Team:Grenoble/Biology/Notebook/August/week 32

From 2012.igem.org

(Difference between revisions)
(Created page with "{{:Team:Grenoble/Templates/Biology}} <html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en"> <body id="Biology"> <div id="cadre"> <section> <h1>August</h1> <a href=...")
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<h1> Week 32: August 06<span class="exposant">th</span> to 12<span class="exposant">th</span> </h1>
<h1> Week 32: August 06<span class="exposant">th</span> to 12<span class="exposant">th</span> </h1>
<h2> Goal of the week: </h2>
<h2> Goal of the week: </h2>
 +
Assembly of pAra/Bad_RBS_GFP and RBS_Cya.
</section>
</section>
<section>
<section>
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<li><b>Lane 12:</b> pAra/Bad_RBS_GFP_RBS_Cya (pSB4C5) <b>2</b> digestion product</li>
<li><b>Lane 12:</b> pAra/Bad_RBS_GFP_RBS_Cya (pSB4C5) <b>2</b> digestion product</li>
<li><b>Lane 13:</b> pAra/Bad_RBS_GFP_RBS_Cya (pSB4C5) <b>2</b> miniprep product</li></ul></div>
<li><b>Lane 13:</b> pAra/Bad_RBS_GFP_RBS_Cya (pSB4C5) <b>2</b> miniprep product</li></ul></div>
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<center><img src="http://2012.igem.org/wiki/images/3/3e/120806.jpg" alt="photo_gel_29"/></center>
<center><img src="http://2012.igem.org/wiki/images/3/3e/120806.jpg" alt="photo_gel_29"/></center>
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<li><b>Lane 7:</b> pAra/Bad_RBS_GFP_RBS_Cya (pSB4C5) <b>2</b> miniprep product</li>
<li><b>Lane 7:</b> pAra/Bad_RBS_GFP_RBS_Cya (pSB4C5) <b>2</b> miniprep product</li>
<li><b>Lane 8:</b> DNA ladder 80pb-10kb (fermentas)</li>
<li><b>Lane 8:</b> DNA ladder 80pb-10kb (fermentas)</li>
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</ul></div>
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<br/>
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The digestions showed no significant results.
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  </section>
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<section>
 +
<h2> Thursday, August 07<span class="exposant">th</span>:</h2>
 +
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
 +
constructions.<br/>
 +
<br/>
 +
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/>
 +
Migration conditions = 100V during 30 min.<br/>
 +
In order to reveal the DNA fragments, we used <a href="http://2012.igem.org/Team:Grenoble/Biology/Protocols/EtBr">EtBr</a>.<br/>
 +
<br/>
 +
The PCR showed no significant results (data not shown).
</section>
</section>
 +
<section>
<section>
 +
<h2> Wednesday, August 08<span class="exposant">th</span>:</h2>
 +
We did some digestions (<a href="http://2012.igem.org/Team:Grenoble/Biology/Protocols/Restriction">protocol</a>) in order to check the
 +
construction: pAra/Bad_RBS_GFP_RBS_Cya and to do the construcion: pompC_mcherry.<br/>
 +
<br/>
 +
We did an overlapping PCR on miniprep with HF Phusion enzyme (<a href="http://2012.igem.org/Team:Grenoble/Biology/Protocols/PCR_2">protocol</a>) in order to do the construction: pAra/Bad_RBS_GFP_RBS_Cya.<br/>
 +
<br/>
 +
To separate (<a href="http://2012.igem.org/Team:Grenoble/Biology/Protocols/Gel">protocol</a>) the PCR products and the digestion products, we prepared 1.8% TAE agarose gels.<br/>
 +
Migration conditions = 100V during 30 min.<br/>
 +
In order to reveal the DNA fragments, we used <a href="http://2012.igem.org/Team:Grenoble/Biology/Protocols/EtBr">EtBr</a>.<br/>
 +
<br/>
 +
The digestions experiments worked well, the PCR didn't work (data not shown).
 +
</section>
 +
<section>
 +
  <h2> Tuesday, August 09<span class="exposant">th</span>:</h2>
 +
    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 amplify RBS_Cya and pAra/Bad_RBS_GFP.<br/>
 +
<br/>
 +
We realised a Gibson Assembly (<a href="http://2012.igem.org/Team:Grenoble/Biology/Protocols/GA">protocol</a>) to build: pSB3C5 with pAra/Bad_RBS_GFP and RBS_Cya.<br/>
 +
With our Gibson Assembly products, we transformed (new <a href="http://2012.igem.org/Team:Grenoble/Biology/Protocols/Transformation_2">protocol</a>) BW25113 Cya<span class="exposant">-</span>
 +
competent cells (<a href="http://2012.igem.org/Team:Grenoble/Biology/Protocols/Competence">protocol</a>).<br/>
 +
<br/>
 +
We did an overlapping PCR on miniprep with HF Phusion enzyme (<a href="http://2012.igem.org/Team:Grenoble/Biology/Protocols/PCR_2">protocol</a>) in order to do the construction: pAra/Bad_RBS_GFP_RBS_Cya.<br/>
 +
It showed no significant result (data not shown).
 +
</section>
 +
 +
<section>
 +
  <h2> Friday, August 10<span class="exposant">th</span>:</h2>
 +
    We relaunched in fresh LB the cultures of transformed cells made the day before (12/08/09).<br/>
 +
    <br/>
 +
    We did a miniprep (<a href="http://2012.igem.org/Team:Grenoble/Biology/Protocols/Miniprep">protocol</a>) on these transformed strains.
 +
</section>
 +
 +
<section>
 +
<h2> Conclusion of the week:</h2>
 +
We didn't achieved to construct: pAra/Bad_RBS_GFP_RBS_Cya.
 +
</section>
 +
</div>
</div>
</body>
</body>

Revision as of 10:33, 26 September 2012

iGEM Grenoble 2012

Project

August

Week 31Week 32Week 33Week 34Week 35

Week 32: August 06th to 12th

Goal of the week:

Assembly of pAra/Bad_RBS_GFP and RBS_Cya.

Monday, August 06th:

We wanted to check if the Gibson Assemblies (12/08/01) worked well.

To separate (protocol) the GA miniprep (12/08/03) 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.

photo_gel_27

Migration result for a 1.8% TAE agarose gel
(the DNA ladder scale is in kb)

  • Lane 1: pLAC_rsmY (pSB1A3) 3 miniprep product
  • Lane 2: pLAC_rsmY (pSB1A3) 2 miniprep product
  • Lane 3: pLAC_rsmY (pSB1A3) 1 miniprep product
  • Lane 4: pLAC_fha1_eCFP (pSB4C5) 2 miniprep product
  • Lane 5: pLAC_fha1_eCFP (pSB4C5) 1 miniprep product
  • Lane 6: DNA ladder 1kb (biolabs)
  • Lane 7: pAra/Bad_RBS_GFP_RBS_Cya (pSB3C5) 1 miniprep product
  • Lane 8: pAra/Bad_RBS_GFP_RBS_Cya (pSB3C5) 2 miniprep product
  • Lane 9: pAra/Bad_RBS_GFP_RBS_Cya (pSB4C5) 4 miniprep product
  • Lane 10: pAra/Bad_RBS_GFP_RBS_Cya (pSB4C5) 3 miniprep product
  • Lane 11: pAra/Bad_RBS_GFP_RBS_Cya (pSB4C5) 1 miniprep product
  • Lane 12: pAra/Bad_RBS_GFP_RBS_Cya (pSB4C5) 2 miniprep product
  • Lane 13: DNA ladder 1kb (biolabs)


We did some digestions (protocol) on GA miniprep (12/08/03). The digestions were achieved with XbaI during 10 minutes.

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

photo_gel_28

Migration result for a 1.8% TAE agarose gel
(the DNA ladder scale is in kb)

  • Lane 1: data not shown pAra/Bad_RBS_GFP_RBS_Cya (pSB3C5) 1 digestion product
  • Lane 2: data not shown pAra/Bad_RBS_GFP_RBS_Cya (pSB3C5) 1 miniprep product
  • Lane 3: pAra/Bad_RBS_GFP_RBS_Cya (pSB3C5) 2 digestion product
  • Lane 4: pAra/Bad_RBS_GFP_RBS_Cya (pSB3C5) 2 miniprep product
  • Lane 5: pAra/Bad_RBS_GFP_RBS_Cya (pSB4C5) 4 digestion product
  • Lane 6: pAra/Bad_RBS_GFP_RBS_Cya (pSB4C5) 4 miniprep product
  • Lane 7: DNA ladder 1kb (biolabs)
  • Lane 8: pAra/Bad_RBS_GFP_RBS_Cya (pSB4C5) 3 digestion product
  • Lane 9: pAra/Bad_RBS_GFP_RBS_Cya (pSB4C5) 3 miniprep product
  • Lane 10: pAra/Bad_RBS_GFP_RBS_Cya (pSB4C5) 1 digestion product
  • Lane 11: pAra/Bad_RBS_GFP_RBS_Cya (pSB4C5) 1 miniprep product
  • Lane 12: pAra/Bad_RBS_GFP_RBS_Cya (pSB4C5) 2 digestion product
  • Lane 13: pAra/Bad_RBS_GFP_RBS_Cya (pSB4C5) 2 miniprep product

photo_gel_29

Migration result for a 1.8% TAE agarose gel
(the DNA ladder scale is in kb)

  • Lane 1: DNA ladder 1kb (biolabs)
  • Lane 2: pAra/Bad_RBS_GFP_RBS_Cya (pSB4C5) 4 digestion product
  • Lane 3: pAra/Bad_RBS_GFP_RBS_Cya (pSB4C5) 4 miniprep product
  • Lane 4: pAra/Bad_RBS_GFP_RBS_Cya (pSB4C5) 1 digestion product
  • Lane 5: pAra/Bad_RBS_GFP_RBS_Cya (pSB4C5) 1 miniprep product
  • Lane 6: pAra/Bad_RBS_GFP_RBS_Cya (pSB4C5) 2 digestion product
  • Lane 7: pAra/Bad_RBS_GFP_RBS_Cya (pSB4C5) 2 miniprep product
  • Lane 8: DNA ladder 80pb-10kb (fermentas)

The digestions showed no significant results.

Thursday, August 07th:

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

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).

Wednesday, August 08th:

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

We did an overlapping PCR on miniprep with HF Phusion enzyme (protocol) in order to do the construction: pAra/Bad_RBS_GFP_RBS_Cya.

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

The digestions experiments worked well, the PCR didn't work (data not shown).

Tuesday, August 09th:

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

We realised a Gibson Assembly (protocol) to build: pSB3C5 with pAra/Bad_RBS_GFP and RBS_Cya.
With our Gibson Assembly products, we transformed (new protocol) BW25113 Cya- competent cells (protocol).

We did an overlapping PCR on miniprep with HF Phusion enzyme (protocol) in order to do the construction: pAra/Bad_RBS_GFP_RBS_Cya.
It showed no significant result (data not shown).

Friday, August 10th:

We relaunched in fresh LB the cultures of transformed cells made the day before (12/08/09).

We did a miniprep (protocol) on these transformed strains.

Conclusion of the week:

We didn't achieved to construct: pAra/Bad_RBS_GFP_RBS_Cya.