Team:UC Davis/protocols/ligation.html
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<p> You will need: <br /> | <p> You will need: <br /> | ||
<ul> | <ul> | ||
- | <li> | + | <li><a href="https://2010.igem.org/Team:UC_Davis/protocols/gelextraction.html" class="help">Purified vector (plasmid) DNA sample</a></li> |
- | <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> | ||
+ | <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><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 <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 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> | ||
+ | |||
+ | 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> | ||
+ | |||
+ | <p> | ||
<a name="purpose"><h1>Purpose</h1></a> | <a name="purpose"><h1>Purpose</h1></a> |
Latest revision as of 20:32, 10 September 2010
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