Team:Warsaw/Calendar-Main/22 April 2009

Cloning of hly gene into pKSII+ vector Kama Tasks:  Amplification of hly 

Methods:  PCR mixture's composition: 2,5&mu;l pfu buffer (Fermentas) 2,5&mu;l MgSO4 (Fermentas) 1,5&mu;l primers 1,5&mu;l dNTPs (10 mM) 0,5&mu;l pfu turbo polymerase (od Antka, ale KNGiE też powinna działać) 1&mu;l template DNA from Listeria Solution was topped up with H2O to 25&mu;l. 1 repeat of every sample was made (2 different programs). Additionally for each sample was made version with 10&lowast; dilution of template.    PCR programs: hly 4min 95&deg;C (30s 95&deg;C, 35s 42&deg;C, 1min20s 72&deg;C)x3 (30s 95&deg;C, 35s 47&deg;C, 1min20s 72&deg;C)x28 10min 72&deg;C ~ 7&deg;C random program   Electrophoretic separation on 1% agarose gel 

Results: <img src="http://2009.igem.org/wiki/images/5/5e/2009.04.22_-_PCR_hly_opisany.jpg"/>  Gel (from left)</li> </ul> <ol> GeneRuler DNA Ladder Mix #SM0333 (Fermentas)</li> hly control -</li> hly</li> hly (10x diluted template)</li> hly control -*</li> hly*</li> hly (10x diluted template)*</li> </ol> * random program hly was succesfully amplified (termocykler z gradientem w pokoju 145)