Team:UC Chile2/General Protocols

From 2012.igem.org

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<p>It is important to dilute the T5 Exonuclease stock from 10U/uL to 1U/ul to measure the volume correctly</p>
<p>It is important to dilute the T5 Exonuclease stock from 10U/uL to 1U/ul to measure the volume correctly</p>
<li>50uL Taq DNA Ligase 2000 U/uL (cat N° M208S) from NEB</li>
<li>50uL Taq DNA Ligase 2000 U/uL (cat N° M208S) from NEB</li>
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<li>216.75uL of nuclease free H20</li>
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<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">
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</div>
</div>
<h1>Gibson Assembly</h1>
<h1>Gibson Assembly</h1>
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<h2>Design primers</h2>
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<p>The easiest way to design primers to obtain amplicons with the required overlaps (40bp final overlaps) is to make an <i>in sillico</i> design of the final construct</p>
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<ul>
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<li>Design forward primer of right amplicon of joint in 5'->3' direction</li>
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<li>Calculate length of annealing part of primer as to reach a Tm of approximately 63°C</li>
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<li>Add 20 bp of overlap</li>
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<li>Design reverse primer of left amplicon of joint in 5'->3' direction</li>
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<li>To do this, select full joint and "reverse complement" it (be sure to be able to discriminate between right and left parts of joint)</li>
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<li>Calculate length of annealing part of primer as to reach a Tm of approximately 63°C</li>
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<li>Add 20 bp of overlap</li>
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<li>Apply same principle in all joints</li>
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</ul>
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<h2>Obtaining parts</h2>
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<ul>
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<li>PCR parts using Phusion Polymerase datasheet indications. It is important to use a low ammount of template plasmid (10pg) as to reduce possibility of carry-over during band purification</li>
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<li>Run agarose gel electrophoresis on a adecuate gel (50bp to 200bp parts should be run on a 3% w/v agarose gel, larger parts should be run on a 1% w/v to 1.5% w/v agarose gel) until clear distinction of bands is achieved. It is recommended that gels should be exposed as little as possible to UV transilluminators as UV damages DNA.</li>
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<li>Cut band and proceed with band purification. Elute in smallest volume as possible according to your kit specification</li>
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<li>Quantify purified DNA</li>
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</ul>

Revision as of 03:40, 13 September 2012

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