Team:UC Chile2/General Protocols

From 2012.igem.org

(Difference between revisions)
Line 80: Line 80:
<b>Alicuot 9uL in 0.2mL PCR tubes. This will yield about 42 reactions</b>
<b>Alicuot 9uL in 0.2mL PCR tubes. This will yield about 42 reactions</b>
</ul>
</ul>
-
<div id="DNA assembly">
+
<div id="DNA assembly protocols">
-
<h1>DNA assembly</h1>
+
<h1>DNA assembly protocols</h1>
</div>
</div>
 +
<h2>Standard Assembly</h2>
 +
<p>The standard assembly reaction relies on the standarization of Biobricks to join different DNA parts. Plasmids from the registry of standard parts allow joining of DNA parts by using a combination of specific enzymes to cut a Upstream Biobrick with the restriction enzymes EcoRI and SpeI, and a Downstream Biobrick with the restriction enzymes XbaI and PstI into a Destination plasmid which has been cut with the restriction enzymes EcoRI and PstI. The reaction yields the Upstream and Downstream part joined by a mixed restriction site and allows further elongation of the construct by the same strategy. It is important to notice that such a reaction requires purification of the digested parts if any of the plasmids (Upstream, Downstream or Destination) share a resistance marker.</p>
 +
<ul>
 +
<li>Upstream part enzymes: EcoRI & SpeI</li>
 +
<li>Downstream part enzymes: XbaI & PstI</li>
 +
<li>Destination backbone enzymes: EcoRI & PstI</li>
 +
</ul>
 +
<h3>Digestion reaction</h3>
 +
<ul>
 +
<li>X volume of DNA to 500ng of plasmid</li>
 +
<li>(42.5 - X)uL of nuclease free water</li>
 +
<li>5uL of NEB buffer 2</li>
 +
<li>0.5uL of BSA 100X</li>
 +
<li>1uL of Enzyme 1</li>
 +
<li>1uL of Enzyme 2</li>
 +
<li>Incubate at 37°C for 2 hours</li>
 +
<li>Heat inactivate enzymes at 80°C for 20 minutes</li>
 +
</ul>
 +
<h3>Ligation reaction</h3>
 +
<ul>
 +
<li>2uL of digested Upstream part</li>
 +
<li>2uL of digested Downstream part</li>
 +
<li>2uL of digested Destination plasmid</li>
 +
<li>2uL of T4 DNA ligase buffer</li>
 +
<li>11uL of nuclease free water</li>
 +
<li>1uL of T4 DNA ligase</li>
 +
</ul>
 +
<h2>Gibson Assembly</h2>
<h2>Gibson Assembly</h2>
<h3>Design primers</h3>
<h3>Design primers</h3>

Revision as of 04:49, 13 September 2012

Project: Luxilla - Pontificia Universidad Católica de Chile, iGEM 2012