Team:Warsaw/Calendar-Main/3 August 2009

Cloning of p53 coding sequence Marcin

Task 1:  Isolation  pSB1A3 plasmid containing  BBa_E0010   Methods:  Plasmids were isolated using the A&A plasmid mini kit. Detailed procedure of the isolation is described here. Task 2:  Clean-up the PCR product - amplified p53 CDS</li> </ul> Methods:  DNA was purified using the A&A clean-up kit. Detailed procedure is described here</a></li></ul> Task 3:  Restriction digest of insert and vector sequence.</li> </ul> Comment I decide that p53 coding sequence will be cloned to plasmid which is compatible with the Biobrick standard. Used plasmid contains CDS of RFP. After digest with SpeI and XbaI the sequence will be cut and the plasmid may be ligated with p53 which also will be digested using aforementioned enzymes. However the maximum effectiveness of the ligation is 50% because of the possibility of cloning the insert in opposite direction. Methods:  Digest for subsequent cloning using XbaI and SpeI</li> Reaction mixture composition: 25 &mu;l purified PCR product (or, 10 &mu;l plasmid), 1 &mu;l XbaI (Fermentas), 1 &mu;l SpeI (Fermentas), 5 &mu;l Buffer Tango (Fermentas), required &mu;l MQ water to reach final volume of 50 &mu;l</li></ul> Both reaction were perform in the same condition:</li> </ul> Program of digest: 1. 37&deg;C - 6 hours 2. 80&deg;C - 15 minutes 3. 4&deg;C - hold  Purification of digested products via gel-out</li> </ul> Procedure:  Fragments of agarose gel were carefully cut out and in the next step DNA was extracted from the gel using the A&A gel-out kit. Detailed procedure is described here</a></li> Quantification of amount of p53 DNA after restriction digest:</li> </ul> 1 &mu;l of the digest mixture was diluted to 10 &mu;l and loaded into the gel. Results: <img src="http://2009.igem.org/wiki/images/d/dc/P53_pSB_gel_out_03_08_09.png" height="60%"> <font face="Times New Roman" size="3"> verification of the restriction patterns Task 4: <ul> <li>Cloning p53 coding sequence to the <a href="http://partsregistry.org/Part:pSB1A3"> pSB1A3</a> vector</li> </ul> Methods: <ul> <li> Ligation mixture composition</li>: 11 &mu;l digested p53 11 &mu;l digested pSB1A3 2.5 &mu;l ligation buffer (Fermentas) 1 &mu;l ligase T4 (Fermentas) <li>Duration of ligation was about 12 hours</li> </ul>

Invasine amplification and cloning Justyna

Task: <ul> <li> Amplification of invasine by PCR and cloning into pKS plasmid, for further preparation of biobrick. </li> </ul> Comment: No positive results, it seems primers don't bind specifically to genomic DNA, as only non-specific products (or none) appear.