Team:Grenoble/Biology/Notebook/July/week 30

From 2012.igem.org

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Lane 12: DNA ladder 80bp-10kb (fermentas)<br/></div>
Lane 12: DNA ladder 80bp-10kb (fermentas)<br/></div>
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We saw primer dimer bands and the bands corresponding to the amplified fha1 sequence (lane 2 & 5). For the other, there was a PCR condition problem.<br/>
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<br/>
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We realised a DNA extraction (protocol kit: Nucleospin extract II) from the two fha PCR products (PCR product code = 120723AM_PCR_020).<br/>
 +
<br/>
 +
We did touchdown PCRs with HF Phusion enzyme (protocol) on digestion product (eCFP E0022 and E0422) (12/07/20) in order to amplify eCFP. We did the PCR both with and without DMSO.<br/>
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<br/>
 +
Using iGEM 2012 biobricks we transformed (new protocol) BW25113 WT competent cells (protocol) with pSB4K5.<br/>
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<br/>
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<h2> Tuesday, July 24<span class="exposant">th</span>:</h2>
 +
To separate the PCR products (12/07/23), we prepared a 1.3% TAE agarose gel.<br/>
 +
Migration conditions = 100V during 30 min.<br/>
 +
In order to reveal the DNA fragments, we used EtBr.<br/>
 +
<br/>
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<center><img src="https://static.igem.org/mediawiki/2012/e/e0/120724_%282%29.jpg" alt="photo_gel_17"/></center>
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<div class="legend"><u>Migration result for a 1.3% TAE agarose gel</u><br/>
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  <i>(the DNA ladder scale is in kb)</i><br/>
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Lane 1: DNA ladder 80bp-10kb (fermentas)<br/>
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Lane 2: E0022 PCR product<br/>
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Lane 3: E0022 PCR product (DMSO)<br/>
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Lane 4: E0422 PCR product<br/>
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Lane 5: E0422 PCR product (DMSO)<br/>
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Lane 6: DNA ladder 80bp-10kb (fermentas)<br/></div>
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<br/>
</section>
</section>
</body>
</body>

Revision as of 15:42, 7 August 2012

iGEM Grenoble 2012

iGEM Grenoble iGEM
iGEM 2012
main page
Project

July

week 27 week 28 week 29 week 30

Week 30: July 23rd to 29th

Goal of the week:

Test the Gibson Assembly and the transformation protocols, recover and amplify some biobricks involved in our genetic networks :
  • pAra/Bad_RBS_GFP (1300bp)
  • RBS_Cya (2600bp)
  • fha (80bp)
  • eCFP (800bp)
  • pSB4K5 (2400bp)

Monday, July 23rd:

We did PCRs with HF Phusion enzyme (protocol) on miniprep in order to amplify pAra/Bad_RBS_GFP, and on colony to amplify RBS_Cya. We did the PCR both with and without DMSO.

To separate the PCR products (12/07/23 and 12/07/20), 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_16
Migration result for a 1.3% TAE agarose gel
(the DNA ladder scale is in kb)
Lane 1: DNA ladder 80pb-10kb (fermentas)
Lane 2: pSB4K5 PCR product (DMSO)
Lane 3: fha1 PCR product (DMSO)
Lane 4: fha1 PCR product
Lane 5: pSB4K5 PCR product
Lane 6: pAra/Bad_RBS_GFP PCR product (DMSO)
Lane 7: pAra/Bad_RBS_GFP PCR product (DMSO)
Lane 8: RBS_Cya PCR product (DMSO)
Lane 9: RBS_Cya PCR product
Lane 10: pAra/Bad_RBS_GFP PCR product
Lane 11: pAra/Bad_RBS_GFP PCR product
Lane 12: DNA ladder 80bp-10kb (fermentas)

We saw primer dimer bands and the bands corresponding to the amplified fha1 sequence (lane 2 & 5). For the other, there was a PCR condition problem.

We realised a DNA extraction (protocol kit: Nucleospin extract II) from the two fha PCR products (PCR product code = 120723AM_PCR_020).

We did touchdown PCRs with HF Phusion enzyme (protocol) on digestion product (eCFP E0022 and E0422) (12/07/20) in order to amplify eCFP. We did the PCR both with and without DMSO.

Using iGEM 2012 biobricks we transformed (new protocol) BW25113 WT competent cells (protocol) with pSB4K5.

Tuesday, July 24th:

To separate the PCR products (12/07/23), we prepared a 1.3% TAE agarose gel.
Migration conditions = 100V during 30 min.
In order to reveal the DNA fragments, we used EtBr.

photo_gel_17
Migration result for a 1.3% TAE agarose gel
(the DNA ladder scale is in kb)
Lane 1: DNA ladder 80bp-10kb (fermentas)
Lane 2: E0022 PCR product
Lane 3: E0022 PCR product (DMSO)
Lane 4: E0422 PCR product
Lane 5: E0422 PCR product (DMSO)
Lane 6: DNA ladder 80bp-10kb (fermentas)

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