Team:Grenoble/Biology/Notebook/August

From 2012.igem.org

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We did some minipreps (protocol kit: Nucleospin plasmid Quick Pure) on the transformed strains (12/07/25) with pOmpC, mcherry, eCFP and pSB4C5 ; in order to set up the test on the receptor with a reporter<br/>
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We did some minipreps (protocol kit: Nucleospin plasmid Quick Pure) on the transformed strains (12/07/25) with pOmpC, mcherry, eCFP and pSB4C5 ; in order to set up the test on the receptor with a reporter.<br/>
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<br/>
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Then, we did some digestions (protocol) on these minipreps. The digestions were achieved with two restriction enzymes :
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<ul>
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<li> mcherry and eCFP with XbaI and SpeI during 10 minutes</li>
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<li> pSB4C5 with EcoRI and PstI during 10 minutes</li>
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</ul>
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<br/>
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To separate 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 EtBr.<br/>
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<br/>
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<center><img src="https://static.igem.org/mediawiki/2012/0/05/120801_%282%29.jpg" alt="photo_gel_25"/></center>
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<div class="legend"><u>Migration result for a 1.8% 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 80pb-10kb (fermentas)<br/>
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Lane 2: pOmpC miniprep product<br/>
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Lane 3: pOmpC miniprep product<br/>
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Lane 4: DNA ladder 80pb-10kb (fermentas)<br/>
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Lane 5: eCFP digestion product<br/>
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Lane 6: eCFP digestion product<br/>
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Lane 7: mcherry digestion product<br/>
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Lane 8: mcherry digestion product<br/>
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Lane 9: pSB4C5 digestion product<br/>
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Lane 10: pSB4C5 digestion product<br/>
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Lane 11: DNA ladder 1kb (biolabs)<br/></div>
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<br/>
</section>
</section>

Revision as of 12:01, 13 August 2012

iGEM Grenoble 2012

iGEM Grenoble iGEM
iGEM 2012
main page
Project

August

week 31 week 32 week 33 week 34 week 35

Week 31: July 30th to August 05th

Goal of the week:

We wanted to recover and amplify some biobricks involved in our genetic networks :
  • pSB4C5 (2400bp)
  • pompC (100bp)
  • mcherry (900bp)
  • eCFP (800bp)
  • GFP (100bp)

Tuesday, July 31th:

We did a miniprep (protocol kit: Nucleospin plasmid Quick Pure) on the transformed strain with pSB4C5 (12/07/25), on which we wanted to recover pSB4C5.

We did some colony PCRs (on 12/07/25 colony) and PCRs on miniprep with HF Phusion enzyme (protocol) in order to amplify pSB4C5.
Annealing temperature = 60°C.

To separate 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.

photo_gel_22
Migration result for a 1.8% TAE agarose gel
(the DNA ladder scale is in kb)
Lane 1: DNA ladder 1kb (biolabs)
Lane 2: pSB4C5 (miniprep) PCR product (DMSO)
Lane 3: pSB4C5 (miniprep) PCR product (DMSO)
Lane 4: pSB4C5 (miniprep) PCR product
Lane 5: pSB4C5 (miniprep) PCR product
Lane 6: pSB4C5 (colony) PCR product (DMSO)
Lane 7: pSB4C5 (colony) PCR product (DMSO)
Lane 8: pSB4C5 (colony) PCR product
Lane 9: pSB4C5 (colony) PCR product
Lane 10: DNA ladder 1kb (biolabs)

We only saw primer dimer bands, there was a PCR condition problem.

We did some PCRs on miniprep with HF Phusion enzyme (protocol) in order to amplify pSB4C5.
Annealing temperature = 65°C.

We did some digestions (protocol) on miniprep in order to do the construction: pAra/Bad_RBS_GFP_RBS_Cya. The digestions were achieved with two restriction enzymes : XbaI and SpeI during 10 minutes.

To separate the PCR products 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.

photo_gel_23
Migration result for a 1.8% TAE agarose gel
(the DNA ladder scale is in kb)
Lane 1: DNA ladder 1kb (biolabs)
Lane 2: pSBAC5 digestion product
Lane 3: pSB4C5 digestion product
Lane 4: pSB4C5 (miniprep) PCR product (DMSO)
Lane 5: pSB4C5 (miniprep) PCR product (DMSO)
Lane 6: pSB4C5 (miniprep) PCR product
Lane 7: pSB4C5 (miniprep) PCR product
Lane 8: pSB4C5 (colony) PCR product (DMSO)
Lane 9: pSB4C5 (colony) PCR product (DMSO)
Lane 10: pSB4C5 (colony) PCR product
Lane 11: pSB4C5 (colony) PCR product
Lane 12: DNA ladder 1kb (biolabs)

For the PCR results, we only saw primer dimer bands, there was a PCR condition problem.
The digestion worked well, we realised a DNA extraction (protocol kit: Nucleospin extract II) from the two digestion products (code = 120731AM_DIG_024).

Wednesday, August 01st:

We did a touchdown PCR (from 70°C to 60°C) from miniprep with HF Phusion enzyme (protocol) in order to amplify pSB4C5.

To separate the PCR products 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.

photo_gel_24
Migration result for a 1.8% TAE agarose gel
(the DNA ladder scale is in kb)
Lane 1: DNA ladder 1kb (biolabs)
Lane 2: pSBAC5 (digestion) PCR product
Lane 3: pSB4C5 (digestion) PCR product (DMSO)
Lane 4: pSB4C5 (miniprep) PCR product
Lane 5: pSB4C5 (miniprep) PCR product (DMSO)
Lane 6: DNA ladder 1kb (biolabs)

We only saw primer dimer bands, there was a PCR condition problem.

We realised four Gibson Assemblies (protocol) to build four plasmids:

  • pSB4C5 (fha1) (120726AM_PCR_021) with pLAC (fha1) (120713PP_PCR_009), fha1 (120723AM_PCR_020) and eCFP (120720_DIG_019)
    => 120801PP_GA_003

  • pSB1A3 (120713PP_PCR_014) with pLAC (rsmY) (120713PP_PCR_011) and rsmY (120713PP_PCR_013)
    => 120801PP_GA_004

  • pSB3C5 (Cya) (120713PP_PCR_015) with pAra/Bad_RBS_GFP (120727AM_PCR_023) and RBS_Cya (120727AM_PCR_022)
    => 120801PP_GA_005

  • pSB4C5 (120731AM_DIG_024) with pAra/Bad_RBS_GFP (120727AM_PCR_023) and RBS_Cya (120727AM_PCR_022)
    => 120713PP_GA_006

We did some minipreps (protocol kit: Nucleospin plasmid Quick Pure) on the transformed strains (12/07/25) with pOmpC, mcherry, eCFP and pSB4C5 ; in order to set up the test on the receptor with a reporter.

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

To separate 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.

photo_gel_25
Migration result for a 1.8% TAE agarose gel
(the DNA ladder scale is in kb)
Lane 1: DNA ladder 80pb-10kb (fermentas)
Lane 2: pOmpC miniprep product
Lane 3: pOmpC miniprep product
Lane 4: DNA ladder 80pb-10kb (fermentas)
Lane 5: eCFP digestion product
Lane 6: eCFP digestion product
Lane 7: mcherry digestion product
Lane 8: mcherry digestion product
Lane 9: pSB4C5 digestion product
Lane 10: pSB4C5 digestion product
Lane 11: DNA ladder 1kb (biolabs)

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