Team:Toulouse/Notebook/Protocols

From 2014.igem.org

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<B> BOTH PARTS HAVE THE SAME ANTIBIOTIC RESISTANCE </B>
<B> BOTH PARTS HAVE THE SAME ANTIBIOTIC RESISTANCE </B>
<br> 1) Digestion mix
<br> 1) Digestion mix
 +
<br> For the vector :
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<br>- 5 µL of miniprep plasmid
<br>
<br>
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- 10 µL of miniprep plasmid  
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- 2 µL of each restriction enzymes
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<br>
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- 2 µL of Green Buffer
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<br>
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- 9 µL of Milli-Q water
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<br>
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<br> For the insert :
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<br>- 10 µL of miniprep plasmid  
<br>
<br>
- 2 µL of each restriction enzymes
- 2 µL of each restriction enzymes
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- 2 µL of Green Buffer
- 2 µL of Green Buffer
<br>
<br>
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- 6 µL of water  
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- 4 µL of Milli-Q water  
<br>
<br>
 Incubate 15 minutes at 37°C  
 Incubate 15 minutes at 37°C  
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<p>
<p>
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2) Make an electrophoresis
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2) Gel extraction
<br>
<br>
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- Prepare 1% of 2% electrophoresis agarose gel with 0.5x TAE buffer
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- Prepare a 1% of 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
<br>
<br>
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- Migration 30 min at 100V
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- Migration for 30 min at 100V or 1hour at 50V.
<br>
<br>
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- The revelation is made in BET (10minutes) and then 5minutes in water
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- The revelation is made in BET (10minutes) and then 5minutes in water.
<br>
<br>
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- The gel extraction is realized thanks to the THERMO SCIENTIFIC GeneJET Gel Extraction and DNA Clean Up Microkit.
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<p>
<p>
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3) Gel extraction with Thermofisher kit
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4) Inactivation of the enzymes for the vector
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<br>
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<br>There are two ways to inactivate the enzymes :
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- Remove quickly under UV the DNA fragment from BET to avoid any kind of mutation. Place the fragment in a 1.5mL Eppendorf tube. Make sure to excise as close as possible from the fragment.
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<br>- Use of DNA Clean up kit for the DNA fragment above 200 pb
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Take a picture under UV before and after the excision.
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<br>- Heat inactivation at 95°C for 10 minutes.
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<br>
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- Add 200µL of extraction buffer and mix everything
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<br>
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- Incubate 10-15 minutes at 50-58°C until the gel is dissolved completely.
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<br>
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- Add 200µL of ethanol and mix
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<br>
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- Transfer into a column tube and centrifuge 1minute at 14 000 g and remove the liquid
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<br>
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- Add 200µL of Prewash buffer and centrifuge 1minute at 14 000 g and remove the liquid
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<br>
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- Add 700µL of Wash Buffer and centrifuge 1minute at 14 000 g and remove the liquid. Repeat this step twice.
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<br>- Centrifuge the tubes without any liquid 1minute at 14 000 g
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- Transfer the column in another 1.5mL Eppendorf tube
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<br>- Add 10µL of Elution Buffer and centrifuge 1minute at 14000 g
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- Remove the column  and keep the tubes at -20°C
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<br>
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<P>
<P>
<br>
<br>

Revision as of 11:10, 9 October 2014