Team:Grenoble/Biology/Notebook/July

From 2012.igem.org

(Difference between revisions)
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<dd>Lane 8 and 9: rsmY PCR product<br/>
<dd>Lane 8 and 9: rsmY PCR product<br/>
<dd>Lane 10: DNA ladder 100bp (biolabs)<br/>
<dd>Lane 10: DNA ladder 100bp (biolabs)<br/>
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<br/>
 
<br/>
<br/>
<center><img src="https://static.igem.org/mediawiki/2012/6/67/120705.jpg" alt="photo_gel_2"/></center>
<center><img src="https://static.igem.org/mediawiki/2012/6/67/120705.jpg" alt="photo_gel_2"/></center>
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<dd>Lane 8 and 9: pAra/Bad_RBS_GFP PCR product<br>
<dd>Lane 8 and 9: pAra/Bad_RBS_GFP PCR product<br>
<dd>Lane 10: DNA ladder 1kb (biolabs)<br>
<dd>Lane 10: DNA ladder 1kb (biolabs)<br>
 +
<br/>
 +
There is no visible migration on the second gel (big fragments). We realized a DNA extraction from the three small fragments (protocol kit: Nucleospin extract II):
 +
<ul>
 +
<li>fha1 (PCR product code = 120705PP_PCR_001 and 120705PP_PCR_002)</li>
 +
<li>RsmA (PCR product code = 120705PP_PCR_003 and 120705PP_PCR_004)</li>
 +
<li>rsmY (PCR product code = 120705PP_PCR_005 and 120705PP_PCR_006)</li>
 +
</ul>
 +
<br/>
 +
Precultured cells are prepared:
 +
<ul>
 +
<li>Strains = iGEM Grenoble 2011 glycerol stock with fha, RsmA and rsmY.</li>
 +
<li>Conditions = LB liquid medium, 37°C, 200rpm, overnight.</li>
 +
</ul>
 +
<br/>
 +
We did colony PCR with a GoTaq enzyme (protocol) to amplify:
 +
<ul>
 +
<li>RBS_Cya from BW25113 WT strain</li>
 +
<li>pSB1A3 from iGEM 2011 glycerol stock</li>
 +
</ul>
 +
<br/>
 +
To separate the PCR products, we prepared a 0.8% TAE agarose gel.<br/>
 +
Migration conditions = 50V during 1h35.<br/>
 +
The DNA fragments were revealed with EtBr.<br/>
</section>
</section>

Revision as of 14:03, 6 August 2012

iGEM Grenoble 2012

Project
week 27 week 28 week 29 week 30

Week 27: July 02nd to July 08st

Goal of the week

We wanted to test the PCR conditions, recover and amplify the biobricks involved in our genetic networks :
  • pAra/Bad_RBS_GFP (1300bp)
  • RBS_Cya (2600bp)
  • pLAC (100bp)
  • fha (80bp)
  • eCFP (800bp)
  • pLAC_RBS (120bp)
  • RsmA (200bp)
  • rsmY (170bp)
  • pSB1A3 (2400bp)
  • pSB4K5 (2400bp)
  • pSB3C5 (2400bp)

Tuesday, July 03rd:

Precultured cells are prepared:
  • Strains = BW25113 WT, BW25113 cya- and BW25113 cya- pAra/Bad
  • Conditions = LB liquid medium, 37°C, 200rpm, overnight

Wednesday, July 04th:

We did PCRs on colony with a GoTaq enzyme (protocol) to amplify:
  • fha1, RsmA, rsmY and pSB1A3 from iGEM Grenoble 2011 glycerol stock.
  • pAra/Bad_RBS_GFP from BW25113 cya- pAra/Bad precultured cells.
  • RBS_Cya from BW25113 WT precultured cells.

Using iGEM 2012 biobricks we transformed BW25113 WT cells (protocol). We obtained four transformed strains with four different biobricks:
  • BBa_I13601: pLAC_RBS
  • BBa_E0422: eCFP
  • pSB3C5 plasmid
  • psB4K5 plasmid

Thursday, July 5th:

The transformations (12/07/04) show no growth on LB Agar plate + antibiotics.

In order to separate the PCR products (12/07/04), we prepared two gels:
  • a 1.3% Tris base Acetic acid EDTA (TAE) agarose gel, for the small fragments: RsmA, fha1 and rsmY (length < 1000bp)
  • a 0.8% TAE agarose gel, for the big fragments: pSB1A3, RBS_Cya, and pAra/Bad_RBS_GFP (length > 1000bp)

Migration conditions = 100V during 35 min.
In order to reveal the fragments, we used Ethidium Bromide (EtBr).

photo_gel_1
Migration results for the 1.3% TAE agarose gel (small fragments)
(the DNA ladder scale is in kb)
Lane 1: DNA ladder 100bp (biolabs)
Lane 2 and 3: not iGEM PCR products
Lane 4 and 5: RsmA PCR product
Lane 6 and 7: fha1 PCR product
Lane 8 and 9: rsmY PCR product
Lane 10: DNA ladder 100bp (biolabs)

photo_gel_2
Migration results for the 0.8% TAE agarose gel (big fragments)
(the DNA ladder scale is in kb)
Lane 1: DNA ladder 1kb (biolabs)
Lane 2 and 3: not iGEM PCR products
Lane 4 and 5: pSB1A3 PCR product
Lane 6 and 7: RBS_Cya PCR product
Lane 8 and 9: pAra/Bad_RBS_GFP PCR product
Lane 10: DNA ladder 1kb (biolabs)

There is no visible migration on the second gel (big fragments). We realized a DNA extraction from the three small fragments (protocol kit: Nucleospin extract II):
  • fha1 (PCR product code = 120705PP_PCR_001 and 120705PP_PCR_002)
  • RsmA (PCR product code = 120705PP_PCR_003 and 120705PP_PCR_004)
  • rsmY (PCR product code = 120705PP_PCR_005 and 120705PP_PCR_006)

Precultured cells are prepared:
  • Strains = iGEM Grenoble 2011 glycerol stock with fha, RsmA and rsmY.
  • Conditions = LB liquid medium, 37°C, 200rpm, overnight.

We did colony PCR with a GoTaq enzyme (protocol) to amplify:
  • RBS_Cya from BW25113 WT strain
  • pSB1A3 from iGEM 2011 glycerol stock

To separate the PCR products, we prepared a 0.8% TAE agarose gel.
Migration conditions = 50V during 1h35.
The DNA fragments were revealed with EtBr.