Team:Warsaw/Calendar-Main/5 May 2009

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)</li> </ul> <ol> GeneRuler DNA Ladder Mix #SM0333 (Fermentas) (3ul)</li> sample no. 14 (5ul)</li> sample no. 15 (5ul)</li> sample no. 16 (5ul)</li> sample no. 18 (5ul)</li> pKS plasmid (control) (5ul)</li>

</ol>

Discussion:  The expected fragments size for the pKS plasmid are 142, 491, 1097 and 1171 base pairs.</li> The expected fragments size for the insert containing plasmid are 142, 535, 1097, 1171 and 1538

base pairs.</li> The samples no. 14, 15 and 16 were supposed to conatin the hly insert.</li> The sample no. 18 did not contain the hly insert and was used as a second control.</li> </ul>