Team:UC Chile2/General Protocols

From 2012.igem.org

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{{UC_Chile4}}
{{UC_Chile4}}
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<div id=" Growth Media">
<div id=" Growth Media">
<h1>Growth Media</h1>
<h1>Growth Media</h1>
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<li>100uL 5X Gibson Assembly Isothermal Buffer</li>
<li>100uL 5X Gibson Assembly Isothermal Buffer</li>
<li>6.25uL Phusion Polymerase 2 U/uL (cat N° F-350S) from Thermo Scientific </li>
<li>6.25uL Phusion Polymerase 2 U/uL (cat N° F-350S) from Thermo Scientific </li>
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<li>2uL Epicentre T5 Exonuclease 1U/uL (cat N° T5E4111K) from Illumina</li>
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<li>2uL T5 Exonuclease 1U/uL (cat N° T5E4111K) from Epicentre</li>
<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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<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>
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<div id="DNA assembly">
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<div id="DNA assembly protocols">
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<h1>DNA assembly</h1>
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<h1>DNA assembly protocols</h1>
</div>
</div>
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<h1>Gibson Assembly</h1>
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<h2>Standard Assembly</h2>
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<h2>Design primers</h2>
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<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>
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<ul>
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<li>Upstream part enzymes: EcoRI & SpeI</li>
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<li>Downstream part enzymes: XbaI & PstI</li>
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<li>Destination backbone enzymes: EcoRI & PstI</li>
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</ul>
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<h3>Digestion reaction</h3>
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<ul>
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<li>X volume of DNA to 500ng of plasmid</li>
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<li>(42.5 - X)uL of nuclease free water</li>
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<li>5uL of NEB buffer 2</li>
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<li>0.5uL of BSA 100X</li>
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<li>1uL of Enzyme 1</li>
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<li>1uL of Enzyme 2</li>
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<li>Incubate at 37°C for 2 hours</li>
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<li>Heat inactivate enzymes at 80°C for 20 minutes</li>
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</ul>
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<h3>Ligation reaction</h3>
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<ul>
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<li>2uL of digested Upstream part</li>
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<li>2uL of digested Downstream part</li>
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<li>2uL of digested Destination plasmid</li>
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<li>2uL of T4 DNA ligase buffer</li>
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<li>11uL of nuclease free water</li>
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<li>1uL of T4 DNA ligase</li>
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<li>Incubate at room temperature for 10 minutes</li>
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<li>Heat inactivate at 80°C for 20 minutes</li>
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<li>[[#E.coli Transformation|Transform]]</li>
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</ul>
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<h2>Gibson Assembly</h2>
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<p>Gibson Assembly is a DNA assembly method created by Daniel Gibson during the development of the first Synthetic Genome (Synthia) (reference YYY). Its adaptation to a cloning method allows fast and accurate production of increasingly complex constructions. The strategy behind the method relies on PCR to obtain different parts which share a 40bp homology region, and a 3 enzyme reaction which produces cohesive ends, fills the gaps between the parts and ligates the resulting construct into a scarless assembly of various (>2) parts.</p>
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<h3>Design primers</h3>
<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>
<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>
<ul>
<ul>
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<li>Apply same principle in all joints</li>
<li>Apply same principle in all joints</li>
</ul>
</ul>
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<h2>Obtaining parts</h2>
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<h3>Obtaining parts</h3>
<ul>
<ul>
<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>
<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>Quantify purified DNA</li>
<li>Quantify purified DNA</li>
</ul>
</ul>
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<h2>Assembly reaction</h2>
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<h3>Assembly reaction</h3>
<p>The assembly reaction is composed of 9uL of 1.33X Gibson assembly master mix + 3uL of purified parts DNA</p>
<p>The assembly reaction is composed of 9uL of 1.33X Gibson assembly master mix + 3uL of purified parts DNA</p>
<ul>
<ul>
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<li>Thoroughly mix the 3uL of purified parts DNA with the 9uL of 1.33X Gibson assembly master mix in ice</li>
<li>Thoroughly mix the 3uL of purified parts DNA with the 9uL of 1.33X Gibson assembly master mix in ice</li>
<li>Directly incubate the reaction at 50°C for 1 hour</li>
<li>Directly incubate the reaction at 50°C for 1 hour</li>
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<li>Transform competent cells with 8uL of assembled DNA (that leaves 4uL to check in an agarose gel if the reaction fails)</li>
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<li>[[#E.coli Transformation| Transform]] competent cells with 8uL of assembled DNA (that leaves 4uL to check in an agarose gel if the reaction fails)</li>
</ul>
</ul>
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<h2Checking assembly</h2>
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<h3>Checking assembly</h3>
<ul>
<ul>
<li>Check transformant colonies by colony PCR using primers for whole insert</li>
<li>Check transformant colonies by colony PCR using primers for whole insert</li>
<li>Grow positive colonies in media with corresponding antibiotic</li>
<li>Grow positive colonies in media with corresponding antibiotic</li>
<li>Miniprep colonies and digest plasmid with EcoRI & PstI restriction enzymes</li>
<li>Miniprep colonies and digest plasmid with EcoRI & PstI restriction enzymes</li>
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<li>If checking protocols validate until know, proceed by sequencing plasmid</li>
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<li>If checking protocols validate until now, proceed by sequencing to corroborate</li>
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</ul>
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<div id="E.coli Transformation">
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<h1>E.coli Transformation</h1>
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</div>
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<ul>
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<li>Keep 50uL of [http://openwetware.org/wiki/TOP10_chemically_competent_cells chemically competent cells] in ice (for no more than 10 minutes until transformation)</li>
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<li>Mix 0.01 to 1 total ng of plasmid with competent cells (depending on size of plasmid and competence of your cells)</li>
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<p>Flick very softly to mix (competent cells are very fragile)</p>
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<li>Leave on ice for 30 minutes</li>
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<li>Heat shock at 42°C for 60 seconds</li>
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<li>Add 250uL of [[#Growth Media| LB media]]</li>
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<li>Incubate at 37°C in a rotating shaker for 1 hour (Ampicillin) or 2 hours (Chloramphenicol and Kanamycin)</li>
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<li>Plate 50uL of transformed cells to a LB agar petri dish with corresponding antibiotic (30ug/mL Kanamycin or 30ug/mL Chloramphenicol or 100ug/mL Ampicillin)</li>
</ul>
</ul>

Latest revision as of 05:42, 21 September 2012

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