Team:Grenoble/Biology/Notebook/July

From 2012.igem.org

(Difference between revisions)
Line 106: Line 106:
</ul>
</ul>
<br/>
<br/>
-
To separate the PCR products, we prepared a 0.8% TAE agarose gel.<br/>
+
To separate (<a href="http://2012.igem.org/Team:Grenoble/Biology/Protocols/Gel">protocol</a>) the PCR products, we prepared a 0.8% TAE agarose gel.<br/>
Migration conditions = 50V during 1h35.<br/>
Migration conditions = 50V during 1h35.<br/>
-
The DNA fragments were revealed with EtBr.<br/>
+
The DNA fragments were revealed with <a href="http://2012.igem.org/Team:Grenoble/Biology/Protocols/EtBr">EtBr</a>.<br/>
<br/>
<br/>
<center><img src="http://2012.igem.org/wiki/images/c/c1/120705_%283%29.jpg" alt="photo_gel_3"/></center>
<center><img src="http://2012.igem.org/wiki/images/c/c1/120705_%283%29.jpg" alt="photo_gel_3"/></center>
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As our PCR results were not conclusive, we decided to test the PCR conditions.<br/>
As our PCR results were not conclusive, we decided to test the PCR conditions.<br/>
<br/>
<br/>
-
We did a miniprep (protocol kit: Nucleospin plasmid Quick Pure) on BW25113 cya<span class="exposant">-</span> pAra/Bad strain, in order to recover the plasmid with pAra/Bad_RBS_GFP on it.<br/>
+
We did a miniprep (<a href="http://2012.igem.org/Team:Grenoble/Biology/Protocols/Miniprep">protocol</a>) on BW25113 cya<span class="exposant">-</span> pAra/Bad strain, in order to recover the plasmid with pAra/Bad_RBS_GFP on it.<br/>
<br/>
<br/>
To test the PCR conditions, we separated the sample in two groups (to check the Green buffer effect) subdivised into five samples according to the amount of template DNA:
To test the PCR conditions, we separated the sample in two groups (to check the Green buffer effect) subdivised into five samples according to the amount of template DNA:
<ul>
<ul>
-
<li>1<span class="exposant">st</span> group: PCR with GoTaq (protocol) and Green Buffer 5X</li>
+
<li>1<span class="exposant">st</span> group: PCR with GoTaq (<a href="http://2012.igem.org/Team:Grenoble/Biology/Protocols/PCR_1">protocol</a>) and Green Buffer 5X</li>
-
<li>2<span class="exposant">nd</span> group: PCR with GoTaq (protocol) and Colorless Buffer 5X (+ loading dye for the migration)</li>
+
<li>2<span class="exposant">nd</span> group: PCR with GoTaq (<a href="http://2012.igem.org/Team:Grenoble/Biology/Protocols/PCR_1">protocol</a>) and Colorless Buffer 5X (+ loading dye for the migration)</li>
</ul>
</ul>
<br/>For each group, there were fivz samples (different concentration of template DNA):
<br/>For each group, there were fivz samples (different concentration of template DNA):
Line 152: Line 152:
<ul>
<ul>
<li>the 1<span class="exposant">st</span>: no digestion</li>
<li>the 1<span class="exposant">st</span>: no digestion</li>
-
<li>the 2<span class="exposant">nd</span>: one hour digestion (protocol) by a restriction enzyme (=XbaI)</li>
+
<li>the 2<span class="exposant">nd</span>: one hour digestion (<a href="http://2012.igem.org/Team:Grenoble/Biology/Protocols/Restriction">protocol</a>) by a restriction enzyme (=XbaI)</li>
-
<li>the 3<span class="exposant">rd</span>: 10 min digestion (protocol) by a restriction enzyme (=XbaI)</li>
+
<li>the 3<span class="exposant">rd</span>: 10 min digestion (<a href="http://2012.igem.org/Team:Grenoble/Biology/Protocols/Restriction">protocol</a>) by a restriction enzyme (=XbaI)</li>
</ul>
</ul>
<br/>For each group, there were five samples with different concentration of DNA:
<br/>For each group, there were five samples with different concentration of DNA:
Line 163: Line 163:
<li>5<span class="exposant">th</span> with 0μL of DNA template</li>
<li>5<span class="exposant">th</span> with 0μL of DNA template</li>
</ul>
</ul>
-
<br/>To separate the PCR products and the digestion products, we prepared a 0.8% TAE agarose gel.<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 a 0.8% TAE agarose gel.<br/>
Migration conditions = 50V during 1h35.<br/>
Migration conditions = 50V during 1h35.<br/>
-
The DNA fragments were revealed with EtBr.<br/>
+
The DNA fragments were revealed with <a href="http://2012.igem.org/Team:Grenoble/Biology/Protocols/EtBr">EtBr</a>.<br/>
<br/>
<br/>
There was no visible migration on the gel (data not shown).
There was no visible migration on the gel (data not shown).

Revision as of 08:33, 25 September 2012

iGEM Grenoble 2012

Project

July

Week 27Week 28Week 29Week 30

Week 27: July 02nd to 08th

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 (protocol) 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)
Lanes 2 and 3: not iGEM PCR products
Lanes 4 and 5: RsmA PCR product
Lanes 6 and 7: fha1 PCR product
Lanes 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)
Lanes 2 and 3: not iGEM PCR products
Lanes 4 and 5: pSB1A3 PCR product
Lanes 6 and 7: RBS_Cya PCR product
Lanes 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 (protocol) from the three small fragments (protocol kit: Nucleospin extract II):
  • fha1 120705PP_PCR_001 // 120705PP_PCR_002
  • RsmA 120705PP_PCR_003 // 120705PP_PCR_004
  • rsmY 120705PP_PCR_005 // 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 (protocol) the PCR products, we prepared a 0.8% TAE agarose gel.
Migration conditions = 50V during 1h35.
The DNA fragments were revealed with EtBr.

photo_gel_3
Migration results for a 0.8% TAE agarose gel
(the DNA ladder scale is in kb)
Lane 1: DNA ladder 1kb (biolabs)
Lanes 2 and 3: RBS_Cya PCR product
Lanes 4 and 5: not iGEM PCR product
Lanes 6 and 7: not iGEM PCR product
Lanes 8 and 9: pSB1A3 PCR product
Lane 10: DNA ladder 1kb (biolabs)

There is no visible migration on the gel, we only saw primer dimer bands. There was a PCR condition problem.

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

Friday, July 6th:

As our PCR results were not conclusive, we decided to test the PCR conditions.

We did a miniprep (protocol) on BW25113 cya- pAra/Bad strain, in order to recover the plasmid with pAra/Bad_RBS_GFP on it.

To test the PCR conditions, we separated the sample in two groups (to check the Green buffer effect) subdivised into five samples according to the amount of template DNA:
  • 1st group: PCR with GoTaq (protocol) and Green Buffer 5X
  • 2nd group: PCR with GoTaq (protocol) and Colorless Buffer 5X (+ loading dye for the migration)

For each group, there were fivz samples (different concentration of template DNA):
  • 1st with 1μL of DNA template (= plasmid from the miniprep)
  • 2nd with 2μL of DNA template
  • 3rd with 3μL of DNA template
  • 4th with 4μL of DNA template
  • 5th with 0μL of DNA template


On the same plasmid (from the miniprep to check its efficiency), we separated the sample in three groups ; each group is subdivised into five samples to test the different amount of template DNA:
  • the 1st: no digestion
  • the 2nd: one hour digestion (protocol) by a restriction enzyme (=XbaI)
  • the 3rd: 10 min digestion (protocol) by a restriction enzyme (=XbaI)

For each group, there were five samples with different concentration of DNA:
  • 1st with 1μL of DNA template (= plasmid from the miniprep)
  • 2nd with 2μL of DNA template
  • 3rd with 3μL of DNA template
  • 4th with 4μL of DNA template
  • 5th with 0μL of DNA template

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

There was no visible migration on the gel (data not shown).

Conclusion of the week:

As we had no conclusive results, we have decided to change the protocols.