Team:Grenoble/Biology/Notebook/July/week 33

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<h1>August</h1>
 
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<a href="http://2012.igem.org/Team:Grenoble/Biology/Notebook/August">Week 31</a> •
 
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<a href="http://2012.igem.org/Team:Grenoble/Biology/Notebook/August/week_32">Week 32</a> •
 
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<a href="http://2012.igem.org/Team:Grenoble/Biology/Notebook/August/week_33">Week 33</a> •
 
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<a href="http://2012.igem.org/Team:Grenoble/Biology/Notebook/August/week_34">Week 34</a> •
 
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<a href="http://2012.igem.org/Team:Grenoble/Biology/Notebook/August/week_35">Week 35</a>
 
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<section>
 
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<h1> Week 33: August 13<span class="exposant">th</span> to 19<span class="exposant">th</span> </h1>
 
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<h2> Goal of the week: </h2>
 
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Assembly of:
 
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<ul><li>pAra/Bad_RBS_GFP and RBS_Cya.</li>
 
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    <li>pompC and mcherry</li>
 
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    <li>Tap and EnvZ</li>
 
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</ul>
 
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<section>
 
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    <h2> Monday, August 13<span class="exposant">th</span>:</h2>
 
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We did some verification PCRs on miniprep with HF Phusion enzyme (<a href="http://2012.igem.org/Team:Grenoble/Biology/Protocols/PCR_2">protocol</a>) in order to check the
 
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Gibson Assembly products (12/09/09>.<br/>Annealing temperature = 55°C.<br/>
 
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<br/>
 
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To separate (<a href="http://2012.igem.org/Team:Grenoble/Biology/Protocols/Gel">protocol</a>) the PCR products, we prepared a 1.8% TAE agarose gel.<br/>
 
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Migration conditions = 100V during 30 min.<br/>
 
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In order to reveal the DNA fragments, we used <a href="http://2012.igem.org/Team:Grenoble/Biology/Protocols/EtBr">EtBr</a>.<br/>
 
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<br/>
 
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It showed no significant results (data not shown).
 
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</section>
 
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<section>
 
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    <h2> Tuesday, August 14<span class="exposant">th</span>:</h2>
 
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    We did an experiment (<a href="http://2012.igem.org/Team:Grenoble/Biology/Protocols/AND_test">protocol</a>) in order to test the "AND" gate (CRP-cAMP & araC) without the amplifier module. <br/>
 
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    <br/>
 
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    We transformed (new <a href="http://2012.igem.org/Team:Grenoble/Biology/Protocols/Transformation_2">protocol</a>) BW25113 WT
 
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competent cells (<a href="http://2012.igem.org/Team:Grenoble/Biology/Protocols/Competence">protocol</a>) with pSB3C5.<br/>
 
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</section>
 
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<section>
 
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    <h2> Wednesday, August 15<span class="exposant">th</span>:</h2>
 
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    We did an experiment (<a href="http://2012.igem.org/Team:Grenoble/Biology/Protocols/AND_test">protocol</a>) in order to test the "AND" gate (CRP-cAMP & araC) without the amplifier module. The M9 medium used was complemented: 0.1% acetate.
 
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</section>
 
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<section>
 
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    <h2>Thursday, August 16<span class="exposant">th</span>:</h2>
 
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We did some colony PCRs with HF Phusion enzyme (<a href="http://2012.igem.org/Team:Grenoble/Biology/Protocols/PCR_2">protocol</a>) in order to amplify pSB1A3 (araC), pSB3C5 (araC), pSB3C5 (pAra/Bad_RBS_GFP_RBS_Cya) and pSB4C5 (pAra/Bad_RBS_GFP_RBS_Cya)<br/>
 
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<br/>
 
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Then, we did some digestions (<a href="http://2012.igem.org/Team:Grenoble/Biology/Protocols/Restriction">protocol</a>). The digestions were achieved with two restriction enzymes:
 
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<ul>
 
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<li> pOmpC with EcoRI and SpeI during 10 minutes </li>
 
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<li> pOmpC with EcoRI</li>
 
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<li> pSB4K5 with EcoRI and SpeI during 10 minutes</li>
 
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<li> pSB4K5 with EcoRI during 10 minutes</li>
 
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</ul>
 
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<br/>
 
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To separate (<a href="http://2012.igem.org/Team:Grenoble/Biology/Protocols/Gel">protocol</a>) the PCR and the digestion products, we prepared a 1.8% TAE agarose gel.<br/>
 
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Migration conditions = 100V during 30 min.<br/>
 
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In order to reveal the DNA fragments, we used <a href="http://2012.igem.org/Team:Grenoble/Biology/Protocols/EtBr">EtBr</a>.<br/>
 
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<br/>
 
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The PCR and the digestion worked well, we realised a DNA extraction (<a href="http://2012.igem.org/Team:Grenoble/Biology/Protocols/DNA_extract">protocol</a>) from the pSB1A3 (araC) and pSB4C5 (pAra/Bad_RBS_GFP_RBS_Cya) PCR products.<br/>
 
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Latest revision as of 22:54, 26 September 2012