Team:Warsaw/Calendar-Main/18 August 2008

Purification of proteins: Z-alpha and Z-omega Piotr

Measurement of concentration of proteins (BCA method).

Cloning of protein A DNA to GeneArt plasmid Antoni Inoculation to liquid LB + ampicilin with bacteria carrying pGeneart+Z and pKS+A plasmids.

Cloning of alpha to pACYC177+OmpA_A_omega and pACYC177+OmpA_Z_omega Michał K.

PCR on pUC19 plasmid using

AlphaL+SacI</a> and AlphaPlinkSac</a>

primers (gradient of annealing temperature from 55&deg;C to 75&deg;C; elongation length 60s; 35 cycles).</li> <li>Gel electrophoresis and chioce of proper PCR conditions.</li> <li>PCR</a> on pUC19</a> plasmid using

AlphaL+SacI</a> and AlphaPlinkSac</a>

primers (annealing temperature - 65&deg;C; elongation length 60s; 20 and 25 cycles (Fig. 1.</a>)).</li> <li>Gel electrophoresis and gel-out</a> of 550 bp band.</li> <li> Overnight digest</a> of isolated PCR product with SacI (SacI buffer).</li></ol>

<img src="http://2008.igem.org/wiki/images/0/07/Go20.jpg" width=300/></a> Fig. 1.PCR to obtain alpha with various number of cycles. 1. Marker 2. Alpha PCR, 25 cycles 3. Alpha PCR, 20 cycles