragments size for the pKS plasmid are 142, 491, 1097 and 1171 | ||
| + | base | ||
| + | pairs.</li> | ||
| + | <li>The expected fragments size for the insert containing plasmid are 142, 535, 1097, 1171 and 1538 | ||
| + | |||
| + | base pairs.</li> | ||
| + | <li>The samples no. 14, 15 and 16 were supposed to conatin the hly insert.</li> | ||
| + | <li>The sample no. 18 did not contain the hly insert and was used as a second control.</li> | ||
| + | </ul> | ||
| + | <br /> | ||
</html> | </html> | ||
Latest revision as of 15:10, 17 September 2009
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Cloning of hly gene into pKSII+ vector
Kamil
Tasks:
- Verify the correctness of the pKS/hly constructs
Methods:
- The BanI enzyme was selected for the test because the hly insert bears an additional digest site.
Digest mixture's composition:
2ul Orange buffer (Fermentas) 1ul BanI enzyme 5ul plasmid solution
The solution was topped up with H2O to 20ul.- The digest was kept at 37 °C overnight (~18h) and then inactivated at 65 °C for 10 min.
- The electrophoretic separation was carried out on 1% agarose gel
Results:
- Gel (from left)
- GeneRuler DNA Ladder Mix #SM0333 (Fermentas) (3ul)
- sample no. 14 (5ul)
- sample no. 15 (5ul)
- sample no. 16 (5ul)
- sample no. 18 (5ul)
- pKS plasmid (control) (5ul)
Discussion:
- The expected fragments size for the pKS plasmid are 142, 491, 1097 and 1171 base pairs.
- The expected fragments size for the insert containing plasmid are 142, 535, 1097, 1171 and 1538 base pairs.
- The samples no. 14, 15 and 16 were supposed to conatin the hly insert.
- The sample no. 18 did not contain the hly insert and was used as a second control.
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