Team:Warsaw/Calendar-Main/29 September 2008

MutD5 testing Piotr Sequencing results: there weren't any mutations in plasmids isolated from MutD5 - maybe this strain is too weak as a mutator.

Preparation of OmpA-linker-omega-linker (BBa_K103016) Michał K.

 PCR on pACYC177+OmpA_omega_&Delta;A_alpha plasmid using OmLNXNEB and LinPBSNP primers (annealing temperature 58 &deg;C; elongation length 90s) to obtain OmpA-linker-omega-linker (BBa_K103016) fragment.   Gel electrophoresis of PCR products and gel-out</a> of proper bands (OmpA-linker-omega-linker - 900 bp). Fig. 1</a>. </li></li> </ol>

<img src="http://2008.igem.org/wiki/images/e/ef/Go_29_09_2008.jpg"width=150/> Fig. 1.  PCR products: lane 2 - Omega_Link, lane 3 - OmpA_Omega_Link.

Preparation of OmpA-linker (BBa_K103006)</a> Michał K.

<ol> Digest</a> of <A href=http://partsregistry.org/wiki/index.php?title=Part:pSB1A3>pSB1A3</a> carrying &Delta;A (BBa_K103003)</a> plasmid with NdeI and SacI (BamHI buffer). Dephosphorylation</a> (CIAP) of plasmid.</li>

 Gel electrophoresis of digested plasmids and gel-out</a> of proper band (<A href=http://partsregistry.org/wiki/index.php?title=Part:pSB1A3>pSB1A3</a> - 2200 bp). Fig. 2</a>. </li>

 Overnight ligation</a> of isolated DNA fragments: <A href=http://partsregistry.org/wiki/index.php?title=Part:pSB1A3>pSB1A3</a> + OmpA_linker (BBa_K103006)</a> (from 18 September). </li></ol>

Preparation of omega_linker under PT7 (BBa_K103020)</a> Michał K. <ol> PCR</a> on <a href="http://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-omega">pACYC177 + OmpA_omega</a> plasmid using <a href="http://2008.igem.org/Wiki/Team:Warsaw/primers#OmegLNde">OmegLNde</a> and <a href="http://2008.igem.org/Wiki/Team:Warsaw/primers#LinP_BS">LinP_BS</a> primers (annealing temperature 55 &deg;C; elongation length 60s) to obtain omega_linker fragment. </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 band (omega_linker - 350 bp). <a href="http://2008.igem.org/Team:Warsaw/Calendar-Main/29_September_2008#fig1">Fig. 1</a>.</li></ol>

<img src="http://2008.igem.org/wiki/images/e/ef/Go_29_09_2008.jpg"width=180/></a> Fig. 1.  PCR products: lane 2 - Omega_Link, lane 3 - OmpA_Omega_Link.

Preparation of <a href=http://partsregistry.org/wiki/index.php?title=Part:BBa_K103004>Z(BBa_K103004)</a> Michał K. <ol> <li><a href="http://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">Digest</a> of <a href=http://2008.igem.org/Wiki/Team:Warsaw/vectors/pGeneart_Z>pGeneArt-Z</a> and <A href=http://partsregistry.org/wiki/index.php?title=Part:pSB1A3>pSB1A3</a> carrying <a href=http://partsregistry.org/wiki/index.php?title=Part:BBa_K103003>&Delta;A (BBa_K103003)</a> plasmids with NdeI and BcuI (BamHI buffer). <a href="http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#removing">Dephosphorylation</a> (CIAP) of plasmid. </li>

<li> Gel electrophoresis of digested plasmids 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 (Z - 200 bp and  <A href=http://partsregistry.org/wiki/index.php?title=Part:pSB1A3>pSB1A3</a> - 2200 bp). <a href="http://2008.igem.org/Team:Warsaw/Calendar-Main/29_September_2008#fig2">Fig. 2</a> and <a href="http://2008.igem.org/Team:Warsaw/Calendar-Main/29_September_2008#fig3">Fig. 3</a>.</li> <li> Overnight <a href="http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#ligation">ligation</a> of isolated DNA fragments: <A href=http://partsregistry.org/wiki/index.php?title=Part:pSB1A3>pSB1A3</a> + <a href=http://partsregistry.org/wiki/index.php?title=Part:BBa_K103004>Z (BBa_K103004)</a>. </li></ol>

<img src="http://2008.igem.org/wiki/images/d/d1/Go2_30_09_2008na29_09.jpg" width=160/></a> Fig. 2. Digests of plasmid pSB1A3 1. Marker 2. pSB1A3 digested NdeI/SacI 3. pSB1A3 digested NdeI/BcuI <img src="http://2008.igem.org/wiki/images/e/e5/Go_Z_25_09_2008.jpg" width=150/></a> Fig. 3. Digest of plasmid pGeneArt-Z 1. Marker 2. pGeneArt-Z digested NdeI/BcuI

Preparation of <a href=http://partsregistry.org/Part:BBa_K103018>OmpA_linker_omega_linker under Plac (BBa_K103018)</a> Michał K. <ol>

<li><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/pACYC177%2BompA-omega-A-alpha>pACYC177+OmpA_omega_&Delta;A_alpha</a> plasmid using <a href="http://2008.igem.org/Wiki/Team:Warsaw/primers#PlacL_XNE">PlacL_XNE</a> and <a href="http://2008.igem.org/Wiki/Team:Warsaw/primers#LinP_BS">LinP_BS</a> primers (annealing temperature 58 &deg;C; elongation length 90s) to obtain <a href=http://partsregistry.org/Part:BBa_K103018>OmpA_linker_omega_linker under Plac (BBa_K103018)</a> fragment. </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 band (BBa_K103018 - 1200 bp). <a href="http://2008.igem.org/Team:Warsaw/Calendar-Main/29_September_2008#fig4">Fig. 4</a>.</li>

</ol> <img src="http://2008.igem.org/wiki/images/1/19/Go22_29_09.jpg" width=180/></a> Fig. 4.Result of PCR on pLac_OmpA_omega_ 1. Marker 2. PCR product

Preparation of vectors for Biobricks Piotr Inoculation of bacteria received from iGEM HQs, carrying <a href=http://partsregistry.org/wiki/index.php?title=Part:pSB2K3>pSB2K3</a> and <a href=http://partsregistry.org/Part:BBa_I739204>BBa_I739204 (pACYC177 converted into BioBrick vector)</a> plasmids.