Team:Groningen/protocols/Digestion

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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>
 +
 
 +
<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>Ligation 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 preparation:</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>50&#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 50&#956;l</td>
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<td></td>
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  <tr>
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</tr>
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    <td>MQ water</td>
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<tr><td>5x Phusion Buffer</td>
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    <td>up to 20 &micro;l</td>
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<td>10</td>
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    <td></td>
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<td>1x</td>
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  </tr>
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</tr>
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<tr><td>10mM dNTPs</td>
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  <tr>
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<td>1</td>
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    <td>10x fast digestion buffer</td>
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<td>200&#956;M</td>
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    <td>2 &micro;l</td>
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</tr>
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    <td>1x</td>
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<tr><td>F-primer</td>
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  </tr>
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<td>1</td>
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<td>0.2&#956;M</td>
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  <tr>
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</tr>
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    <td>restriction enzyme(s)</td>
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<tr><td>R-primer</td>
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    <td>1 &micro;l</td>
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<td>1</td>
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    <td>1U</td>
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<td>0.2&#956;M</td>
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  </tr>
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</tr>
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<tr><td>Template DNA</td>
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  <tr>
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<td>1&#956;l</td>
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    <td>DNA</td>
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<td>~1ng</td>
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    <td>4 &micro;l</td>
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</tr>
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    <td>up to 1 &micro;g</td>
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<tr><td>Phusion Pol.</td>
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  </tr>
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<td>0.5&#956;l</td>
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<td>0.02U/&#956;l</td>
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</table>
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</tr>
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-
</table>
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 +
<br>When the reaction mix is ready, the content is mixed and incubated at 37&deg;C for 1h [1].
 +
 
 +
 
 +
<p><br>
 +
<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>
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<h3>Cycling instructions:</h3>
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</table>  
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<table border = "1">
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-
<tr><td>Temperature</td>
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<td>Time</td>
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<td>Number of cycles</td>
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</tr>
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<tr><td>98&deg;C</td>
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<td>30sec</td>
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<td>1</td>
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</tr>
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<tr><td>98&deg;C</td>
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<td>10sec</td>
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<td>30</td>
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</tr>
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<tr><td>primer annealing temperature</td>
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<td>30sec</td>
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<td>30</td>
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</tr>
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<tr><td>72&deg;C</td>
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<td>30sec/kb</td>
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<td>30</td>
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</tr>
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<tr><td>72&deg;C</td>
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<td>10min</td>
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<td>1</td>
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</tr>
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<tr><td>4&deg;C</td>
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<td>&infin;</td>
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<td>1</td>
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-
</tr>
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-
</table>
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 +
<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