Team:Warsaw/Calendar-Main/24 September 2008

MutD5 testing Emilia  Inoculation MutD5 from previous day to new medium (liquid LB + 0.25mM IPTG + kanamycin + tetracyclin + 50 μl/ml + prey) 6 hours culture Inoculation MutD5 from the morning to new medium (liquid LB + 0.25mM IPTG + kanamycin + tetracyclin + 50 μl/ml + prey)



Mutagenesis of protein A Paweł

Results of mutagenesis: no colonies on any plate.  Mutagenesis repeated with modified conditions: fresh stock of dNTPs used and additional MgCl2 added.

Preparation of alpha_A construct Antoni   <a href="http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> on <a href=http://2008.igem.org/Wiki/Team:Warsaw/vectors/pKSII+A>pKS plasmid containing protein A</a> with <a href="http://2008.igem.org/Wiki/Team:Warsaw/primers#AL+link10+homo2">AL+link10+homo2</a> and <a href="http://2008.igem.org/Wiki/Team:Warsaw/primers#AP+NotI">AP+NotI</a> primers and 10% DMSO(20 cycles, elongation 40 s, annealing temperature 72&deg;C). </li>  <a href="http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> on <a href=http://www.fermentas.com/techinfo/nucleicacids/mappuc1819.htm>pUC19</a> plasmid with <a href="http://2008.igem.org/Wiki/Team:Warsaw/primers#AlphaL+SacI">AlphaL+SacI</a> and <a href="http://2008.igem.org/Wiki/Team:Warsaw/primers#AlphaP+link10+homo2">AlphaP+link10+homo2</a> primers (20 cycles, elongation 45 s, annealing temperature 63&deg;C). As a result we got two PCR products (alpha-linker and linker-A) wich will be utilized as templates in next PCR. </li>  Gel electrophoresis of PCR products 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 (alpha_linker - 570 bp and linker_A - 470 bp ).</li> Measurment of concentration of both isolated products.</li> </ol>

Preparation of <a href=http://partsregistry.org/wiki/index.php?title=Part:BBa_K103003>&Delta;A (BBa_K103003)</a> Piotr  E. coli <a href="http://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">TOP10</a> <a href="http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#chemotransform">transformation</a> with overnight ligation of <a href=http://partsregistry.org/wiki/index.php?title=Part:pSB1A3>pSB1A3</a> and <a href=http://partsregistry.org/wiki/index.php?title=Part:BBa_K103003>&Delta;A</a> DNA fragments. </li> Plating on LB + ampicillin.</li>

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