Team:Warsaw/Calendar-Main/14 May 2008

Preparation of pMPMT5+AID construct Michał K.   Isolation of hypothetical pMPM-T5+AID plasmids from cultures inoculated on previous day. 

 Restriction digest of plasmids with HindIII and NcoI (1xTango buffer) - construct control.

 Gel electrophoresis - choice of proper clones (all checked colonies). Fig. 1.</li></ol>

<img src="http://2008.igem.org/wiki/images/e/e7/Ligation_test_WAW.jpg" width=350/></a> Fig. 1. 1-DNA ladder; 2 to 9-plasmids isolated from colonies of transformants and digested with HindIII and NcoI

Preparation of pMPMT5-AID+T7</a> and pMPMT5+AID-T7</a> Michał K.

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 Optimization of conditions for PCR</a> - T7 RNA polymerase for translation fusion; annealing temperature gradient (from 62&deg;C to 82&deg;C); Taq polymerase with supplemented buffer (Łukasz Banasiak helped us with that problematic PCR).

Primers:

T7lLinkB</a> T7pXbSal</a>

Template DNA: E. coli Rosetta</a> genomic DNA

Elongation time: 4 minutes

20 cycles

 Gel electrophoresis of PCR products. </li>

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