Team:Warsaw/Calendar-Main/14 July 2008

Cloning of protein Z DNA to OmpA constructs Michał K.    Digest of Geneart_Z and pACYC177+OmpA_omega with SacI and NotI (BamHI buffer), pACYC177 was also dephosphorylated with CIAP (3 hr).  Gel electrophoresis and gel-out of proper bands 220 bp (for Geneart_Z</a> lane) and 4050 bp (pACYC177+OmpA_omega</a> lane). </li>  Ligation</a> of pACYC177+OmpA_omega</a> and Z fragment DNA (1 hr). </li>  Transformation of E. coli TOP10</a> strain with ligation. </li>  Transformants plating on LB + kanamycin.</li> </ol>

Preparation of alpha+A conctruct Antoni <ol> PCR</a> on pKS plasmid containing protein A</a> with AL+link10+homo2</a> and AP+NotI</a> primers (20 cycles, elongation 40 s, annealing temperature 72&deg;C). </li>  PCR</a> on 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> <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> <li>PCR on alpha+A PCR products 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#AP+NotI">AP+NotI</a> primers.</li> <li>Gel electrophoresis reveal lack of proper 1000 bp band. </li> </ol>

Cloning omega-A fusion on <a href=http://2008.igem.org/Wiki/Team:Warsaw/vectors/pKSII%2B>pKS</a> (second attempt) Michał L., Ewa, Marcin

We had to start form scratch with this one. <ol> <li> <a href="http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> A in 50 µl template DNA - <A href=http://2008.igem.org/Wiki/Team:Warsaw/vectors/pKSII+A>pKS-A4</a> 1 µl primer <a href="http://2008.igem.org/Wiki/Team:Warsaw/primers#AP+NotI">AP+NotI</a> - 2 µl primer <a href="http://2008.igem.org/Wiki/Team:Warsaw/primers#AL+link10+homo2">AL+link10+homo2</a> - 2 µl Pfu polymerase buffer + Mg2+ (from Fermentas) - 5 µl dNTPs - 1 µl Pfu turbo - 0.5 µl H2o - 38.5 µl Program: <ol> <li> 95&deg;C 3'</li> <li> 95&deg;C 30"</li> <li>62&deg;C 45"</li> <li>72&deg;C 45"</li> <li>72&deg;C 10'</li> <li>keeping in 4&deg;C</li></ol> </li> <li> <a href="http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> omega in 50 µl template DNA - <a href=http://www.fermentas.com/techinfo/nucleicacids/mappuc1819.htm>pUC19</a> 1 µl primer <a href="http://2008.igem.org/Wiki/Team:Warsaw/primers#OmegaL+SacI">OmegaL+SacI</a> - 2 µl primer <a href="http://2008.igem.org/Wiki/Team:Warsaw/primers#OmegaP+link10+homo2">OmegaP+link10+homo2</a> - 2 µl Pfu polymerase buffer + Mg2+ (from Fermentas) - 5 µl dNTPs - 1 µl Pfu turbo - 0.5 µl H2o - 38.5 µl

Program: <ol> <li> 95&deg;C 3'</li> <li> 95&deg;C 30"</li> <li> 62&deg;C 45"</li> <li> 72&deg;C 45"</li> <li> 72&deg;C 10'</li> <li> keeping in 4&deg;C</li></ol> 25 cycles </li> <li> Gel electrophoresis</li>

<li><a href="http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">Reisolation</a> from agarose gel</li> </ol>