Difference between revisions of "Team:Tuebingen/Experiments"

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             <ul>
 
             <ul>
 
             <li>Following electrophoresis, excise DNA band from gel and place gel slice in a 1,5ml microcentrifuge tube.</li>
 
             <li>Following electrophoresis, excise DNA band from gel and place gel slice in a 1,5ml microcentrifuge tube.</li>
             <li>Add 10&mu;</li>
+
             <li>Add 10&mu;l Membrane Binding Solution per 10mg of gel slice. Vortex and incubate at 50-60&deg;C until gel slice is completely dissolved.</li>
             <li></li>
+
             <li>Insert SV Minicolumn into Collection Tube.</li>
             <li></li>
+
             <li>Transfer dissolved gel mixture to the Minicolumn assembly. Incubate at room temp. for 1 minute.</li>
             <li></li>
+
             <li>Centrifuge at 16,000xg for 1 min. Discard flowthrough and reinsert Minicolumn into Collection tube.</li>
             <li></li>
+
             <li>Add 700&mu;l Membrane Wash Solution (ethanol added). Centrifuge at 16,000xg for 1 min. Discard flowthrough and reinsert Minicolumn into Collection tube. </li>
             <li></li>
+
             <li>Repeat this step with 500&mu;l Membrane Wash Solution. Centrifuge at 16,000xg for 5 min.</li>
             <li></li>
+
             <li>Empty the Collection Tube and recentrifuge the column assembly for 1,5 min.</li>
 +
            <li>Leave the tubes open for 10 min (to let any rest of ethanol evaporate).</li>
 +
            <li>Carefully transfer Minicolumn to a clean 1.5ml microcentrifuge tube.</li>
 +
            <li>Add 30&mu;l of Nuclease-Free-Water (65&deg;C) to the Minicolumn. Incubate at 65&deg;C for 5 min. Centrifuge at 16,000xg for 1 min.</li>
 +
            <li>Discard Minicolumn and store DNA at 4&deg;C or -20&deg;C.</li>
 
             </ul>
 
             </ul>
 
</div>
 
</div>

Revision as of 09:04, 25 August 2015

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Protocols

Prep

Colony-PCR

3A-Assembly

Restriction

Transformation

PCR

Ligation

Testrestriktion

Gel extraction

DNA Purification using Promega Wizard SV Gel and PCR Clean-Up System Kit

This is a modified protocol for the Wizard SV Gel and PCR Clean-Up System Kit from Promega

  • Following electrophoresis, excise DNA band from gel and place gel slice in a 1,5ml microcentrifuge tube.
  • Add 10μl Membrane Binding Solution per 10mg of gel slice. Vortex and incubate at 50-60°C until gel slice is completely dissolved.
  • Insert SV Minicolumn into Collection Tube.
  • Transfer dissolved gel mixture to the Minicolumn assembly. Incubate at room temp. for 1 minute.
  • Centrifuge at 16,000xg for 1 min. Discard flowthrough and reinsert Minicolumn into Collection tube.
  • Add 700μl Membrane Wash Solution (ethanol added). Centrifuge at 16,000xg for 1 min. Discard flowthrough and reinsert Minicolumn into Collection tube.
  • Repeat this step with 500μl Membrane Wash Solution. Centrifuge at 16,000xg for 5 min.
  • Empty the Collection Tube and recentrifuge the column assembly for 1,5 min.
  • Leave the tubes open for 10 min (to let any rest of ethanol evaporate).
  • Carefully transfer Minicolumn to a clean 1.5ml microcentrifuge tube.
  • Add 30μl of Nuclease-Free-Water (65°C) to the Minicolumn. Incubate at 65°C for 5 min. Centrifuge at 16,000xg for 1 min.
  • Discard Minicolumn and store DNA at 4°C or -20°C.

Experiments & Protocols

Describe the experiments, research and protocols you used in your iGEM project.

What should this page contain?
  • Protocols
  • Experiments
  • Documentation of the development of your project

Inspiration