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Team:Warsaw/Calendar-Main/5 May 2009
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| - | <h3> | + | <h3>Cloning of hly gene into pKSII+ vector</h3> |
<h4><font color="black">Kamil</font></h4> | <h4><font color="black">Kamil</font></h4> | ||
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<ul> | <ul> | ||
<li> The BanI enzyme was selected for the test because the hly insert bears an additional digest site.</li> | <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> 2ul Orange buffer (Fermentas) | + | <li><p>Digest mixture's composition:</p> |
| - | + | <pre>2ul Orange buffer (Fermentas) | |
| - | solution | + | 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 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> | <li>The electrophoretic separation was carried out on 1% agarose gel</li> | ||
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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