Team:Warsaw/Calendar-Main/21 September 2009

Assembly of endosome detection operon Marcin Task 1: Isolation of pSB1A3 plasmid containing BBa_K177044

Methods:
 * Plasmids were isolated using the A&A plasmid mini kit. Detailed procedure of the isolation is described here

Task 2: Restriction digest of previously isolated plasmids:

Methods: 3 μl purified plasmid DNA product 1 μl XbaI (Fermentas) 1 μl PstI (Fermentas) 2 μl Buffer Tango (Fermentas) 13 μl MQ water
 * Reaction mixture composition:
 * Reactions were carried out 2 hour

Results:

Some sequences 1500 bp length are visible on the gel but I need to determine whether it is expected product or contamination (bacteria containing plasmid with listeriolysin were also presented on the same plate)

Task 3: PCR reaction to determine the identity of isolated (Task 1) DNA

Commment:

I decided to perform PCR reaction using primers for listeriolysin. If the plasmids contain BBa_K177044 there will be no PCR product.

Methods: 0.1 μl purified plasmid DNA product 1.4 μl Opti Taq Polymerase (EURx) 1 μl dNTPs (EURx) 2 μl Opti Taq Buffer (Fermentas) 15.5 μl MQ water
 * Reaction mixture composition:

Results:

All isolated plasmid contain listeriolysin. It is necessary to carry out ligation one more time

Task 4: Construct to transform: Methods:
 * Transformation of chemocompetent E. coli TOP10 strain
 * BBa_K177044
 * Ligation mixture was thermally inactivated in 80 &deg;C for 20 minutes
 * Detailed protocol of transformation is described here

Cloning of the mgtc promoter into the pSB1A3 plasmid Kamil

Tasks:  Selection of the colonies 

Methods:  The appropriate colonies were transferred to a fresh agarose plate and liquid cultures were established. The plate and the liquid cultures were incubated overnight in 37&deg;C 

Cloning of the cro-box into the pSB1A3 plasmid Kamil

Tasks:  Selection of the colonies 

Methods:  The appropriate colonies were transferred to a fresh agarose plate and liquid cultures were established.</li> The plate and the liquid cultures were incubated overnight in 37&deg;C</li> </ul>