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| - | {{:Team:Grenoble/Templates/Biology}}
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| - | <html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">
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| - | <body id="Biology">
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| - | <section>
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| - | <a href="https://2012.igem.org/Team:Grenoble/Biology/Notebook/July">week 27</a>
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| - | <a href="https://2012.igem.org/Team:Grenoble/Biology/Notebook/June/week_28">week 28</a>
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| - | <a href="https://2012.igem.org/Team:Grenoble/Biology/Notebook/June/week_29">week 29</a>
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| - | <a href="https://2012.igem.org/Team:Grenoble/Biology/Notebook/June/week_30">week 30</a>
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| - | <br/>
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| - | <br/>
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| - | <h1> Week 28: July 09<span class="exposant">th</span> to July 15<span class="exposant">th</span> </h1>
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| - | <h2> Goal of the week </h2>
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| - | we wanted to recover and amplify the biobricks involved in our genetic networks:
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| - | <ul>
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| - | <li>pAra/Bad_RBS_GFP (1300bp)</li>
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| - | <li>RBS_Cya (2600bp)</li>
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| - | <li>pLAC (100bp)</li>
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| - | <li>fha (80bp)</li>
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| - | <li>eCFP (800bp)</li>
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| - | <li>pLAC_RBS (120bp)</li>
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| - | <li>RsmA (200bp)</li>
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| - | <li>rsmY (170bp)</li>
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| - | <li>pSB1A3 (2400bp)</li>
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| - | <li>pSB4K5 (2400bp)</li>
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| - | <li>pSB3C5 (2400bp)</li>
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| - | </ul>
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| - | We also planned to realise the gibson assemblies for the first constructions.
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| - | <br/><br/>
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| - | <h2> Monday, July 09<span class="exposant">th</span>:</h2>
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| - | Precultured cells are prepared:
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| - | <ul>
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| - | <li>Strains = BW25113 WT and BW25113 cya- pAra/Bad </li>
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| - | <li>Conditions = LB liquid medium, 37°C, 200rpm, overnight </li>
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| - | </ul>
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| - | <br/>
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| - | <h2>Tuesday, July 10<span class="exposant">th</span>:</h2>
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| - | For the PCRs, we changed the enzyme from GoTaq to High Fidelity (HF) Phusion.<br/>
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| - | We did some colony PCR with a HF Phusion enzyme (protocol) to amplify:
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| - | <ul>
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| - | <li>fha1, RsmA, rsmY and pSB1A3 from iGEM Grenoble 2011 glycerol stock.</li>
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| - | <li>pAra/Bad_RBS_GFP from BW25113 cya- pAra/Bad precultured cells.</li>
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| - | <li>RBS_Cya from BW25113 WT precultured cells.</li>
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| - | </ul>
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| - | We did the PCR both with and without DMSO (Amplification of difficult targets, such as those with GC-rich sequences or secondary structure, may be improved by the presence of additives such as DMSO).<br/>
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| - | <br/>
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| - | To separate the PCR products, we prepared two gels:
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| - | <ul>
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| - | <li>a 1.3% TAE agarose gel, for the small fragments: RsmA, fha1 and rsmY (length < 1000bp)</li>
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| - | <li>a 0.8% TAE agarose gel, for the big fragments: pSB1A3, RBS_Cya, and pAra/Bad_RBS_GFP (length > 1000bp)</li>
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| - | </ul>
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| - | <br/>Migration conditions = 50V during 1h15.<br/>
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| - | We used EtBr to reveal the DNA fragments.<br/>
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| - | </section>
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| - |
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| - | </body>
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| - | </html>
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| - | {{:Team:Grenoble/script}}
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| - | {{:Team:Grenoble/menu}}
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