Team:Grenoble/Biology/Notebook/August/week 35

From 2012.igem.org

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constructions with pompC_mcherry and pAra/Bad_RBS_GFP_RBS_Cya.<br/>
constructions with pompC_mcherry and pAra/Bad_RBS_GFP_RBS_Cya.<br/>
<br/>
<br/>
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</section>
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<section><h2>Friday, August 31<span class="exposant">th</span>:</h2>
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We did some PCRs with HF Phusion enzyme (<a href="http://2012.igem.org/Team:Grenoble/Biology/Protocols/PCR_2">protocol</a>) in order to amplify pSB4C5 (mcherry),  pSB4C5 (pAra/Bad_RBS_GFP), RBS_Cya and pAra/Bad_RBS_GFP.<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> pSB4C5 with EcoRI and PstI during 10 minutes</li>
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<li> pSB4K5 with EcoRI and PstI during 10 minutes</li>
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<li> mcherry with PstI and XbaI during 10 minutes</li>
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<li> mcherry with PstI and XbaI 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 Cya and pAra/Bad_RBS_GFP PCR products and from the mcherry digestion products.<br/>
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<br/>
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</section>

Latest revision as of 03:25, 27 September 2012

iGEM Grenoble 2012

Project

August

Week 31Week 32Week 33Week 34Week 35

Week 35: August 27th to September 02nd

Goal of the week:

We wanted to construct (protocol) the double mutant strains we need for our experiments.
We also wanted to assemble pAra/Bad_RBS_GFP_RBS_Cya, pompC_mcherry, TapZ and tu put these constructions on pSB1C3.

Tuesday, August 28th:

We did some verification PCRs on miniprep with HF Phusion enzyme (protocol) in order to check the constructions with pompC_mcherry.

To separate (protocol) the PCR products, we prepared a 1.8% TAE agarose gel.
Migration conditions = 100V during 30 min.
In order to reveal the DNA fragments, we used EtBr.

The PCR showed no significant results (data not shown).

We did some digestions (protocol) in order to do the construction: pAra/Bad_RBS_GFP_RBS_Cya and the construcion: pompC_mcherry.

Wednesday, August 29th:

We did some ligations (protocol) in order to do the construction: pAra/Bad_RBS_GFP_RBS_Cya and the construcion: pompC_mcherry.

Thursday, August 30th:

We did some verification PCRs on miniprep with HF Phusion enzyme (protocol) in order to check the constructions with pompC_mcherry and pAra/Bad_RBS_GFP_RBS_Cya.

Friday, August 31th:

We did some PCRs with HF Phusion enzyme (protocol) in order to amplify pSB4C5 (mcherry), pSB4C5 (pAra/Bad_RBS_GFP), RBS_Cya and pAra/Bad_RBS_GFP.

Then, we did some digestions (protocol). The digestions were achieved with two restriction enzymes:
  • pSB4C5 with EcoRI and PstI during 10 minutes
  • pSB4K5 with EcoRI and PstI during 10 minutes
  • mcherry with PstI and XbaI during 10 minutes
  • mcherry with PstI and XbaI during 10 minutes

To separate (protocol) the PCR and the digestion products, we prepared a 1.8% TAE agarose gel.
Migration conditions = 100V during 30 min.
In order to reveal the DNA fragments, we used EtBr.

The PCR and the digestion worked well, we realised a DNA extraction (protocol) from the Cya and pAra/Bad_RBS_GFP PCR products and from the mcherry digestion products.