Team:Warsaw/Calendar-Main/29 July 2008

Purification of proteins: Z-alpha and Z-omega Piotr

pET15b+Z_alpha and pET15b+Z_omega in Rosetta strain inoculated in liquid LB with chloramphenicol and ampicillin. Cloning of omega_&Delta;A DNA fragment to pACYC177+OmpA_alpha Michał K.   Isolation of pKS+omega_&Delta;A from culture inoculated on previous day. Digest</a> of pKS+omega_&Delta;A</a> with SacI and NotI (BamHI buffer).</li> Digest</a> (SacI and NotI) and dephosphorylation</a> (CIAP) of pACYC177+OmpA_alpha</a>. </li> Gel electrophoresis and gel-out</a> of 4300 bp (pACYC177+OmpA_alpha</a>) and 550 bp (omega_&Delta;A) bands (Fig. 1.</a>).</li> Ligation</a> of DNA fragments from 2. and 3. (1 hr).</li> Transformation</a> of E. coli TOP10</a> strain with ligation.</li> Transformants plating on LB + kanamycin.</li></ol> <img src="http://2008.igem.org/wiki/images/6/6a/Po_go.jpg" width=200/></a> Fig. 1.  SacI/NotI digests of isolated plasmids 1. Marker 2. digested pKS_omega_&Delta;A 3. digested pACYC_Omp_alpha