Team:UC Chile2/Protocols

From 2012.igem.org

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<li>Adjust pH to 7.5 with NaOH 1M</li>
<li>Adjust pH to 7.5 with NaOH 1M</li>
</ul>
</ul>
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<h3>BG-11</h3>
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<br>
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For 1L:
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<ul>
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<li>1.5gr NaNO3</li>
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<li>1L distilled H2O </li>
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<li>10mL Concentrated salts solution 100X (4°C)</li>
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<li>200µL H2PO4 1M (Room T°)</li>
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<li>10gr BactoAgar -optional-</li>
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</ul>
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<h3>BG-110</h3>
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<br>
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For 1L:
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<ul>
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<li>1L distilled H2O </li>
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<li>10mL Concentrated salts solution 100X (4°C)</li>
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<li>200µL H2PO4 1M (Room T°)</li>
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<li></li>
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<li>10gr BactoAgar -optional-</li>
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</ul>
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<h3>BG-11C</h3>
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<br>
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For 1L:
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<ul>
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<li>1.5gr NaNO3</li>
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<li>1L distilled H2O </li>
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<li>10mL Concentrated salts solution 100X (4°C)</li>
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<li>200µL H2PO4 1M (Room T°)</li>
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<li>0.84gr NaHCO3</li>
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<li>10gr BactoAgar -opcional-</li>
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</ul>
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<h3>Concentrated salts solution 100X</h3>
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For 1L:
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<ul>
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<li>7.5gr SO4Mg * 7H2O</li>
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<li>3.6gr CaCl2 * 2H2O</li>
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<li>0.6gr Citric Acid</li>
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<li>0.6gr Ammonium Ferric Citrate</li>
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<li>0.093gr EDTA-Na2 </li>
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<li>2.0gr CO3 Na2 </li>
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<li>0.286gr BO3H3</li>
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<li>0.181gr MnCl2 * 4H2O</li>
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<li>0.022gr ZnSO4 * 7H2O</li>
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<li>0.039gr NaMoO4 * 2H2O</li>
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<li>0.0079gr CuSO4 * 5H2O</li>
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<li>0.0049gr CoCl2 * 6H2O</li>
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<li>1L distilled H2O </li>
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</ul>
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<br>
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Autoclave and store at 4°C.
<div id="Buffers">
<div id="Buffers">
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<h3>Gibson Assembly</h3>
<h3>Gibson Assembly</h3>
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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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<p>Gibson Assembly is a DNA assembly method created by Daniel Gibson during the development of the first Synthetic Genome (Synthia) [[#2|2]]. 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>
<h4>Primer design</h4>
<h4>Primer design</h4>
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<br><br>
<br><br>
<i>Colonies should appear between 1 to 2 weeks after the transformation. First you will see most of the cellular suspension dissapear and slowly small colonies should appear in the plate.</i>
<i>Colonies should appear between 1 to 2 weeks after the transformation. First you will see most of the cellular suspension dissapear and slowly small colonies should appear in the plate.</i>
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<div id="Reinoculation">
<div id="Reinoculation">
<h2>Reinoculation</h2>
<h2>Reinoculation</h2>
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Protocol right here!
</div>
</div>
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<div id="Cyanobacterial DNA extraction">
<div id="Cyanobacterial DNA extraction">
<h2>Cyanobacterial DNA extraction</h2>
<h2>Cyanobacterial DNA extraction</h2>
</div>
</div>
<p>Protocol goes here!</p>
<p>Protocol goes here!</p>
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<div id="Bactomithril Protocol">
<div id="Bactomithril Protocol">
<h1>Bactomithril Protocols</h1>
<h1>Bactomithril Protocols</h1>
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<h1>Characterization Protocols</h1>
<h1>Characterization Protocols</h1>
</div>
</div>
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<div id="SyneGrowth">
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<h2>Methods for the characterization of Synechocystis PCC 6803 growth curve</h2>
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This are the methods we used for setting up the growth curve experiment of our Synechocystis PCC 6803 which is further described [[Team:UC_Chile2/Characterization | here ]].
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<h3>Materials</h3>
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<ul>
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<li>4 sterile (autoclaved) 250 mL Erlenmeyer flasks</li>
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<li>1 liter of BG-11</li>
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<li>250 mL of BG-110</li>
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<li>Axenic culture of Synechocystis PCC 6803</li>
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<li>Sterile filtered 1 mL tips</li>
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<li>Spectrophotometer</li>
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<li>5 1 mL Cuvettes</li>
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</ul>
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<br>
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All procedures should be realized under sterile conditions, preferably in a laminar flow hood.
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<ul>
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<li>Measure 150 mL of BG-11 with a sterile 50 mL Falcon tube and put into each of the 3 Erlenmeyer flasks.</li>
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<li>Measure 150 mL of BG-110 with a sterile 50 mL Falcon tube and put into the remaining Erlenmeyer flask.</li>
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<li>Inoculate 1 mL of axenic Synechocystis PCC 6803 with and OD730 of X to each flask (including the one with BG-110). </li>
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<li>Place flasks in rotating incubator and try to avoid leaving them out of the incubator for long when taking measurements</li>
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<li>Measure OD600 and OD730 twice a day (preferably with at least 8 hours of difference between measurements)</li>
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<li>Repeat measurements for 14 days</li>
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</ul>
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</div>
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<div id="LuxBrick Characterization">
<div id="LuxBrick Characterization">
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The experiment for the characterization of T5 exonuclease concentration in Gibson Assembly for small parts was set as follows:
The experiment for the characterization of T5 exonuclease concentration in Gibson Assembly for small parts was set as follows:
<ul>
<ul>
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<li>After the design of the segment to be assembled as described in the [[/Team:UC_Chile2/Results/Characterization#Gibson Assembly for small parts Characterization section]], amplification of the parts was done using standard Phusion Polymerase PCR conditions but with a total amount of template of 0.05ng</li>
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<li>After the design of the segment to be assembled as described in the [[Team:UC_Chile2/Results/Gibson#Gibson_Assembly_for_small_parts | Characterization section]], amplification of the parts was done using standard Phusion Polymerase PCR conditions but with a total amount of template of 0.05ng</li>
<li>Bands where cut and purification of DNA from agarose gel was realized using a Promega's Wizard SV Gel and PCR Clean-Up System, final elution volume of 20 ul</li>
<li>Bands where cut and purification of DNA from agarose gel was realized using a Promega's Wizard SV Gel and PCR Clean-Up System, final elution volume of 20 ul</li>
<li>Purified DNA was quantified using a Nanodrop 2000</li>
<li>Purified DNA was quantified using a Nanodrop 2000</li>
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pmoles of DNA = weigth in ng * 1000 (conversion factor from nano to pico) / (650 Daltons * base pair length of part)
pmoles of DNA = weigth in ng * 1000 (conversion factor from nano to pico) / (650 Daltons * base pair length of part)
<li>Volumes of each part were calculated to obtain a final amount of 0.0125 pmoles of the small part and 0.0025 pmoles of the backbone in a final volume of 3 uL (add nuclease-free water to reach 3 uL) for each reaction</li>
<li>Volumes of each part were calculated to obtain a final amount of 0.0125 pmoles of the small part and 0.0025 pmoles of the backbone in a final volume of 3 uL (add nuclease-free water to reach 3 uL) for each reaction</li>
-
<li>For the preparation of Gibson Assembly Master Mixes, different volumes of 1.33X Gibson Assembly Master Mix were mixed with T5 exonuclease-free 1.33X Gibson Assembly Master Mix until appropriate concentrations were reached. If mixing volumes of any of the Master Mixes was lower than 10 uL, then one of the diluted T5 exonuclease 1.33X Master Mixes were used for the mix.</li>
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<li>For the preparation of Gibson Assembly Master Mixes, different volumes of a 10X T5 exonuclease 1.33X Gibson Assembly Master Mix were mixed with T5 exonuclease-free 1.33X Gibson Assembly Master Mix until appropriate concentrations were reached. If mixing volumes of any of the Master Mixes was lower than 1 uL, then one of the diluted T5 exonuclease 1.33X Master Mixes was used for the mix.</li>
<li>Gibson Assembly was done as described [[#Assembly_reaction| here]], and transformation proceeded as described [[#E.coli_Transformation| here]]</li>
<li>Gibson Assembly was done as described [[#Assembly_reaction| here]], and transformation proceeded as described [[#E.coli_Transformation| here]]</li>
<li>Each of the concentration points of the experiment was done in triplicate</li>
<li>Each of the concentration points of the experiment was done in triplicate</li>
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<li>Scoring was done 20 hours post-transformation</li>
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<li>Red colonies and white colonies were counted 20 hours post-transformation</li>
</ul>
</ul>
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<h1>References</h1>
<h1>References</h1>
<div id="1">
<div id="1">
-
[1] Julian J. Eaton-Rye , “Construction of Gene Interruptions and Gene Deletions in the Cyanobacterium Synechocystis sp. Strain PCC 6803 ”, Photosynthesis Research Protocols, Methods in Molecular Biology, vol. 684, DOI 10.1007/978-1-60761-925-3_22  
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[1]Julian J. Eaton-Rye , “Construction of Gene Interruptions and Gene Deletions in the Cyanobacterium Synechocystis sp. Strain PCC 6803 ”, Photosynthesis Research Protocols, Methods in Molecular Biology, vol. 684, DOI 10.1007/978-1-60761-925-3_22  
</div>
</div>
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<div id="2">
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[2]Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA 3rd, Smith HO. (2009). "Enzymatic assembly of DNA molecules up to several hundred kilobases". Nature Methods 6 (5): 343–345. doi:10.1038/nmeth.1318
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</div>
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{{UC_Chilefooter}}

Latest revision as of 11:06, 25 September 2012

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