Team:Warsaw/Calendar-Main/22 August 2008

Tests for ampicillin resistance given by protein added to medium Piotr Purification of pACYC177+OmpA_alpha from antibiotic resistance contamination: Transformation of E. coli TOP10 strain with pACYC177+OmpA_alpha.

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

  Ligation of digested vectors from 14 August</a> and alpha DNA fragment from 19 August</a> (more vectors added).</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 GeneArt plasmid Antoni <ol> <li> Control digest</a> of <A href=http://2008.igem.org/Wiki/Team:Warsaw/vectors/pGeneart-A>pGeneart+A</a> plasmids with NdeI and NotI (Orange buffer). </li> <li>Gel electrophoresis (Fig. 1.</a>). Choice of proper clone. </li></ol>

<img src="http://2008.igem.org/wiki/images/d/d0/PGA%2BAtraw.jpg" width=150></a> Fig. 1.Control digest of freshly ligated pKS+A (lanes 1-3). Lane 4 - 0.5&mu;g of DNA ladder mix.

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

<li> Digest</a> of pGeneart+A</a> with NdeI and NotI (Orange buffer). </li> <li> Gel electrophoresis of digested plasmid and gel-out</a> of 470 bp band. </li> <li> Overnight ligation</a> of pET15b+alpha and A</a> (pET15b+alpha from 19 August</a>). </li> </ol>