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| - | <h3>Preparation of construct pKS with A protein</h3>
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| - | <p><ol>
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| - | <li> Colony PCR on colonies from plates with transformations pKS-A <br>
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| - | Primers used:
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| - | <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AL+SacI">AL+SacI</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AP+NotI">AP+NotI</a>
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| - | </li>
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| - | <li> Confirmed transformant colonies inoculated to liquid LB with ampicillin</li></ol></p>
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| - | <h3>Cloning omega-A fusion on pKS (second attempt)</h3>
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| - | <h4>Michał L., Ewa, Marcin:</h4>
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| - | <p>We had to start form scratch with this one.
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| - | <ol>
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| - | <li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> A in 50 µl<br>
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| - | template DNA - pKS-A4 1 µl<br>
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| - | primer <html>
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| - | <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AP+NotI_N">AP+NotI_N</a> - 2 µl<br>
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| - | primer
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| - | <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AL+link10+homo2_N">AL+link10+homo2_N</a> - 2 µl<br>
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| - | Pfu polymerase buffer + Mg<sup>2+</sup> (from Fermentas) - 5 µl<br>
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| - | dNTPs - 1 µl <br>
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| - | Pfu turbo - 0.5 µl<br>
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| - | H2o - 38.5 µl<br>
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| - | <br>
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| - | Program:<br>
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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>62°C 45"</li>
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| - | <li>72°C 45"</li>
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| - | <li>72°C 10'</li>
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| - | <li>keeping in 4°C</li></ol>
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| - | </li>
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| - | <li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> omega in 50 µl<br>
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| - | template DNA - pUC19 1 µl<br>
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| - | primer OmegaLS - 2 µl<br>
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| - | primer AOmegaPli - 2 µl<br>
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| - | Pfu polymerase buffer + Mg<sup>2+</sup> (from Fermentas) - 5 µl<br>
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| - | dNTPs - 1 µl <br>
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| - | Pfu turbo - 0.5 µl<br>
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| - | H2o - 38.5 µl<br>
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| - |
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| - | Program:<br>
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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> 62°C 45"</li>
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| - | <li> 72°C 45"</li>
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| - | <li> 72°C 10'</li>
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| - | <li> keeping in 4°C</li></ol>
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| - | 25 cycles
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| - | </li>
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| - | <li> Gel electrophoresis</li>
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| - |
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| - | <li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">Reisolation</a> from agarose gel</li>
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| - | </ol>
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| - | </p>
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| - |
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| - |
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| - | <h3>Preparation of construct pKS with A protein</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 plasmid DNA</a> from cultures inocluated on previous day. </li>
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| - | <li> Control <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">digest</a> of isolated plasmid with SacI and NotI; 2 h</li>
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| - | <li> Gel electrophoresis of digested DNA</li>
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| - | <li> We are glad to find the proper clone ;-) and inoculate liquid LB with ampicillin to freeze bacteria carrying pKS-A construct</li></ol>
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| - |
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| - | </p>
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"
