Team:Warsaw/Calendar-Main/21 April 2009

Cloning of hly gene into pKSII+ vector Kamil

Tasks:  Amplification of hly   Measurement of concentration of isolates from Yersinia and Listeria 

Methods:   PCR mixture's composition: 2ul pfu buffer (Fermentas) 1ul MgSO4 (Fermentas) 0,5ul primers 1,5ul dNTPs (10 mM, 01,ul pfu polymerase (Fermentas) 1ul template DNA from Listeria  Solution was topped up with H2O to 20ul. 	PCR program:	 hly 300s 95&deg;C (30s 95&deg;C, 35s 42&deg;C, 150s 72&deg;C)x2 (30s 95&deg;C, 35s 47&deg;C, 150s 72&deg;C)x28 600s 72&deg;C ~ 4&deg;C   Electrophoretic separation on 1% agarose gel</li> Measurement of concentration of isolates on NanoDrop</li> </ul> Results: <img src="http://2009.igem.org/wiki/images/7/76/2009.04.21_-_PCR_hly_opisany.jpg"/>  Gel (from left)</li> </ul> <ol>	GeneRuler DNA Ladder Mix #SM0333 (Fermentas)</li>	hly</li>	hly control -</li> </ol>  Measurement from NanoDrop:	+ Yersinia - 29,6 ng/ul	+ Listeria - 50,7 ng/ul</li> </ul> Conclusions:  200ng of DNA should be used on sample</li>	Time of anealing should be prolonged to 45s</li>	Concentration of Mg should be increased</li> </ul>