Team:UC Davis/protocols/ligation.html

From 2010.igem.org

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               <p> You will need: <br />
               <p> You will need: <br />
                 <ul>
                 <ul>
-
                 <li>Vector (plasmid) DNA sample</li>
+
                 <li><a href="https://2010.igem.org/Team:UC_Davis/protocols/gelextraction.html" class="help">Purified vector (plasmid) DNA sample</a></li>
-
                 <li>Insert DNA sample</li>
+
                 <li><a href="https://2010.igem.org/Team:UC_Davis/protocols/gelextraction.html" class="help">Purified insert DNA sample</a></li>
                 <li>Ligation buffer </li>
                 <li>Ligation buffer </li>
                 <li>DNA ligase </li>
                 <li>DNA ligase </li>
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<a name="extranotes"><h1>Extra Notes</h1></a>
<a name="extranotes"><h1>Extra Notes</h1></a>
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Use of a microsoft excel sheet is highly recommended for easy calculations.<p>
+
<ul>
 +
<li>Use of a microsoft excel sheet is highly recommended for easy calculations.</li>
 +
<li>It is recommended that in addition to an experimental ligation, that you have a vector and insert controls. </li>
 +
<li>This is a quick ligation protocol. It is highly recommended that <a href="https://2010.igem.org/Team:UC_Davis/protocols/transformation.html" class="help">transformations</a> are done simultaneously. In 20 minutes (after the addition of ligase), the ligation is ready.  This means competent cells should be taken out in 10 minutes after ligase is added so that ligase and cells are ready at the same time. </li>
 +
</ul>
 +
<p>
<a name="procedure"><h1>Procedure</h1></a>
<a name="procedure"><h1>Procedure</h1></a>
Determining how much vector, insert, and milliQ water should be used <br />
Determining how much vector, insert, and milliQ water should be used <br />
<ul>
<ul>
-
<li>Measure concentrations of the vector and insert samples to be used via nanodrop, spectrophotometer, etc. </li>
+
<li>Measure concentrations of the <a href="https://2010.igem.org/Team:UC_Davis/protocols/gelextraction.html" class="help">purified vector and insert samples</a> to be used via nanodrop, spectrophotometer, etc. </li>
<li>The vector volume = [desired vector mass (ng)]/[vector concentration] </li>
<li>The vector volume = [desired vector mass (ng)]/[vector concentration] </li>
<li>The insert volume = [(desired vector mass (ng))/(vector length)]*[(insert length)/(insert concentration)]*[desired insert:vector ratio] </li>
<li>The insert volume = [(desired vector mass (ng))/(vector length)]*[(insert length)/(insert concentration)]*[desired insert:vector ratio] </li>
<li>milliQ water will be used as a filler to ensure that the end reaction volume is 20μL.  This means milliQ volume = 20μL - 2μL (from DNA ligase buffer) - 1μL (from DNA ligase) - vector volume - insert volume. </li>
<li>milliQ water will be used as a filler to ensure that the end reaction volume is 20μL.  This means milliQ volume = 20μL - 2μL (from DNA ligase buffer) - 1μL (from DNA ligase) - vector volume - insert volume. </li>
 +
</ul>
 +
 +
Ligation (Experimental) <br />
 +
<ul>
 +
<li>Add previously calculated amount of vector.</li>
 +
<li>Add previously calculated amount of insert. </li>
 +
<li>Add 2μL DNA ligation buffer. </li>
 +
<li>Add 1μL DNA ligase. </li>
 +
<li>Add previously calculated milliQ water. </li>
 +
</ul>
 +
 +
Controls <br />
 +
<ul>
 +
<li>Add previously calculated amount of vector OR insert </li>
 +
<li>Add 2μL DNA ligation buffer. </li>
 +
<li>Add 1μL DNA ligase. </li>
 +
<li>Fill total reaction volume to 20μL with milliQ water. </li>
</ul>
</ul>

Latest revision as of 20:32, 10 September 2010

Ligation

Materials

You will need:

Extra Notes

  • Use of a microsoft excel sheet is highly recommended for easy calculations.
  • It is recommended that in addition to an experimental ligation, that you have a vector and insert controls.
  • This is a quick ligation protocol. It is highly recommended that transformations are done simultaneously. In 20 minutes (after the addition of ligase), the ligation is ready. This means competent cells should be taken out in 10 minutes after ligase is added so that ligase and cells are ready at the same time.

Procedure

Determining how much vector, insert, and milliQ water should be used
  • Measure concentrations of the purified vector and insert samples to be used via nanodrop, spectrophotometer, etc.
  • The vector volume = [desired vector mass (ng)]/[vector concentration]
  • The insert volume = [(desired vector mass (ng))/(vector length)]*[(insert length)/(insert concentration)]*[desired insert:vector ratio]
  • milliQ water will be used as a filler to ensure that the end reaction volume is 20μL. This means milliQ volume = 20μL - 2μL (from DNA ligase buffer) - 1μL (from DNA ligase) - vector volume - insert volume.
Ligation (Experimental)
  • Add previously calculated amount of vector.
  • Add previously calculated amount of insert.
  • Add 2μL DNA ligation buffer.
  • Add 1μL DNA ligase.
  • Add previously calculated milliQ water.
Controls
  • Add previously calculated amount of vector OR insert
  • Add 2μL DNA ligation buffer.
  • Add 1μL DNA ligase.
  • Fill total reaction volume to 20μL with milliQ water.

Purpose

To ligate the desired insert fragment to the desired plasmid together.

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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