Team:Warsaw/Calendar-Main/29 June 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 One of the samples also contained 1 ul of DMSO    PCR programs: hly 4min 95&deg;C (30s 95&deg;C, 40s 43&deg;C, 1min30s 72&deg;C)x3 (30s 95&deg;C, 40s 45&deg;C, 1min30s 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/5/50/LLO9(failed).JPG"/>  Gel (from left)</li> </ul> <ol> GeneRuler DNA Ladder Mix #SM0333 (Fermentas)</li> sample with DMSO</li> sample without DMSO</li> no products were observed 

Cloning of the mgtc promoter into the pKSII+ plasmid Kamil

Tasks:  Purification of the PCR product</li> Plasmid assembly</li> </ul>

Methods:  The purification was carried out using Montage PCR Centrifugal Filter Device P36461 (Milipore) and according to the protocol supplied.</li> Plasmid digest mix was prepared as follows: 2ul Tango buffer (Fermentas) 1ul pKSII+ plasmid 1ul XbaI enzyme 1ul SmaI enzyme The solution was topped up with H2O to the final volume of 20 ul.</li> mgtc promoter digest mix was prepared as follows: 2ul Tango buffer (Fermentas) 2ul purified gene 1ul XbaI enzyme the solution was topped up with H2O to the final volume of 20 ul.</li> The digest was kept for 3h at 37&deg;C, and then the enzyme was inactivated for 15min. at 80&deg;C.</li>  The ligation mix was prepared as follows: 2ul digested plasmid 6ul mgtc digest product 2ul ligation buffer G (Fermentas) 2ul T4 DNA ligase (Fermentas), 2ul 30% PEG 2ul 10mM ATP The solution was topped up with H2O to the final volume of 20 ul. The ligation was carried out in 18&deg;C overnight (~12h) and then inactivated for 10min. at 65&deg;C.</li>  A 200ul batch of chemocompetent bacteria was transformed with 500ul of ligation mix and incubated on petri dishes containing LB medium supplemented with ampicilin, X-gal and IPTG.</li> </ul>

Results: Due to a technological error the bacteria used were electrocompetent rather than chemocompetent. This resulted in there being not a single colony on the petri dishes. UPDATE: After another transformation and a second negative result a gel electrophoresis was performed revealing that the plasmid has not properly ligated. Electrophoresis results: <img src=http://2009.igem.org/wiki/images/c/c0/False.jpg /> Prom left: <ol> GeneRuler DNA Ladder Mix #SM0333 (Fermentas)</li> <li>mgtc digest</li> <li>pKSII+ plasmid digest</li> <li>"ligation"</li>