Team:Toulouse/Notebook/Protocols

From 2014.igem.org

(Difference between revisions)
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- Let the culture grow overnight at 37°C in a shaking incubator</p>  
- Let the culture grow overnight at 37°C in a shaking incubator</p>  
-
<p class="texte"><B> Day 1 </B></p>
+
<p class="texte"><B> Day 1 </B>
<br>- Use the QIAprep spin Miniprep Kit for each culture tube. The last step consisting in the elution of the DNA is made with a hot elution buffer or water at 55°C.
<br>- Use the QIAprep spin Miniprep Kit for each culture tube. The last step consisting in the elution of the DNA is made with a hot elution buffer or water at 55°C.
<br/>
<br/>
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<p class="title1" id="select4"> Cloning </p>
<p class="title1" id="select4"> Cloning </p>
<p class="texte">
<p class="texte">
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<br>After taking the competent cells, transforming the Biobricks and making the miniprep, make the digestion mix.
+
<br>After taking the competent cells, transforming the BioBricks and making the miniprep, make the digestion mix.
<br>
<br>
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<p class="texte"><B> First Step </B>
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<p class="title2">First step</p>
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<br> <B> BOTH PARTS HAVE THE SAME ANTIBIOTIC RESISTANCE </B>
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<p class="title3">Both parts have the same antibiotic resistance</p>
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<br> 1) Digestion mix
+
<p class="texte">1) Digestion mix
<br> For the vector :
<br> For the vector :
<br>- 5 µL of miniprep plasmid  
<br>- 5 µL of miniprep plasmid  
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- Incubate 15 minutes at 37°C  
- Incubate 15 minutes at 37°C  
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<p class="texte">2) Gel extraction
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<br><br>2) Gel extraction
<br>
<br>
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- Prepare a 1% of 2% electrophoresis agarose gel with 0.5x TAE buffer
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- Prepare a 1% or 2% electrophoresis agarose gel with 0.5x TAE buffer
<br>
<br>
- Put 20µL of sample + 6µL of marker (1kb for 1% gel and 100pb for 2%) into the well
- Put 20µL of sample + 6µL of marker (1kb for 1% gel and 100pb for 2%) into the well
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- Migration for 30 min at 100V or 1hour at 50V.
- Migration for 30 min at 100V or 1hour at 50V.
<br>
<br>
-
- The revelation is made in BET (10minutes) and then 5minutes in water.
+
- The revelation is made in BET (10 minutes) and then 5 minutes in water.
<br>
<br>
- The gel extraction is realized thanks to the THERMO SCIENTIFIC GeneJET Gel Extraction and DNA Clean Up Microkit.
- The gel extraction is realized thanks to the THERMO SCIENTIFIC GeneJET Gel Extraction and DNA Clean Up Microkit.
-
<p class="texte">3) Inactivation of the enzymes for the vector
+
<br><br>3) Inactivation of the enzymes for the vector
<br>There are two ways to inactivate the enzymes :
<br>There are two ways to inactivate the enzymes :
<br>- Use of DNA Clean up kit for the DNA fragment above 200 pb
<br>- Use of DNA Clean up kit for the DNA fragment above 200 pb
-
<br>- Heat inactivation at 95°C for 10 minutes.
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<br>- Heat inactivation at 95°C for 10 minutes.</p>
-
<p class="texte">
+
<p class="title3">The two parts have a different antibiotic resistance</p>
-
<B> THE TWO PARTS HAVE A DIFFERENT ANTIBIOTIC RESISTANCE </B>
+
<p class="texte">1) Digestion mix  
-
<br>1) Digestion mix  
+
<br>For each part, add :  
<br>For each part, add :  
<br>- 5 µL of miniprep plasmid  
<br>- 5 µL of miniprep plasmid  
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<br>There are two ways to inactivate the enzymes :
<br>There are two ways to inactivate the enzymes :
<br>- Use of DNA Clean up kit for the DNA fragment above 200 pb
<br>- Use of DNA Clean up kit for the DNA fragment above 200 pb
-
<br>- Heat inactivation at 95°C for 10 minutes.
+
<br>- Heat inactivation at 95°C for 10 minutes.</p>
-
<p class="texte">
+
<p class="title2">Second step</p>
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<B> Second step </B>
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<p class="title3">Ligation</p>
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<br><B> Ligation </B>
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<p class="texte">- Mix 10 µL of insert + 4µL of vector + 2µL of 10x T4 buffer + 0.5µL of T4 ligase + 3.5µL of Milli-Q water
-
<br/>
+
-
- Mix 10 µL of insert + 4µL of vector + 2µL of 10x T4 buffer + 0.5µL of T4 ligase + 3.5µL of Milli-Q water
+
<br/>
<br/>
A control without insert must be made
A control without insert must be made
<br/>
<br/>
-
- Incubate the ligation mix 15 minutes at room temperature (22°C) and keep the tubes in ice or at -20°C to prepare the transformation
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- Incubate the ligation mix 15 minutes at room temperature (22°C) and keep the tubes in ice or at -20°C to prepare the transformation</p>
-
<br/>
+
-
</p>
+
 +
<p class="title3">Transformation</p>
<p class="texte">
<p class="texte">
-
2) Transformation
+
- Take 5µL of the ligation mix for 50µL of competent cells and use the <a href="https://2014.igem.org/Team:Toulouse/Notebook/Protocols#select2">Toulouse iGEM Team 2014 transformation protocol</a>.
-
<br/>
+
-
- Take 5µL of the ligation mix for 50µL of competent cells and use the Toulouse iGEM Team 2014 transformation protocol.
+
-
<br/>
+
-
- Plate the solution on selective medium overnight at 37°C.
+
<br/>
<br/>
-
</p>
+
- Plate the solution on selective medium overnight at 37°C.</p>
<p class="title1 " id="select5">Checking of the genetic constructions  </p>
<p class="title1 " id="select5">Checking of the genetic constructions  </p>

Revision as of 18:30, 16 October 2014