Team:Groningen/protocols/Digestion

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<h1>Digestion</h1>
 +
 
 +
<h3>Materials:</h3>
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<ul type="square">
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<li>H<sub>2</sub>O</li>
 +
<li>1.5ml tubes</li>
 +
<li>Ligation buffer</li>
 +
<li>Restriction enzymes</li>
 +
<li>DNA</li>
 +
</ul>
 +
 
 +
<h3>Reaction preparation:</h3>
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<table border = "1">
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<tr><td>Component</td>
 +
<td>50&#956;l reaction</td>
 +
<td>Final concentration</td>
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</tr>
 +
<tr><td>H<sub>2</sub>O</td>
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<td>up to 50&#956;l</td>
 +
<td></td>
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</tr>
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<tr><td>5x Phusion Buffer</td>
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<td>10</td>
 +
<td>1x</td>
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</tr>
 +
<tr><td>10mM dNTPs</td>
 +
<td>1</td>
 +
<td>200&#956;M</td>
 +
</tr>
 +
<tr><td>F-primer</td>
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<td>1</td>
 +
<td>0.2&#956;M</td>
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</tr>
 +
<tr><td>R-primer</td>
 +
<td>1</td>
 +
<td>0.2&#956;M</td>
 +
</tr>
 +
<tr><td>Template DNA</td>
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<td>1&#956;l</td>
 +
<td>~1ng</td>
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</tr>
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<tr><td>Phusion Pol.</td>
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<td>0.5&#956;l</td>
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<td>0.02U/&#956;l</td>
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</tr>
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</table>
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<h3>Cycling instructions:</h3>
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<table border = "1">
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<tr><td>Temperature</td>
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<td>Time</td>
 +
<td>Number of cycles</td>
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</tr>
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<tr><td>98&deg;C</td>
 +
<td>30sec</td>
 +
<td>1</td>
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</tr>
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<tr><td>98&deg;C</td>
 +
<td>10sec</td>
 +
<td>30</td>
 +
</tr>
 +
<tr><td>primer annealing temperature</td>
 +
<td>30sec</td>
 +
<td>30</td>
 +
</tr>
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<tr><td>72&deg;C</td>
 +
<td>30sec/kb</td>
 +
<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>
 +
<td>1</td>
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</tr>
 +
<tr><td>4&deg;C</td>
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<td>&infin;</td>
 +
<td>1</td>
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</tr>
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</table>
</div>
</div>

Revision as of 09:36, 26 July 2013

Digestion

Materials:

  • H2O
  • 1.5ml tubes
  • Ligation buffer
  • Restriction enzymes
  • DNA

Reaction preparation:

Component 50μl reaction Final concentration
H2O up to 50μl
5x Phusion Buffer 10 1x
10mM dNTPs 1 200μM
F-primer 1 0.2μM
R-primer 1 0.2μM
Template DNA 1μl ~1ng
Phusion Pol. 0.5μl 0.02U/μl

Cycling instructions:

Temperature Time Number of cycles
98°C 30sec 1
98°C 10sec 30
primer annealing temperature 30sec 30
72°C 30sec/kb 30
72°C 10min 1
4°C ∞ 1