Team:UC Davis/protocols/ligation.html

From 2010.igem.org

(Difference between revisions)
Line 33: Line 33:
<a name="extranotes"><h1>Extra Notes</h1></a>
<a name="extranotes"><h1>Extra Notes</h1></a>
 +
Use of a microsoft excel sheet is highly recommended for easy calculations.<p>
-
<a name="procedure"><h1>Procedure</h1></a><p>
+
<a name="procedure"><h1>Procedure</h1></a>
 +
Determining how much vector, insert, and milliQ water should be used <br />
 +
<ul>
 +
<li>Measure concentrations of the vector and insert samples to be used via nanodrop, spectrophotometer, etc. </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>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>
 +
 
 +
<p>
<a name="purpose"><h1>Purpose</h1></a>
<a name="purpose"><h1>Purpose</h1></a>

Revision as of 20:19, 10 September 2010

Ligation

Materials

You will need:

  • Vector (plasmid) DNA sample
  • Insert DNA sample
  • Ligation buffer
  • DNA ligase
  • MilliQ water

Extra Notes

Use of a microsoft excel sheet is highly recommended for easy calculations.

Procedure

Determining how much vector, insert, and milliQ water should be used
  • Measure concentrations of the 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.

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! :)

Retrieved from "http://2010.igem.org/Team:UC_Davis/protocols/ligation.html"