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Team:Warsaw/Calendar-Main/8 July 2008
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<li> Transformants plating on LB + kanamycin.</li></ol></p> | <li> Transformants plating on LB + kanamycin.</li></ol></p> | ||
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| + | <h3>Cloning omega-A fusion on pKS (second attempt)</h3> | ||
| + | <h4>Michał L., Ewa, Marcin:</h4> | ||
| + | <ol> | ||
| + | <li>Gel electrophoresis of PCR products</li> | ||
| + | <li>Gel-out of proper bands</li> | ||
| + | <li>PCL reaction to create omega-A fusion:<br> | ||
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| + | <br/> | ||
| + | <table id="result"> | ||
| + | <tr><th>Product</th><th>Templates</th><th>Primers</th><th>Product length</th></tr> | ||
| + | <tr><th>Omega-A fusion</th><td>Omega-linker + linker-A</td><td>OmegaL+SacI + AP+NotI </td><td>1440 bp</td></tr> | ||
| + | </table> | ||
| + | <br> | ||
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| + | <table id="result"> | ||
| + | <tr><th colspan="3"><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcl">PCL</a> program for omega-A fusion</th></tr> | ||
| + | <tr><th>Temperature</th><th>Time</th></tr> | ||
| + | <tr><td>94°C</td><td>4:00</td></tr> | ||
| + | <tr><td>94°C</td><td>0:30</td><td rowspan="3">28 cycles</td></tr> | ||
| + | <tr><td>48°C-58°C</td><td>0:45</td></tr> | ||
| + | <tr><td>68°C</td><td>2:00</td></tr> | ||
| + | <tr><td>68°C</td><td>10:00</td></tr> | ||
| + | <tr><td>4°C</td><td>infinite</td></tr> | ||
| + | </table> | ||
| + | <br/></li> | ||
| + | <li>Gel electrophoresis of PCL products</li> | ||
| + | <li>We have obtained no PCL product (weird?)</li> | ||
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| + | </ol> | ||
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| + | </html> | ||
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<h3>Preparation of construct pKS with A protein<br> | <h3>Preparation of construct pKS with A protein<br> | ||
Michał L., Marcin:</h3> | Michał L., Marcin:</h3> | ||
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Preparation of constructs with OmpA protein fusions
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| Product | Templates | Primers | Product length |
|---|---|---|---|
| Omega-A fusion | Omega-linker + linker-A | OmegaL+SacI + AP+NotI | 1440 bp |
| PCL program for omega-A fusion | ||
|---|---|---|
| Temperature | Time | |
| 94°C | 4:00 | |
| 94°C | 0:30 | 28 cycles |
| 48°C-58°C | 0:45 | |
| 68°C | 2:00 | |
| 68°C | 10:00 | |
| 4°C | infinite | |
Preparation of construct pKS with A protein
Michał L., Marcin:
- Inactivation of digestion enzymes and CIAP.
- <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#ligation">Ligation</a> of digested PCR product and pKS 2h at room temperature.
- <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#electrotransform">Transformation</a> of E. coli <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">TOP10</a> strain with 7 µl of ligation mix.
- Transformants plating on LB + ampicillin + X-gal + IPTG.
</html>
