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| - | <h3>Cloning of protein Z DNA to OmpA constructs</h3>
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| - | <p><ol>
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| - | <li> <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">Digest</a> of Geneart_Z and pACYC177+OmpA_omega with SacI and NotI (pACYC177 was also dephosphorylated with CIAP)(3 hr).</li>
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| - | <li> Gel electrophoresis 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 220 bp (for Geneart_Z lane)and 4050 bp (pACYC177+OmpA_omega lane). </li>
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| - | <li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#ligation">Ligation</a> of pACYC177+OmpA_omega and Z (1 hr). </li>
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| - | <li> Transformation of <i>E. coli</i> <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">TOP10</a> strain with ligation. </li>
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| - | <li> Transformants plating on LB + kanamycin.</li>
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| - | </ol>
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| - | </p>
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| - | <h3>Cloning of protein Z DNA to OmpA constructs</h3>
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| - | <p> 2 colonies was inoculated to liquid LB broth with kanamycin</p>
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| - | <h3>Preparation of constructs with OmpA protein fusions and Cloning of protein Z DNA to OmpA constructs</h3>
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| - | <p><ol>
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| - | <li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#plasmid_DNA_isolation">Isolation of plasmids</a> from cultures inocluated on previous day (pCACYC177 + OmpA_alpha and pACYC177+OmpA_Z_omega). </li>
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| - | <li> Control
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| - | <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">digest</a> of isolated plasmids with BamHI and SacI (we found good clones for both ligations).</li>
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| - | </ol></p>
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| - | <h3><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcl">Polymerase Chain Ligation</a> on linker-A and omega-linker</h3>
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| - | <h4>Michał L., Ewa, Marcin</h4>
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| - | <p>
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| - | <ul>
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| - | <li>reisolated PCR product omega-linker - 4 µl<br>
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| - | <li>reisolated PCR product linker-A - 13.5 µl<br>
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| - | <li>primer
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| - | <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#OmegaL+SacI_N">OmegaL+SacI_N</a> - 2 µl</li>
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| - | <li>primer
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| - | <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AP+NotI_N">AP+NotI_N</a> - 2 µl</li>
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| - | <li>Pfu buffer with Mg<sup>2+</sup> - 5 µl</li>
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| - | <li>dNTPs - 1 µl</li>
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| - | <li>H<sub>2</sub>o - 22 µl</li>
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| - | <li>Program:</li>
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| - | <ol>
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| - | <li> 95°C - 3'</li>
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| - | <li> 95°C - 30"</li>
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| - | <li> 55°C - 45"</li>
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| - | <li> 68°C - 1'</li>
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| - | <li> go to step 2 25 x</li>
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| - | <li> 68° - 10'<br>
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| - | <li> keep in 4°</li>
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| - | </ol></li>
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| - | <li>gel electrophoresis of products</li></ul>
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| - | </p>
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