Team:Warsaw/Calendar-Main/15 September 2009

Cloning of the mgtc promoter into the pSB1A3 plasmid Kamil

Tasks:  PCR product digest 

Methods:  The digest mix was prepared as follows: 40&mu;l plasmid isolation 5&mu;l Buffer O (Fermentas) 1&mu;l EcoRI enzyme 1&mu;l PstI enzyme 3&mu;l H2O  The digest was carried out in 37&deg;C overnight and inactivated for 15 min. in 80&deg;C. The product was later purified using the Montage PCR Centrifugal Filter Device. 

Cloning of the cro-box into the pSB1A3 plasmid Kamil

Tasks:  Creation of the cro-box double stranded DNA Cro-box digest</li> </ul>

Methods:  The mixture of equal volumes (10&mu;l) of both ssDNA was heated to 94&deg;C for 15min. and left to cool. </li> The digest mix was prepared as follows: 20&mu;l plasmid isolation 3&mu;l Tango Buffer (Fermentas) 1&mu;l XbaI enzyme 1&mu;l PstI enzyme 5&mu;l H2O </li> The digest was carried out in 37&deg;C overnight and inactivated for 15 min. in 80&deg;C.</li> The product was later purified with dialysis.</li> </ul>

Assembly of fusion proteins Marcin

Task 1: Dialysis of PCR-amplified COX signal peptide

Comment:

Typical purification methods such as usage of gel-out are useless in the case of small fragments of DNA. It is the reason why I chose dialysis to purify the PCR product.

Methods:
 * Dialysis were perform one hour
 * After dialysis samples were elecrophoretically separated to investigate the identity and amount of DNA.

Results:

Task 2: Prepare the backbone plasmid pSB1K3 and COX mitochondrial signal to ligate them

20 μl purified plasmid DNA product (or PCR-amplified signal peptide) 1 μl PstI (Fermentas) 1 μl SpeI (Fermentas) 5 μl Buffer Tango (Fermentas) 23 μl MQ water
 * Reaction mixture composition:
 * Digestion was carry out 7 hours

Results:

Kanamycine-resistant plasmid must have been mistaken with some other construct because no biobrick is cut and the length of the plasmid is longer than expected. I decided to use pSB1A3.

Task 3: Ligation of mitochondrial signal peptide with pSB1A3

8 μl purified plasmid DNA product (or PCR-amplified signal peptide) 10 μl purified PCR product 2.5 μl dNTPs mixture (EurX, concentration 5 mM) 2.5 μl Buffer Tango (Fermentas)
 * Reaction mixture composition:
 * Ligation was carry out 15 hours