Team:Warsaw/Calendar-Main/9 July 2008

Cloning of protein Z DNA to pET15b-OmpA-alpha in place of OmpA Piotr, Antoni Digest of pET15b-OmpA-alpha and Z (in Geneart vector) with NdeI and NotI (Orange buffer). Gel-out of Z (~200 bp band). Overnight ligation</a> of Z into digested pET15b-OmpA-alpha</a>.</li>

</ol>

Preparation of construct pKS with A protein</a> Michał L., Marcin <ol>  Gradient PCR (achieving multiple copies of gene coding A protein): template DNA pDRIVE-TAPtag - 1 µl primer AP+NotI</a> - 2 µl primer AL+SacI</a> - 2 µl Pfu buffer with Mg2+ - 5 µl 10 mM dNTPs - 1 µl Pfu Turbo polymerase - 0.5 µl H2O - 38.5 µl Program: 1. 94&deg;C, 3 min 2. 94&deg;C, 30 sec 3. 62 to 74&deg;C, 45 sec 4. 72&deg;C, 45 sec 5. Repeat of elongation step 25X 6. 72&deg;C, 10 min 7. Hold at 4 &deg;C </li>  Gel electrophoresis of PCR product.</li>  Isolation</a> of proper band (470 bp) from the gel.</li>  Overnight digest</a> of isolated PCR product and pKS</a> vector with NotI and SacI; to the reaction mix with pKS</a> added 0.5 µl of CIAP</a>. </li>

</ol>

Preparation of constructs: OmpA_alpha and OmpA_omega #2 Michał K.  <ol>  Colony PCR on colonies from plates with transformations pACYC177+OmpA_omega</a> and <a href=http://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-alpha>pACYC177+OmpA_alpha</a>. Primers used:

<a href="http://2008.igem.org/Wiki/Team:Warsaw/primers#OmpaL_N">OmpaL_N</a> and <a href="http://2008.igem.org/Wiki/Team:Warsaw/primers#OmpaP_link">OmpaP_link</a>. </li><li> Gel electrophoresis. (<a href="http://2008.igem.org/Team:Warsaw/Calendar-Main/9_July_2008#fig1">Fig. 1. </a> and <a href="http://2008.igem.org/Team:Warsaw/Calendar-Main/9_July_2008#fig2">Fig. 2.</a>).</li>

<li> Confirmed transformant colonies inoculated to liquid LB with kanamycin. </li></ol> <img src="http://2008.igem.org/wiki/images/3/36/Kolonijny_ompa%2Balfa_omegoa_pci1_04_08_2008.jpg" width=300 /></a> Fig. 1. Colony PCR on primers shown above (vector change from pET15b to pACYC177)  Upper gel: 1. Marker 2-12 PCR from colonies carrying probable Omp_A_alpha Lower gel 1. Marker 2-5. PCR from colonies carrying probable Omp_A_alpha 6. negative control 7-12. PCR from colonies carrying probable Omp_A_omega

<img src="http://2008.igem.org/wiki/images/f/fb/July_9_th.jpg" width=300 /></a> Fig. 2. Colony PCR on primers shown above (vector change from pET15b to pACYC177)  1. Marker 2-10 PCR from colonies carrying probable Omp_A_omega