Team:Warsaw/Calendar-Main/13 May 2008

Preparation of pMPMT5+AID construct Michał K.

Setup of separate cultures from colonies of transformants pMPMT5+AID (liquid LB+tetracycline).

Preparation of pMPMT5-AID+T7 and pMPMT5+AID-T7 Michał K.

  Optimization of conditions for PCR to obtain AID DNA fragment - annealing temperature gradient (from 62&deg;C to 82&deg;C). Fig. 1. Primers:

AIDlNrH AIDpLinB</a>

Template DNA: pTrc99A-AID</a>

Elongation time: 60 sec

20 cycles </li>

<li> Optimization of conditions for PCR</a> - T7 RNA polymerase for translational fusion; annealing temperature gradient (from 62&deg;C to 82&deg;C). Fig. 2</a>.

Primers:

T7lLinkB</a> T7pXbSal</a>

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

Elongation time: 4 minutes

20 cycles

</li> <li> Optimization of conditions for PCR</a> - T7 RNA polymerase for transcriptional fusion; annealing temperature gradient (from 62&deg;C to 82&deg;C). Fig. 2</a>.

Primers:

T7lRBSHi</a> T7pXbSal</a>

Template DNA: E. coli Rosetta</a> and TOP10</a> (negative control - Fig. 3</a>.) genomic DNA

Elongation time: 4 minutes

20 cycles

</li> <li> Gel electrophoresis of PCR products (there weren't any products for T7 RNA polymerase for translational fusion).</li> </ol>

<img src="http://2008.igem.org/wiki/images/7/71/Aid_pcr_grad_WAW.jpg" width=350/></a> Fig. 1. Gradient PCR products for AID: 1 -DNA ladder; 2 to 13 -PCR products: In lane 2 is product of PCR reaction with annealing temperature 62&deg;C (the lowest temperature of gradient), in lane 13 is product of PCR reaction with annealing temperature 82&deg;C (the highest temperature of gradient). The lanes between them contain products of PCR with middle annealing temperatures.

<img src="http://2008.igem.org/wiki/images/9/97/T7grad_WAW1.jpg" width=350/></a> Fig. 2. Gradient PCR products: upper - for transcriptional fusion, lower - for translational fusion; 1-DNA ladder; 2 to 12-PCR products-border annealing temperatures of gradient: 62&deg;C (lane 2) to 82&deg;C (lane 12)

<img src="http://2008.igem.org/wiki/images/6/6c/T7grad_WAW2.jpg" width=350/></a> Fig. 3. Gradient PCR products for negative control (Top10 genomic DNA): 1-DNA ladder; 2 to 6-PCR products-border annealing temperatures of gradient: 62&deg;C (lane 2) to 82&deg;C (lane 6).