Team:Warsaw/Calendar-Main/24 July 2009

Cloning of the mgtc promoter into the pKSII+ plasmid Kamil

Tasks:  Plasmid purification Control digest 

Methods:  Despite the fact that all transferred colonies turned more or less blue the purification was carried out using the Plasmid Mini kit (A&A Biotechnology) according to the manufacturers protocol. The digest mix was prepared as follows: 10&mu;l of purified plasmid 2&mu;l Tango buffer (Fermentas) 1&mu;l SpeI enzyme 1&mu;l XbaI enzyme The solution was topped up with H2O to the final volume of 20&mu;l. The digest was incubated for 3h at 37&deg;C. The results were visualised with gel electrophoresis on 1% agarose gel. 

Results:  Gel Electrophoresis:</li> </ul> <img src=http://2009.igem.org/wiki/images/5/57/2009.07.24.jpg /> From left: <ol> GeneRuler DNA Ladder Mix #SM0333 (Fermentas)</li> #1 Digest</li> #2 Digest</li> #3 Digest</li> #4 Digest</li> #5 Digest</li> </ol> NOTE: All of the samples consisted of just a linear plasmid.

Conclusions:  A colony is blue for a reason.</li> </ul>

Construction of <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K177012 ">K177012</a>  operon1_part2

Ania Tasks:

 Set up 20 liquid cultires, plasmid isolation, EcorI/PstI digest. </a> </li></ul>

Assembly of endosomal detection operon Marcin Task 1:  Isolate the plasmids from bacterial cultures and verify the effectivity of the isolation via gel electrophoresis</li> Methods: Plasmids were isolated using the A&A plasmid mini kit. Detailed procedure of the isolation is described <a href="http://aabiot.com/products/dna_purification/plasmid_dna/plasmid_mini/protocol_plasmid_mini.pdf" here>here</a></li> After the isolation 1 ul of each plasmid solution was diluted to 10 ul and loaded into the 1% agarose gel</li> Electrophoresis condition:</li></ul>

voltage - 70V time - 30 min

Task 2:  <li>Digest of isolate plasmids with ligated biobricks to verify the success of ligation </li> </ul> Methods: <ul> <li>Digest of isolate plasmids using EcoRI and PstI</li> <ul><li>Reaction mixture composition: <pre<0.5 &mu;l purified plasmid DNA product 0.5 &mu;l EcoRI (Fermentas) 0.5 &mu;l PstI (Fermentas) 2 &mu;l Buffer Tango (Fermentas) 16.5 &mu;l MQ water </li></ul> <li>The reaction was performed three hours and it was subsequently inactivated via heating in 80&deg;C for 20 minutes.</li> <li>In the next step reaction mixtures were loaded into the agarose gel to analize restriction pattern of the plasmids</li> Comment: The procedure of digest was incorrect due to use Tango Buffer - EcoRI need specific buffer and it may do not work correctly in this buffer. It is obligated to repeat the procedure using modified set on restriction enzymes.

Preparation of the electrocompetent bacteria Marcin Task 1: <ul> <li>Preparation of electrocompetent E.coli strain TOP10</li> Methods: <ul> <li>detailed ptotocol of preparation</li><ul> <ol><li>Prepare new bacterial culture - add 10 ml of previously prepared bacterial inoculum to 1 l of LB liquid medium.</li> <li>Incubate the culture in 37&deg;C until the absorbance of the bacterial solution will be at least 0.5 <li>Cooled tne bacteria on ice for at least 25 minutes.</li> <li>Centrifuge the culture for 15 minutes at 4000g for 4&deg;C minutes. The temperarure must be below 5&deg;C.</li> <li>Carefully remove the supernatant and resuspend the bacteria in 1 l of cold water.</li> <li>Centrifuge the bacteria in the same manner as previously.</li> <li>Resuspend the bacteria in 0.5 l of cold water.</li> <li>Repeated the centrifuge procedure.</li <li>Resuspend carefully last formed pellet in 10% solution of the glycerole</li> <li>Centrifuge the culture for 15 minutes at 4000g for 4&deg;C minutes. The temperarure must be below 5&deg;C</li> <li>Resuspent the bacterial cells in 3 ml of 10% solution of the glycerole and prepare the aliquots of the bacteria which all contains about 200 &mu;l of the solution</li> <li>Freeze the bacteria in liquid nitrogen.</li></ol>