Team:Warsaw/Calendar-Main/19 August 2008

Tests for ampicillin resistance given by protein added to medium Piotr

Inoculation of TOP10 carrying pACYC177+OmpA_A_alpha, pACYC177+OmpA_&Delta;A_alpha, pACYC177+OmpA_&Delta;A_omega, pACYC177+OmpA_A_omega, pACYC177+OmpA_omega_&Delta;A, pACYC177+OmpA_alpha, pACYC177+OmpA_omega and pACYC177+OmpA_omega_&Delta;A_alpha to LB with inductor (0.25 mM IPTG) and kanamycin.

Cloning of alpha to pACYC177+OmpA_A_omega</a> and pACYC177+OmpA_Z_omega</a> Michał K.

<ol><li>Clean-up</a> of overnight digest reaction. </li> <li> Ligation</a> of digested vectors from 14 August</a> and alpha DNA fragment.</li>

<li> Electroporation</a> of E. coli TOP10</a>  with ligations products.</li>

<li> Transformants plating on LB + kanamycin.</li></ol>

Cloning of protein A DNA to pET15b+OmpA-alpha</a> plasmid Antoni

<ol><li> Isolation of plasmid</a> from cultures inocluated on previous day</a> (<A href=http://2008.igem.org/Wiki/Team:Warsaw/vectors/pET15b%2BOmpA-alpha>pET15b+OmpA-alpha</a>). </li> <li>Digest</a> of pET15b+OmpA_alpha</a> with NdeI and NotI (Orange buffer) and  dephosphorylation with CIAP</a>.</li> <li> Gel electrophoresis (Fig. 1.</a>) and gel-out</a> of 6300 bp band. </li> </ol>

<img src="http://2008.igem.org/wiki/images/4/4b/PKS%2BApGApET%2Balfa.JPG"> Fig. 1.Digest product - pGeneart+Z (lanes 1-3), pKS+A (4-6), pET15b+OmpA-A (8-10). Lane 7 - GeneRuler™ DNA Ladder Mix

Cloning of protein A DNA to GeneArt plasmid Antoni <ol>

<li> Isolation</a> of plasmids from bacterial cultures inoculated on previous day (<A href=http://2008.igem.org/Wiki/Team:Warsaw/vectors/pKSII+A>pKS+A</a> and <a href=http://2008.igem.org/Wiki/Team:Warsaw/vectors/pGeneart_Z>pGeneart+Z</a>). </li> <li> <a href="http://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">Digest</a> of <a href=http://2008.igem.org/Wiki/Team:Warsaw/vectors/pKSII+A>pKS+A</a> and <a href=http://2008.igem.org/Wiki/Team:Warsaw/vectors/pGeneart_Z>pGeneart+Z</a> with SacI and NotI (BamHI buffer), <a href=http://2008.igem.org/Wiki/Team:Warsaw/vectors/pGeneart_Z>pGeneart</a><a href="http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#removing"> dephosphorylation</a> with CIAP.</li> <li> Gel electrophoresis (<a href="http://2008.igem.org/Team:Warsaw/Calendar-Main/19_August_2008#fig1">Fig. 1.</a>) of digested plasmids and <a href="http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">gel-out</a> of proper bands (470 bp for protein A lane and 2570 for pGeneart)  </li> <li> Overnight <a href="http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#ligation">ligation</a> of <a href=http://2008.igem.org/Wiki/Team:Warsaw/vectors/pGeneart-A>pGeneart and A</a>. </li> </ol>

<img src="http://2008.igem.org/wiki/images/4/4b/PKS%2BApGApET%2Balfa.JPG"></a> Fig. 1.Digest product - pGeneart+Z (lanes 1-3), pKS+A (4-6), pET15b+OmpA-A (8-10). Lane 7 - GeneRuler™ DNA Ladder Mix