Team:Tuebingen/Notebook/Protocols/preparative-restriction

From 2013.igem.org

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<a href="https://2013.igem.org/Team:Tuebingen/Notebook/Protocols" style="font-size: 16px">Back to Protocols</a>
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<a href="https://2013.igem.org/Team:Tuebingen/Notebook/Protocols" style="font-size: 18px">Back to Protocols</a>
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<p>&nbsp;</p>
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<h2>Digestion</h2>
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<h3>Reagents</h3>
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<table border="0">
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    <col width="80">
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    <col width="300">
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    <td style="text-align: center">10.0 µL</td>
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    <td><a href="https://www.neb.com/products/b7002-nebuffer-2">10x NEBuffer 2</a></td>
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  </tr>
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    <td style="text-align: center">1.0 µL</td>
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    <td>100x BSA</td>
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  <tr>
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    <td style="text-align: center">5 - 10 µg</td>
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    <td>Plasmid DNA</td>
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  </tr>
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  <tr>
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    <td style="text-align: center">2.0 µL</td>
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    <td>EcoRI or XbaI</td>
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  </tr>
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  <tr>
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    <td style="text-align: center">2.0 µL</td>
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    <td>PstI or SpeI</td>
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  </tr>
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  <tr>
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    <td style="text-align: center">to 100 µL</td>
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    <td>Aqua dest.</td>
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  </tr>
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    <td style="text-align: center">1/10 vol</td>
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    <td>10X Antarctic Phosphatase</td>
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<p>&nbsp;</p>
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<h3>Procedure</h3>
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<p>Mix all reagents (except for Antarctic Phosphatase) in an 1.5 mL Eppendorf-tube and incubate at 37 °C over night. On the next day, heat inactivate restriction enzymes at 80°C for 20 min. Add 1/10 volume of 10X Antarctic Phosphatase and mix. Incubate at 37 °C for 15 min. Heat inactivate phosphatase at 70 °C for 5 min. Proceed with ligation (3A-Assembly).</p>
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Revision as of 01:20, 4 October 2013

Return to iGEM Main Page.

Preparative Restriction Digest
Back to Protocols

 

Digestion

Reagents

10.0 µL 10x NEBuffer 2
1.0 µL 100x BSA
5 - 10 µg Plasmid DNA
2.0 µL EcoRI or XbaI
2.0 µL PstI or SpeI
to 100 µL Aqua dest.
1/10 vol 10X Antarctic Phosphatase

 

Procedure

Mix all reagents (except for Antarctic Phosphatase) in an 1.5 mL Eppendorf-tube and incubate at 37 °C over night. On the next day, heat inactivate restriction enzymes at 80°C for 20 min. Add 1/10 volume of 10X Antarctic Phosphatase and mix. Incubate at 37 °C for 15 min. Heat inactivate phosphatase at 70 °C for 5 min. Proceed with ligation (3A-Assembly).


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