Team:Warsaw/Calendar-Main/25 June 2008

Change of the reporter from pZC320 with B-galactosidase to GFP or RFP Piotr, Weronika  Inoculation of 10ml LB with E.coli TOP10 carrying pZC.

Isolation and chemotransformation with pSB1A2 standard plasmids carrying parts: BBa_E0840(GFP genetrator) and BBa_J04450 (RFP generator).

Plating of chemotransformants on LB+ampicillin.



Preparation of constructs: OmpA_alpha and OmpA_omega Michał K. Repetition of PCRs and <a href="http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">gel-out purifications</a> from <a href="http://2008.igem.org/Team:Warsaw/Calendar-Main/16_June_2008">16 June.</a>

Preparation of alpha-A and omega-A fusions

Michał L. Marcin and Ewa  We've run gradient <a href="http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> to obtain Alpha-link and link-A. <ul>DNA template: pDRIVE-TAPTAG</li> Taq polymerase</li>buffer suplemented with (NH4)2SO4</li>MgCl2 concentration gradient (from 1 to 4 mM)</li>annealing temp 55&deg;C</li>elongation time 1 - 2 minutes</li></ul></li>

We have successfully amplified Alpha-link and link-A (<a href="http://2008.igem.org/Team:Warsaw/Calendar-Main/25_June_2008#fig1">Fig. 1</a>). Now it's time for PCL (Polymerase Chain Ligation) to create fusions Alpha-A and Omega-A.</li></ol>

<img src="http://2008.igem.org/wiki/images/8/89/PCR_gradient_Alinker_WAW.jpg" width=400/></a> Fig. 1. PCR product - link-A.