Team:Warsaw/Calendar-Main/24 April 2009

PCR inv Kamil

Tasks:  Amplification of inv 

Methods:  PCR mixture's composition: 2,5ul pfu buffer (Fermentas) 2,5ul MgSO4 (Fermentas) 1,5ul primers 1ul dNTPs (10 mM) 0,5ul pfu turbo polymerase (od Antka) 1ul template DNA from Yersinia Solution was topped up with H2O to 25ul.    PCR program: inv 4min 95&deg;C (30s 95&deg;C, 1min 53&deg;C, 4min15s 72&deg;C)x3 (30s 95&deg;C, 1min 58&deg;C, 4min15s 72&deg;C)x28 10min 72&deg;C ~ 7&deg;C   Electrophoretic separation on 1% agarose gel 

Results: <img src="http://2009.igem.org/wiki/images/f/fa/2009.04.24_-_PCR_inwazyna_%2B_ligacja_opisany.jpg"/>  Gel (from left)</li> </ul> <ol> GeneRuler DNA Ladder Mix #SM0333 (Fermentas)</li> inv</li> inv (template diluted 10x)</li> control -</li> vector</li> </ol>

Nonspecific products

Cloning of hly gene into pKSII+ vector

Marcin

Task:

 Transformation of electrocompetent E. coli strain TOP10 with pKS plasmid containing hemolysin coding sequence</li> </ul> Procedure: <ol> Ligation mixture (9 &mu;l) was dialise 30 minut</li> Half of the volume of the mixture was added to thawed bacteria</li> Bacteria were transfered to the cuvette and electroporated (2500V, 5 sec)</li> After the electroporation 1 ml of SOB medium was added to the cuvette</li> Mixture with bacteria was transfered to the Eppendorf probe and it was incubated for 45 minut in 37 &deg;C</li> Mixture was divided to 2 equal portion which were plated on medium containing ampicillin, X-Gal and IPTG</li> </ol>