Team:Warsaw/Calendar-Main/1 July 2009

Making Llo fusion with secretion signal peptide Kuba

Tasks:  Amplification of llo(hly) for fusion with the secretion signal peptide 

Methods:  PCR mixture's composition: 2,5ul pfu buffer (Fermentas) 2,5ul MgSO4 (Fermentas) 1,5ul primers 1,5ul dNTPs (10 mM) 0,5ul pfu turbo polymerase 1ul template DNA from Listeria, solution was topped up with H2O to 25ul    PCR programs: hly 4min 95&deg;C (30s 95&deg;C, 40s 40-48&deg;C, 1min30s 72&deg;C)x3 (30s 95&deg;C, 40s 44-55&deg;C, 1min30s 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/0/0a/Llo_gradient.JPG"/>  Gel (from left)</li> </ul> <ol> M - GeneRuler DNA Ladder Mix #SM0333 (Fermentas)</li> 1-5 - samples (annealing temperature increases to from the left to the right)</li> many unspecific products were obtained (product of the correct lenght marked with an arrow) 

Quality check (cro PCR product and pKS isolate) Kuba  </ul> http://2009.igem.org/wiki/images/7/75/Cro(wstawki)%2Bpks(test).JPG  Gel (from the left)</li> </ul> <ol> CRO</li> pKS isolate</li> GeneRuler DNA Ladder Mix #SM0333 (Fermentas)</li> </ol>