Team:UC Davis/protocols/gelextraction.html

From 2010.igem.org

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<li>All steps should be carried out at room temperature.</li>
<li>All steps should be carried out at room temperature.</li>
<li>All centrifugations should be carried out in a microcentrifuge at > 12,000xg (10,000-14,000 rpm, depending on the rotor type)</li>
<li>All centrifugations should be carried out in a microcentrifuge at > 12,000xg (10,000-14,000 rpm, depending on the rotor type)</li>
 +
<li>See <a href="http://www.fermentas.com" class="help">Fermentas</a> protocol booklet for more information and detailed instructions. </li>
</ul><p>
</ul><p>
<a name="procedure"><h1>Procedure</h1></a>
<a name="procedure"><h1>Procedure</h1></a>
 +
Pre-binding procedure <br />
<ul>
<ul>
-
<li>Cut out gel slice with the desired DNA fragment and weigh gel. </li>
+
<li>Cut out <a href="https://2010.igem.org/Team:UC_Davis/protocols/gelelectrophoresis.html#procedure" class="help">gel</a> slice with the desired DNA fragment and weigh gel. </li>
<li>Add 1:1 volume of binding buffer to gel slice. (Eg. 100μL of binding buffer for every 100mg of agarose gel.) </li>
<li>Add 1:1 volume of binding buffer to gel slice. (Eg. 100μL of binding buffer for every 100mg of agarose gel.) </li>
<li>Incubate gel solution at 50°C-60°C for 10 minutes.  Inverting tube helps melting process. </li>
<li>Incubate gel solution at 50°C-60°C for 10 minutes.  Inverting tube helps melting process. </li>
-
<li>Transfer solution to spin column. </li>
+
<li>(Optional: only use if DNA fragment is <500bp or >10kb long) If DNA fragment is <500bp, add a 1:2 volume of 100% isopropanol to the solubilized gel solution. Mix thoroughly.  If DNA fragment is >10kb, add a 1:2 volume of water to the solubilized gel solution.  Mix thoroughly. </li>
 +
</ul><p>
 +
 
 +
Binding DNA <br />
 +
<ul>
 +
<li>Transfer (up to 800μL) gel solution to spin column. </li>
<li>Centrifuge for 30-60 seconds. Discard flow-through and place spin column back into same collection tube. </li>
<li>Centrifuge for 30-60 seconds. Discard flow-through and place spin column back into same collection tube. </li>
 +
</ul><p>
 +
 +
Washing column of residue <br />
 +
<ul>
<li>Add 700μL of wash buffer and centrifuge for 1 minute. </li>
<li>Add 700μL of wash buffer and centrifuge for 1 minute. </li>
<li>Discard flow-through and the centrifuge empty column for 1 minute. </li>
<li>Discard flow-through and the centrifuge empty column for 1 minute. </li>
<li>Place the column into a fresh 1.5mL microcentrifuge tube. </li>
<li>Place the column into a fresh 1.5mL microcentrifuge tube. </li>
 +
</ul><p>
 +
 +
Eluting DNA <br />
 +
<ul>
<li>Add 50μL of elution buffer to the column. </li>
<li>Add 50μL of elution buffer to the column. </li>
<li>Centrifuge for 1 minute.  Keep the flow-through. </li>
<li>Centrifuge for 1 minute.  Keep the flow-through. </li>
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<a name="references"><h1>References</h1></a>
<a name="references"><h1>References</h1></a>
<ul>
<ul>
-
<li>Paraphrased from <a href="http://www.fermentas.com" class="help">Fermentas</a> protocol</li>
+
<li>Summarized from <a href="http://www.fermentas.com" class="help">Fermentas</a> protocol</li>
</ul><p>                               
</ul><p>                               
         </td>
         </td>

Latest revision as of 20:05, 10 September 2010

Gel Extraction

Materials

You will need:

  • Fermentas GeneJET Gel Extraction Kit
  • Microcentrifuge tubes
  • Microtiter pipette & tips

Extra Notes

  • All steps should be carried out at room temperature.
  • All centrifugations should be carried out in a microcentrifuge at > 12,000xg (10,000-14,000 rpm, depending on the rotor type)
  • See Fermentas protocol booklet for more information and detailed instructions.

Procedure

Pre-binding procedure
  • Cut out gel slice with the desired DNA fragment and weigh gel.
  • Add 1:1 volume of binding buffer to gel slice. (Eg. 100μL of binding buffer for every 100mg of agarose gel.)
  • Incubate gel solution at 50°C-60°C for 10 minutes. Inverting tube helps melting process.
  • (Optional: only use if DNA fragment is <500bp or >10kb long) If DNA fragment is <500bp, add a 1:2 volume of 100% isopropanol to the solubilized gel solution. Mix thoroughly. If DNA fragment is >10kb, add a 1:2 volume of water to the solubilized gel solution. Mix thoroughly.

Binding DNA

  • Transfer (up to 800μL) gel solution to spin column.
  • Centrifuge for 30-60 seconds. Discard flow-through and place spin column back into same collection tube.

Washing column of residue

  • Add 700μL of wash buffer and centrifuge for 1 minute.
  • Discard flow-through and the centrifuge empty column for 1 minute.
  • Place the column into a fresh 1.5mL microcentrifuge tube.

Eluting DNA

  • Add 50μL of elution buffer to the column.
  • Centrifuge for 1 minute. Keep the flow-through.

Purpose

To purify the DNA from the gel.

References

We would like to take a moment to thank all of our sponsors for their very generous donations, as we could not have done this without your help!

We would also like to thank and acknowledge:
Our Advisors
Marc Facciotti
Ilias Tagkopoulos
Technical Guidance
David Larsen
Andrew Yao
Visiting iGEMer
Jia Li of Zhejiang University (TEAM ZJU-China)
cI Promoter Screen
Drew Endy - Stanford
Thomas Schneider - NIH
Want to sponsor us? Send an email to mtfacciotti@ucdavis.edu to discuss various ways you can help! :)

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