Team:Grenoble/Biology/Notebook/August/week 33

From 2012.igem.org

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<section>
<section>
     <h2>Thursday, August 16<span class="exposant">th</span>:</h2>
     <h2>Thursday, August 16<span class="exposant">th</span>:</h2>
-
We did some colony PCRs with HF Phusion enzyme (<a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/PCR_2">protocol</a>) in order to amplify pSB1A3 (araC), pSB3C5 (araC), pSB3C5 (pAra/Bad_RBS_GFP_RBS_Cya) and pSB4C5 (pAra/Bad_RBS_GFP_RBS_Cya)<br/>
+
We did some colony PCRs with HF Phusion enzyme (<a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/PCR_2">protocol</a>) in order to amplify pSB1A3 (araC), pSB3C5 (araC), pSB3C5 (pAra/Bad_RBS_GFP_RBS_Cya) and pSB4C5 (pAra/Bad_RBS_GFP_RBS_Cya).<br/>
<br/>
<br/>
Then, we did some digestions (<a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/Restriction">protocol</a>). The digestions were achieved with two restriction enzymes:  
Then, we did some digestions (<a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/Restriction">protocol</a>). The digestions were achieved with two restriction enzymes:  
<ul>
<ul>
<li> pOmpC with EcoRI and SpeI during 10 minutes </li>
<li> pOmpC with EcoRI and SpeI during 10 minutes </li>
-
<li> pOmpC with EcoRI</li>
+
<li> pOmpC with EcoRI during 10 minute</li>
<li> pSB4K5 with EcoRI and SpeI during 10 minutes</li>
<li> pSB4K5 with EcoRI and SpeI during 10 minutes</li>
<li> pSB4K5 with EcoRI during 10 minutes</li>
<li> pSB4K5 with EcoRI during 10 minutes</li>
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In order to reveal the DNA fragments, we used <a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/EtBr">EtBr</a>.<br/>
In order to reveal the DNA fragments, we used <a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/EtBr">EtBr</a>.<br/>
<br/>
<br/>
-
The PCR and the digestion worked well, we realised a DNA extraction (<a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/DNA_extract">protocol</a>) from the pSB1A3 (araC) and pSB4C5 (pAra/Bad_RBS_GFP_RBS_Cya) PCR products.<br/>
+
The PCR and the digestion worked well, we realised a DNA extraction (<a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/DNA_extract">protocol</a>) from the pSB1A3 (araC) and pSB4C5 (pAra/Bad_RBS_GFP_RBS_Cya) PCR products and from the pOmpC and pSB4K5 digestion products.<br/>
<br/>
<br/>
 +
We did some PCRs with HF Phusion enzyme (<a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/PCR_2">protocol</a>) in order to amplify pSB4C5 (mcherry),  pSB4C5 (pAra/Bad_RBS_GFP), RBS_Cya and pAra/Bad_RBS_GFP.<br/>
 +
<br/>
 +
Then, we did some digestions (<a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/Restriction">protocol</a>). The digestions were achieved with two restriction enzymes:
 +
<ul>
 +
<li> pSB4C5 with EcoRI and PstI during 10 minutes</li>
 +
<li> pSB4K5 with EcoRI and PstI during 10 minutes</li>
 +
<li> mcherry with PstI and XbaI during 10 minutes</li>
 +
<li> mcherry with PstI and XbaI during 10 minutes</li>
 +
</ul>
 +
<br/>
 +
To separate (<a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/Gel">protocol</a>) the PCR and the digestion 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="https://2012.igem.org/Team:Grenoble/Biology/Protocols/EtBr">EtBr</a>.<br/>
 +
<br/>
 +
The PCR and the digestion worked well, we realised a DNA extraction (<a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/DNA_extract">protocol</a>) from the Cya and pAra/Bad_RBS_GFP PCR products and from the mcherry digestion products.<br/>
 +
<br/>
 +
We did some digestions (<a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/Restriction">protocol</a>) on pSB3C5, pSB4K5 and pSB1A3. The digestions were achieved with two restriction enzymes: EcoRI and PstI during 10 minutes.<br/>
 +
<br/>
 +
To separate (<a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/Gel">protocol</a>) the PCR and the digestion 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="https://2012.igem.org/Team:Grenoble/Biology/Protocols/EtBr">EtBr</a>.<br/>
 +
<br/>
 +
The digestion worked well, we realised a DNA extraction (<a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/DNA_extract">protocol</a>) from the pSB1A3 and pSB3C5 products.<br/>
 +
</section>
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Latest revision as of 03:26, 27 September 2012

iGEM Grenoble 2012

iGEM Grenoble iGEM
iGEM 2012
main page
Project

August

Week 31 • Week 32 • Week 33 • Week 34 • Week 35

Week 33: August 13th to 19th

Goal of the week:

Assembly of:
  • pAra/Bad_RBS_GFP and RBS_Cya.
  • pompC and mcherry
  • Tap and EnvZ

Monday, August 13th:

We did some verification PCRs on miniprep with HF Phusion enzyme (protocol) in order to check the Gibson Assembly products (12/09/09>.
Annealing temperature = 55°C.

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.

It showed no significant results (data not shown).

Tuesday, August 14th:

We did an experiment (protocol) in order to test the "AND" gate (CRP-cAMP & araC) without the amplifier module.

We transformed (new protocol) BW25113 WT competent cells (protocol) with pSB3C5.

Wednesday, August 15th:

We did an experiment (protocol) in order to test the "AND" gate (CRP-cAMP & araC) without the amplifier module. The M9 medium used was complemented: 0.1% acetate.

Thursday, August 16th:

We did some colony PCRs with HF Phusion enzyme (protocol) in order to amplify pSB1A3 (araC), pSB3C5 (araC), pSB3C5 (pAra/Bad_RBS_GFP_RBS_Cya) and pSB4C5 (pAra/Bad_RBS_GFP_RBS_Cya).

Then, we did some digestions (protocol). The digestions were achieved with two restriction enzymes:
  • pOmpC with EcoRI and SpeI during 10 minutes
  • pOmpC with EcoRI during 10 minute
  • pSB4K5 with EcoRI and SpeI during 10 minutes
  • pSB4K5 with EcoRI during 10 minutes

To separate (protocol) the PCR 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.

The PCR and the digestion worked well, we realised a DNA extraction (protocol) from the pSB1A3 (araC) and pSB4C5 (pAra/Bad_RBS_GFP_RBS_Cya) PCR products and from the pOmpC and pSB4K5 digestion products.

We did some PCRs with HF Phusion enzyme (protocol) in order to amplify pSB4C5 (mcherry), pSB4C5 (pAra/Bad_RBS_GFP), RBS_Cya and pAra/Bad_RBS_GFP.

Then, we did some digestions (protocol). The digestions were achieved with two restriction enzymes:
  • pSB4C5 with EcoRI and PstI during 10 minutes
  • pSB4K5 with EcoRI and PstI during 10 minutes
  • mcherry with PstI and XbaI during 10 minutes
  • mcherry with PstI and XbaI during 10 minutes

To separate (protocol) the PCR 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.

The PCR and the digestion worked well, we realised a DNA extraction (protocol) from the Cya and pAra/Bad_RBS_GFP PCR products and from the mcherry digestion products.

We did some digestions (protocol) on pSB3C5, pSB4K5 and pSB1A3. The digestions were achieved with two restriction enzymes: EcoRI and PstI during 10 minutes.

To separate (protocol) the PCR 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.

The digestion worked well, we realised a DNA extraction (protocol) from the pSB1A3 and pSB3C5 products.

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