Team:Groningen/protocols/Digestion

From 2013.igem.org

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<div class="mainContent">
<div class="mainContent">
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<h1>Digestion</h1>
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<h2>Restriction digestion</h2>
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<h3>Materials:</h3>
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<h3>Fast digest</h3>
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<h5>Materials:</h5>
<ul type="square">
<ul type="square">
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<li>H<sub>2</sub>O</li>
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<li>MQ water</li>
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<li>1.5ml tubes</li>
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<li>1.5 ml tubes</li>
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<li>Digestion buffer</li>
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<li>Fast digestion buffer</li>
<li>Restriction enzymes</li>
<li>Restriction enzymes</li>
<li>DNA</li>
<li>DNA</li>
</ul>
</ul>
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<h3>Reaction:</h3>
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<h5>Reaction:</h5>
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<table border = "1">
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<table id="normal" width="60%">
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<tr><td>Component</td>
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  <tr>
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<td>30&#956;l reaction</td>
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    <th>Compound</th>
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<td>Final concentration</td>
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    <th>20 &micro;l</th>
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</tr>
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    <th>Final concentration</th>
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<tr><td>H<sub>2</sub>O</td>
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  </tr>
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<td>up to 30&#956;l</td>
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<td></td>
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</tr>
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<tr><td>Buffer 10x</td>
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<td>3&#956;l</td>
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<td>1x</td>
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</tr>
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<tr><td>DNA</td>
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<td>4&#956;l</td>
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<td>up to 1&#956;g</td>
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</tr>
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<tr><td>Enzyme/s</td>
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<td>1&#956;l</td>
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<td>1U</td>
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</tr>
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</table>
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<br>All the reagents are added following the order listed in the table above.
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  <tr>
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<br>After the reaction is ready mix the content of the tube and spin it down.
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    <td>MQ water</td>
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<br>The tubes are incubated for 1h at 37&deg;C.
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    <td>up to 20 &micro;l</td>
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<br>
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    <td></td>
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<br>Reference: http://www.thermoscientificbio.com/uploadedFiles/Resources/fast-digestion-dna.pdf
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  </tr>
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  <tr>
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    <td>10x fast digestion buffer</td>
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    <td>2 &micro;l</td>
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    <td>1x</td>
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  </tr>
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 +
  <tr>
 +
    <td>restriction enzyme(s)</td>
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    <td>1 &micro;l</td>
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    <td>1U</td>
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  </tr>
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 +
  <tr>
 +
    <td>DNA</td>
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    <td>4 &micro;l</td>
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    <td>up to 1 &micro;g</td>
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  </tr>
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 +
</table>
 +
 +
 +
<br>When the reaction mix is ready, the content is mixed and incubated at 37&deg;C for 1h [1].
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 +
 +
<p><br>
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<h3>Normal digest</h3>
 +
 +
<h5>Materials:</h5>
 +
<ul type="square">
 +
<li>MQ water</li>
 +
<li>1.5 ml tubes</li>
 +
<li>Enzyme digestion buffer</li>
 +
<li>Restriction enzymes</li>
 +
<li>DNA</li>
 +
</ul>
 +
 +
<h5>Reaction:</h5>
 +
<table id="normal" width="60%">
 +
  <tr>
 +
    <th>Compound</th>
 +
    <th>20 &micro;l</th>
 +
    <th>Final concentration</th>
 +
  </tr>
 +
 +
  <tr>
 +
    <td>MQ water</td>
 +
    <td>up to 20 &micro;l</td>
 +
    <td></td>
 +
  </tr>
 +
 +
  <tr>
 +
    <td>10x restriction digestion buffer</td>
 +
    <td>2 &micro;l</td>
 +
    <td>1x</td>
 +
  </tr>
 +
 +
  <tr>
 +
    <td>restriction enzyme(s)</td>
 +
    <td>0.5 &micro;l</td>
 +
    <td>1U</td>
 +
  </tr>
 +
 +
  <tr>
 +
    <td>DNA</td>
 +
    <td>2 &micro;l</td>
 +
    <td>0.2-1.5 &micro;g</td>
 +
  </tr>
 +
 +
</table>
 +
 +
<br>When the reaction mix is ready, the content is mixed and incubated at 37&deg;C for 2h.
 +
<br>
 +
<br> [1] Reference: http://www.thermoscientificbio.com/uploadedFiles/Resources/fast-digestion-dna.pdf
</div>
</div>

Latest revision as of 23:32, 4 October 2013

Restriction digestion

Fast digest

Materials:
  • MQ water
  • 1.5 ml tubes
  • Fast digestion buffer
  • Restriction enzymes
  • DNA
Reaction:
Compound 20 µl Final concentration
MQ water up to 20 µl
10x fast digestion buffer 2 µl 1x
restriction enzyme(s) 1 µl 1U
DNA 4 µl up to 1 µg

When the reaction mix is ready, the content is mixed and incubated at 37°C for 1h [1].


Normal digest

Materials:
  • MQ water
  • 1.5 ml tubes
  • Enzyme digestion buffer
  • Restriction enzymes
  • DNA
Reaction:
Compound 20 µl Final concentration
MQ water up to 20 µl
10x restriction digestion buffer 2 µl 1x
restriction enzyme(s) 0.5 µl 1U
DNA 2 µl 0.2-1.5 µg

When the reaction mix is ready, the content is mixed and incubated at 37°C for 2h.

[1] Reference: http://www.thermoscientificbio.com/uploadedFiles/Resources/fast-digestion-dna.pdf