Team:Warsaw/Calendar-Main/20 May 2008

Preparation of pMPMT5-AID+T7 and pMPMT5+AID-T7

Michał K.   Digest of PCR product for transcriptional fusion (T7 RNA-polymerase) with HindIII and SalI (2x Tango buffer).

 Digest of PCR product for translational fusion (AID+T7 RNA-polymerase) with NcoI and XbaI (1x Tango buffer).</li>

 Clean-up</a> of digested products from p.1 and p.2.</li>

 Digest</a> of pMPM-T5+AID</a> with HindIII and SalI (2x Tango buffer), dephosphorylation</a> (CIAP).</li>

 Digest</a> of pMPM-T5</a> with NcoI and XbaI (1x Tango buffer), dephosphorylation</a> (CIAP).</li>

 Gel-electrophresis and gel-out</a> of proper bands (p.4 - 4850 bp, p.5 - 4250 bp).</li> Electrophoresis to estimate the concentration of purified DNA.</li>  Ligation</a> of purified products from p.1 and p.4.</li>

 <a href="http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#electrotransform">Electroporation</a> of E. coli <a href="http://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">TOP10</a>  with ligation product.</li>

<li> <a href="http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#ligation">Ligation</a> of digested products from p.4 and p.5.</li>

<li> <a href="http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#electrotransform">Electroporation</a> of E. coli <a href="http://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">TOP10</a>  with ligation product.</li>

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

Rifampicin test #1 Michał L., Ewa, Marcin

Plating cultures from the previous day on rifampicin.