Team:Warsaw/Calendar-Main/26 August 2008

Tests for ampicillin resistance given by protein added to medium Piotr  Purification pACYC177+OmpA_alpha from antibiotic resistance contamination: Inoculation of E. coli TOP10 carrying pACYC177+OmpA_alpha to liquid LB with kanamycin and with kanamycin and ampicillin. Inoculation of all pACYC177+OmpA variants of constructs to LB + 0.25 mM IPTG, kanamycin, ampicillin (50 μg/ml) and 50 μg/ml of protein (Z_alpha and Z_omega; negative control without protein). Measurement of growth after 5 hours and on next day.

Cloning of protein A DNA to pET15b+OmpA-alpha plasmid Antoni   Colony <a href="http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> on colonies from plates with transformations <a href=http://2008.igem.org/Wiki/Team:Warsaw/vectors/pGeneart-A>pGeneart+A</a> Primers used:

<a href="http://2008.igem.org/Wiki/Team:Warsaw/primers#AP+NotI">AP+NotI</a> and <a href="http://2008.igem.org/Wiki/Team:Warsaw/primers#AL+SacI">AL+SacI.</a></li>  Lack of confirmed transformant colonies. </li></ol>

Cloning of alpha to <a href=http://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-A-omega>pACYC177+OmpA_A_omega</a> and <a href=http://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-Z-omega>pACYC177+OmpA_Z_omega</a> Michał K.    <a href="http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#plasmid_DNA_isolation">Isolation of plasmids</a> from cultures inocluated on previous day (both potential constructs). </li>  Control <a href="http://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">digest</a> of isolated plasmids with FastBamHI and FastAcc65I (Fast Digest buffer).</li>  Gel electrophoresis - still no proper clones found.</li> </ol>