Team:UC Chile2/Protocols

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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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<h1>Cyanobacteria Protocols</h1>
<h1>Cyanobacteria Protocols</h1>
</div>
</div>
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<br />
 
<div id="Transformation of Synechocystis">
<div id="Transformation of Synechocystis">
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<h2>Transformation of Synechocystis PCC. 6803</h2>
<h2>Transformation of Synechocystis PCC. 6803</h2>
</div>
</div>
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<p>Protocol goes here!</p>
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By iGEM - UC_Chile Team 2012 (Based on [[#1 | 1]])
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<h3>Materials</h3>
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<ul>
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<li>Liquid BG-11 Media</li>
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<li>Solid BG-11 Media</li>
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<li>Sterile 250 mL Erlenmeyer flask</li>
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<li>Sterile 50 mL Falcon tube (2)</li>
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<li>Sterile 1.5ml Eppendorf  tubes</li>
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<li>Petri Dishes</li>
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<li>Millipore Membrane Filters, 0.45um HA (HAWP09000) </li>
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<li>Pasteur pipette</li>
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<li>Antibiotics (according to your selection resistance)</li>
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<li>DNA for transformation</li>
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</ul>
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<h3>To be prepared previously</h3>
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<br>
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Concentrate the DNA to be transformed into Synechocystis to 1µg/µL in 10µL for each transformation (A total of 10 ug of the transformation plasmid).
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<br>
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Inoculate Synechocystis PCC 6803 into a sterile 250mL Erlenmeyer flask with 150mL* of liquid BG-11 and grow culture until you reach an OD730nm  of 0.8 – 1.0 (That should take between 6 to 10 days dependending on the amount of initial inoculum).
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* For each 50 mL of Synechocystis PCC 6803 you will have 5 individual transformations.
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<br>
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Once you have reached the OD730nm and have enough DNA, proceed to transform.
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<br>
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<h3>Transformation</h3>
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All subsequent actions are to be realized under sterile conditions.
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<ul>
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<li>1.-Transfer Synechocystis cells into 50 mL Falcon tubes and centrifuge 10 minutes at 2760g (Room temperature)</li>
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<li>2.- Wash cells: </li>
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Discard supernatant and resuspend pellet in the following manner:
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<ul>- Add 1mL of liquid BG-11 and softly pipet cells until resuspended.</ul>
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<ul>- Add 4mL of liquid BG-11 and pipet cells until completely mixed.</ul>
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<ul>- Add 5ml of BG-11 and pipet cells.</ul>
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* It is important that cells are throroughly resuspended so that recieve fresh media and do not form aggregates.
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<li>3.- Centrifuge 10 minutos at 2760g (Room temperature).</li>
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<li>4.- Discard supernatant and extract any remaining media by pipeting.</li>
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<li>5.- Resuspend pellet with 1mL of BG-11 media. Once resuspended, add 0.5 mL more of BG-11. </li>
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<li>6.- Distribuite 300 uL of resuspended cells into sterile 1,5 mL Eppendorf tube.</li>
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<li>* Make sure to pipet into the bottom of the tube so that none of the cells remain in the walls of the tube.</li>
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<li>7.- Add 10 μL of plasmid DNA (1μg/μL) to the tube containing the 300 uL of the Synechocystis cells.</li>
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<li>Softly mix contents with the point of the pipet. Repeat the procedure to all but the transformation control tube (without DNA). </li>
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<li>8.- Leave the tubes in a light chamber (50 uE/s²/m²) at 30°C for 5 hours.  Flick the tubes at 2.5 hours. </li>
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</ul>
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<h3>Plate preparation</h3>
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<br>
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Prepare 50 mL BG-11 plates with no antibiotic for the transformation day.
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Put Millipore membrane filters on BG-11 plates.
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<ul>
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[[File: Ant_table.jpg| 500px| right]]
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<li>9.- After the 5 hours of recuperation, take the celular suspension and spread over the Millipore membrane. The suspension is to be spreaded carefully over the whole membrane using a sterile bended Pasteur pipete.</li>
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<li>10.- Let the membranes dry before transfering the plates to the growth chamber.</li>
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<li>11.- Transfer plates to the growth chamber putting the membrane facing up and leave at 30°C for 12 hours in light.</li>
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<li>12.- Transfer membranes to BG-11 plates that contain half of the concentration of the antibiotic (12.5 ug/mL of antibiotic) and leave growing for 3 days at 30°C in light. </li>
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<li>13.- After the 3 days have passed, transfer the membrane to BG-11 plates with full concentration of antibiotic (25ug/mL).</li>
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</ul>
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<br><br>
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<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>
 +
<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>
 +
<div id="Bactomithril Protocol">
<div id="Bactomithril Protocol">
<h1>Bactomithril Protocols</h1>
<h1>Bactomithril Protocols</h1>
</div>
</div>
<br />
<br />
-
 
<p>You can find a complete protocol to produce spider-silk fibers in the following paper:
<p>You can find a complete protocol to produce spider-silk fibers in the following paper:
Teulé, F., Cooper, A. R., Furin, W. a, Bittencourt, D., Rech, E. L., Brooks, A., & Lewis, R. V. (2009). A protocol for the production of recombinant spider silk-like proteins for artificial fiber spinning. Nature protocols, 4(3), 341-55. doi:10.1038/nprot.2008.250  
Teulé, F., Cooper, A. R., Furin, W. a, Bittencourt, D., Rech, E. L., Brooks, A., & Lewis, R. V. (2009). A protocol for the production of recombinant spider silk-like proteins for artificial fiber spinning. Nature protocols, 4(3), 341-55. doi:10.1038/nprot.2008.250  
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<br>Other easier protocols are in the following adresses:<br>
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<br><br>Other easier protocols are in the following adresses:<br>
http://www.pnas.org/content/107/32/14059.long<br>
http://www.pnas.org/content/107/32/14059.long<br>
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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>
 +
<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>
 +
<div id="LuxBrick Characterization">
<div id="LuxBrick Characterization">
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</ul>
</ul>
 +
<div id="Gibson Assembly for small parts Characterization">
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<h2>Methods for Gibson Assembly for small parts characterization</h2>
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</div>
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The experiment for the characterization of T5 exonuclease concentration in Gibson Assembly for small parts was set as follows:
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<ul>
 +
<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>
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<li>Purified DNA was quantified using a Nanodrop 2000</li>
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<li>According to concentration of DNA, the amount of pmoles/uL of each part was calculated using the following formulae:</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)
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<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>
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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>
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<li>Gibson Assembly was done as described [[#Assembly_reaction| here]], and transformation proceeded as described [[#E.coli_Transformation| here]]</li>
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<li>Each of the concentration points of the experiment was done in triplicate</li>
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<li>Red colonies and white colonies were counted 20 hours post-transformation</li>
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</ul>
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<h1>References</h1>
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<div id="1">
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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
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</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