Team:Warsaw/Calendar-Main/16 May 2008

Preparation of pMPMT5-AID+T7 and pMPMT5+AID-T7 Michał K. Gel electrophoresis (15 May PCR's) and DNA isolation from proper bands (600 bp for AID lane and 2700 bp for T7 polymerase lanes).

Electrophoresis to estimate the concentration of isolated DNA.  PCR - translational fusion</a>: AID + T7 RNA-polymerase - optimization (annealing temperature gradient 60&deg;C - 80&deg;C). Fig. 1</a>.

Primers:

AIDlNrH</a> and T7pXbSal</a>

Template DNA: purified PCR products from 15 May</a> - AID and T7 RNA-polymerase for translational fusion</a>

Elongation time: 4 minutes

35 cycles </li>  Optimization of PCR</a> - translational fusion: AID + T7 RNA-polymerase - MgCl2 concentration and number of cycles. Fig. 2</a>. Primers:

AIDlNrH</a> and T7pXbSal</a>

Template DNA: purified PCR products from 15 May</a> - AID and T7 RNA-polymerase for translational fusion</a>

Annealing temperature: 73&deg;C Elongation time: 4 minutes </li>  Gel electrophoresis of PCR products.</li> </ol> <img src="http://2008.igem.org/wiki/images/f/f9/Pcr_aid_t7_WAW1.jpg"width=350/></a> Fig. 1. PCR products - optimization of annealing temperature: 1-DNA ladder; 2 to 8 -annealing temperature 60&deg;C (in lane 2) to 80&deg;C (in lane 8). <img src="http://2008.igem.org/wiki/images/d/d1/Pcr_aid_t7_WAW2.jpg"width=350/></a> Fig. 2. PCR products - optimization of MgCl2 concentration and number of cycles: <ol> <li style="font-family: monospace; margin-left: 3cm"> DNA ladder; <li style="font-family: monospace; margin-left: 3cm"> 2 μl MgCl2, 20 cycles; </li> <li style="font-family: monospace; margin-left: 3cm"> 3 μl MgCl2, 20 cycles; </li> <li style="font-family: monospace; margin-left: 3cm"> 2 μl MgCl2, 25 cycles; </li> <li style="font-family: monospace; margin-left: 3cm"> 3 μl MgCl2, 25 cycles; </li> <li style="font-family: monospace; margin-left: 3cm"> 2 μl MgCl2, 30 cycles; </li> <li style="font-family: monospace; margin-left: 3cm"> 3 μl MgCl2, 30 cycles; </li> <li style="font-family: monospace; margin-left: 3cm"> 2 μl MgCl2, 35 cycles; </li> <li style="font-family: monospace; margin-left: 3cm"> 3 μl MgCl2, 35 cycles.</li> </ol>