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Team:Warsaw/Calendar-Main/7 July 2008
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| - | <h3>Preparation of constructs | + | <h3>Preparation of constructs: OmpA_alpha and OmpA_omega #2</h3> |
| - | <h4>Piotr</h4> | + | <h4>Piotr, Weronika</h4> |
| - | <p> | + | <p>It's the second attempt to clone OmpA_alpha and OmpA_omega to pACYC177, but now thank's to Paweł we've got both fusions on pET15b.</p> |
<ol> | <ol> | ||
| - | <li> <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">Digest</a> of confirmed clones of pET15b+OmpA_alpha and pET15b+OmpA_omega with NdeI and BamHI (Tango 2x buffer). </li> | + | <li> <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">Digest</a> of confirmed clones of <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pET15b%2BOmpA-alpha>pET15b+OmpA_alpha</a> and <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pET15b%2BOmpA-omega>pET15b+OmpA_omega</a> with NdeI and BamHI (Tango 2x buffer). </li> |
| - | <li> <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">Digest</a> of pACYC177 plasmid with NdeI and BamHI (Tango 2x buffer), <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#removing">dephosphorylation with CIAP</a>. </li> | + | <li> <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">Digest</a> of <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177>pACYC177</a> plasmid with NdeI and BamHI (Tango 2x buffer), <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#removing">dephosphorylation with CIAP</a>. </li> |
<li> Gel electrophoresis of digested plasmids and <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">gel-out</a> of proper bands (OmpA_alpha- 1000 bp, OmpA_omega - 800 bp). </li> | <li> Gel electrophoresis of digested plasmids and <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">gel-out</a> of proper bands (OmpA_alpha- 1000 bp, OmpA_omega - 800 bp). </li> | ||
<li>Electrophoresis of gel-out products.</li> | <li>Electrophoresis of gel-out products.</li> | ||
| - | <li> Overnight <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#ligation">ligation</a> of pACYC177 and OmpA_alpha. </li> | + | <li> Overnight <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#ligation">ligation</a> of <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177>pACYC177</a> and OmpA_alpha. </li> |
| - | <li> Overnight <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#ligation">ligation</a> of pACYC177 and OmpA_omega. </li> | + | <li> Overnight <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#ligation">ligation</a> of <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177>pACYC177</a> and OmpA_omega. </li> |
</ol> | </ol> | ||
| - | + | ||
<h3>Cloning omega-A fusion on pKS (second attempt)</h3> | <h3>Cloning omega-A fusion on pKS (second attempt)</h3> | ||
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<tr><th>Product</th><th>Template</th><th>Primers</th><th>Product length</th></tr> | <tr><th>Product</th><th>Template</th><th>Primers</th><th>Product length</th></tr> | ||
<tr><th>linker-A</th><td>pDRIVE-TapTag</td><td><a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AL+link10+homo2">AL+link10+homo2</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AP+NotI">AP+NotI</a> </td><td>470 bp</td></tr> | <tr><th>linker-A</th><td>pDRIVE-TapTag</td><td><a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AL+link10+homo2">AL+link10+homo2</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AP+NotI">AP+NotI</a> </td><td>470 bp</td></tr> | ||
| - | <tr><th>omega-linker</th><td>pUC19</td><td><a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#OmegaL+SacI">OmegaL+SacI</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#OmegaP+link10+homo2">OmegaP+link10+homo2</a></td><td>400 bp</td></tr> | + | <tr><th>omega-linker</th><td><a href=http://www.fermentas.com/techinfo/nucleicacids/mappuc1819.htm>pUC19</a></td><td><a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#OmegaL+SacI">OmegaL+SacI</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#OmegaP+link10+homo2">OmegaP+link10+homo2</a></td><td>400 bp</td></tr> |
</table> | </table> | ||
<br> <br> | <br> <br> | ||
Latest revision as of 16:33, 26 October 2008
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Preparation of constructs: OmpA_alpha and OmpA_omega #2Piotr, WeronikaIt's the second attempt to clone OmpA_alpha and OmpA_omega to pACYC177, but now thank's to Paweł we've got both fusions on pET15b.
Cloning omega-A fusion on pKS (second attempt)Michał L., Ewa, MarcinWe have just received corrected version of omegaP-link10-homo2 primer. Let's hope this time everything will be fine. We are repeating the PCRs:
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