Team:Warsaw/Calendar-Main/13 October 2008

Preparation of linker_alpha (BBa_K103009) Michał K. 

 PCR on pACYC177+OmpA_omega_deltaA_alpha plasmid using

LinLSXNE and AlphaPSpe primers (annealing temperature 58 &deg;C; elongation length 60s) to obtain linker_alpha (BBa_K103009) fragment.  Gel electrophoresis and gel-out</a> of proper band 600 bp - linker_alpha (BBa_K103009)</a> PCR product. Fig. 1</a>. </li> Digest</a> of isolated PCR product linker_alpha (BBa_K103009)</a> with EcoRI and BcuI (BamHI buffer). </li>  Clean-up</a> of digested PCR product. </li>  Overnight ligation</a> of DNA fragments: pSB2K3</a> + linker_alpha (BBa_K103009)</a>.</li></ol>

<img src="http://2008.igem.org/wiki/images/0/0a/Go2_13_10_2008.jpg"> Fig. 1.SacI/BcuI digests of PCR to obtain alpha_linker_2 and linker_alpha 1. Marker 2. digested PCR product - alpha_linker 3. digested PCR product - linker_alpha

Preparation of OmpA-linker-omega-linker (BBa_K103016)</a> Michał K. Inoculation of few more colonies from old plate with ligation of pACYC177</a> + OmpA-linker-omega-linker (BBa_K103016)</a> to liquid LB + kanamycin.

Preparation of alpha_linker under PT7 (BBa_K103019)</a> Michał K. Inoculation of colonies from plate with ligation of <A href=http://partsregistry.org/wiki/index.php?title=Part:pSB1A3>pSB1A3</a> carrying  <a href=http://partsregistry.org/Part:BBa_K103019>alpha_linker under PT7 (BBa_K103019)</a> fragment to liquid LB + ampicillin.

Preparation of <a href=http://partsregistry.org/Part:BBa_K103020>omega_linker under PT7 (BBa_K103020)</a> Michał K. Inoculation of colonies from plate with ligation of <a href=http://partsregistry.org/wiki/index.php?title=Part:pSB2K3>pSB2K3</a> +  <a href=http://partsregistry.org/Part:BBa_K103020>omega_linker under PT7 (BBa_K103020)</a> to liquid LB + kanamycin.

Preparation of <a href=http://partsregistry.org/Part:BBa_K103002>AID under pBAD/araC (BBa_K103002)</a> Michał K. Overnight <a href="http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#ligation">ligation</a> of DNA fragments:  <a href=http://partsregistry.org/wiki/index.php?title=Part:pSB1A3>pSB1A3</a> (from <A href=http://2008.igem.org/Team:Warsaw/Calendar-Main/6_October_2008>6 October</a>) + <a href=http://partsregistry.org/Part:BBa_K103002>AID under pBAD/araC (BBa_K103002)</a> (from <a href=http://2008.igem.org/Team:Warsaw/Calendar-Main/10_October_2008>10 October</a>).</li>

Preparation of <a href=http://partsregistry.org/Part:BBa_K103017>OmpA_linker_alpha_linker under Plac (BBa_K103017)</a> Michał K.

<ol> <li><a href=http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest>Digest</a> of <a href=http://partsregistry.org/Part:pSB2K3>pSB2K3</a> + <a href=http://partsregistry.org/Part:BBa_K103018>OmpA_linker_omega_linker under Plac (BBa_K103018)</a> with BcuI and SacI (SacI buffer), <a href="http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#removing">dephosphorylation</a> with CIAP. </li>

<li> <a href="http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> on alpha PCR product from <a href=http://2008.igem.org/Team:Warsaw/Calendar-Main/10_October_2008>10 October</a> using

<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#LinP_BS2">LinP_BS2</a> primers (annealing temperature 58 &deg;C; elongation length 60s) to obtain alpha2 fragment with proper restriction sites (SacI and BcuI). </li> <li>Gel electrophoresis and <a href="http://2008.igem.org/Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">gel-out</a> of proper bands (600 bp for alpha2 PCR product and 3000 bp for <a href=http://partsregistry.org/Part:pSB2K3>pSB2K3</a> (with OmpA_linker under PLac promoter) fragment. </li> <li><a href=http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest>Digest</a> of isolated PCR product (alpha2 fragment) with SacI and BcuI (SacI buffer). </li> <li> <a href="http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_purification_after_enzymatic_reaction">Clean-up</a> of digested PCR product. <a href="http://2008.igem.org/Team:Warsaw/Calendar-Main/13_October_2008#fig1">Fig. 1</a> </li>

<li> Overnight <a href="http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#ligation">ligation</a> of DNA fragments: <a href=http://partsregistry.org/Part:pSB2K3>pSB2K3</a> (with lactose promoter and OmpA_linker)  + alpha2.</li> </ol>

<img src="http://2008.igem.org/wiki/images/0/0a/Go2_13_10_2008.jpg"></a> Fig. 1.SacI/BcuI digests of PCR to obtain alpha_linker_2 and linker_alpha 1. Marker 2. Digested PCR product - alpha_linker 3. Digested PCR product - linker_alpha