Team:Warsaw/Calendar-Main/16 June 2008

Preparation of constructs: OmpA_alpha and OmpA_omega Michał K.   PCR on pB30D plasmid with OmpaL_N and OmpaP_link primers (15 cycles, elongation duration 45 s, annealing temperature 63&deg;C).   PCR on pUC19 plasmid with AlphaL_link</a> and AlphaP_XB</a> primers (20 cycles, elongation duration 45 s, annealing temperature 63&deg;C).</li>  PCR</a> on pUC19</a> plasmid with OmegaL_link</a> and OmegaP_EPB</a> primers (20 cycles, elongation duration 30 s, annealing temperature 58&deg;C). As a result we got three PCR products: OmpA_linker and two fragments of TEM1 beta-lactamese: linker_alpha and linker_omega. </li>  Gel electrophoresis of PCR products (Fig. 1</a>) and gel-out</a> of proper bands (OmpA_linker - 500 bp, linker_alpha - 600 bp and linker_omega - 350 bp).</li> Electrophoresis to estimate the concentration of isolated DNA.</li> </ol> <img src="http://2008.igem.org/wiki/images/a/a3/PCR_Ompa_Alpha_Omega_WAW.jpg" width=300/></a> Fig. 1. PCR products: linker_alpha (lane 2), linker_omega (lane 3), OmpA_linker (lane 4)

Blue/white and rifampicin test #1 Michał L., Ewa <ol> Colony PCR</a>.

Template: DNA isolated from white colonies

Primers: pZCseqL</a> and pZCseqR</a> DNA gel electrophoresis of PCR products. <a href="http://2008.igem.org/wiki/index.php?title=Team:Warsaw/Calendar-Main/16_June_2008#fig2">Fig. 2.</a></li>

<li>Gel-out of proper products (~1200 bp).</li>

<li>Sequencing of proper fragments using primer <a href="http://2008.igem.org/Wiki/Team:Warsaw/primers#pZCseqL">pZCseqL</a>.</li></ol>

<img src="http://2008.igem.org/wiki/images/0/0c/Colony_PCR_pZC_WAW.jpg" width=400/></a> Fig. 2. 1 - DNA ladder; 2 to 11 - colony PCR products (lacZ' gene) from blue and white colonies.