Team:Warsaw/Calendar-Main/7 September 2009

Acquiring the internaline A gene from Listeria monocytogenes st. EDG-e genome
Jarek

Tasks:
 * Electrophoretical segregation of PCR products from previous day in 0,8% agarosis gel (sample containing 10 microL was used).


 * Purifing DNA from agarose block with A&A Gel-Out kit.

Results:
 * Finally I have the correct PCR product (at least I hope it's that one).

Assembly of fusion proteins Marcin

Task 1: Methods: 15 &mu;l purified plasmid DNA product 0.5 &mu;l SmaI (Fermentas) 2 &mu;l Buffer Tango (Fermentas) 12.5 &mu;l MQ water
 * Digest pKSII cloning vector using SmaI:
 * Reaction mixture composition:
 * Digestion was performed 6 hours and in next step enzyme was inactivated in 65 &deg;C for 20 minutes

Task 2: Methods: 15 &mu;l purified plasmid DNA product 2 &mu;l PNK Buffer (NEB) 0.5 &mu;l PNK (NEB) 2.5 &mu;l dNTPs mixture (EurX, concentration 5 mM)
 * Phosphorylation of previously purified sequence of mitochondrial signal peptide from Cox1 :
 * Reaction mixture composition:
 * Reaction was performed 45 minutes and subsequently inactivate via heating (65 &deg;C)

Task 3: Ligate of mitochondrial signal peptide to pKSII vector 10 μl phosphorylated insert 8 μl digested vector 2.5 μl Tango buffer(Fermentas) 2.5 μl dNTPs mixture (EurX, concentration 5 mM) 1 μl ligase T4 (Fermentas) Cloning of the mgtc promoter into the pSB1A3 plasmid Kamil
 * Methods:
 * Ligation mixtures composition:
 * Ligation was carry out about 15 hours

Tasks:  Ligation Bacteria transformation 

Methods:  The ligation mix was prepared as follows: 20&mu;l purified plasmid backbone 10&mu;l purified PCR product 4&mu;l Ligase buffer (Fermentas) 1&mu;l T4 Ligase (Fermentas) 5&mu;l H2O  The ligation was carried out in 18&deg;C for 3h and then inactivated in 80&deg;C for 15 min. A fresh batch of chemocompetent bacteria was transformed with the ligation mix and incubated on agarose plates containing double dose of ampicilin. 

Cloning of the cro-box into the pSB1A3 plasmid Kamil

Tasks:  Ligation to the plasmid</li> Bacteria transformation</li> </ul>

Methods:  The ligation mix was prepared as follows: 10&mu;l purified plasmid backbone 10&mu;l purified PCR product 3&mu;l Ligase buffer (Fermentas) 1&mu;l T4 Ligase (Fermentas) 6&mu;l H2O </li> The ligation was carried out in 18&deg;C for 3h and then inactivated in 80&deg;C for 15 min.</li> A fresh batch of chemocompetent bacteria was transformed with the ligation mix and incubated on agarose plates containing double dose of ampicilin.</li>

Inverting PLacI regulated parts of the switch and assembling the switch on a single plasmid Ania Tasks:   </li> </ul>