Team:Duke/Notebook/Protocols

From 2014.igem.org

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<tr height="10%"><td><div class="protobutt"><a href="#ccec">Preparing Chemically Competent Cells </a></div></td></tr>
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not pictured: Janan
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<tr height="10%"><td><div class="protobutt"><a href="#ligate">Ligation </a></div></td></tr>
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<tr height="10%"><td><div class="protobutt"><a href="#pcrprep">PCR Preparation of Inserts</a></div></td></tr>
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<p class= "big"> Protocols </p>
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<div id="biosection">
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<div class="bio">
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<div id="theteam">
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<h2> Team Duke </h2>
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Click on our faces to learn more about us!
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Basically, the Duke iGEM team is pretty cool. Not pictured is Janan Zhu:
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Janan Zhu is a junior from New York City studying biophysics and math. He is interested in learning more about ways to study biology more quantitatively, and is also involved in computational chemistry research in Weitao Yang's lab. In his free time, he enjoys teaching high school students, playing video games, swimming, and traveling.
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<div class="pro">
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Photo by Karim Ali
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<a id="ccec"><h2> Preparing Chemically Competent Cells </h2></a>
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This protocol is for making chemically competent E. coli for transformations. It is derived from the official iGEM protocol, which can be found <a href="http://parts.igem.org/Help:Protocols/Competent_Cells">here</a>
 +
<ol>
 +
<li> Making CCMB 80 Buffer
 +
<ul>
 +
<li>10 mM KOAc pH 7.0 (10 ml of a 1M stock/L)</li>
 +
<li>80 mM CaCl2.2H2O (11.8 g/L)</li>
 +
<li>20 mM MnCl2.4H2O (4.0 g/L)</li>
 +
<li>10 mM MgCl2.6H2O (2.0 g/L)</li>
 +
<li>10% glycerol (100 ml/L)</li>
 +
<li>adjust pH DOWN to 6.4 with 0.1M HCl if necessary</li>
 +
</ul>
 +
</li>
 +
<li> Culturing Cells
 +
<ol>
 +
<li>Scrape cells from a colony or frozen stock of the desired strain</li>
 +
<li>Inoculate into 5 mL SOC (or LB+Antibiotic for plasmid-containing strains)</li>
 +
<li>Grow for ~8 hrs (morning to late afternoon) in 37C shaker</li>
 +
<li>Add 5 mL of culture to 250 mL SOC (or LB+Antibiotic) in a shaker flask
 +
<ul><li>250 mL culture will yield approximately 50 chemically competent samples. For smaller batches, we add 1 mL into 50 mL.
 +
</li></ul>
 +
</li>
 +
<li>Grow overnight for ~16 hrs in a shaker at room temperature</li>
 +
</ol>
 +
</li>
 +
<li> Treating cells </li>
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<ol>
 +
<li>Transfer culture into 50 mL centrifuge tubes</li>
 +
<li>Pellet cells at 4500 RPM for 10 mins</li>
 +
<li>Pour off supernatant and resuspend cells in 40 mL CCMB 80 Buffer</li>
 +
<li>Incubate on ice for 20 minutes</li>
 +
<li>Pellet cells at 4500 RPM for 10 mins</li>
 +
<li>Pour off supernatant and resuspend cells in 5 mL CCMB 80 Buffer</li>
 +
<li>Incubate on ice for 20 minutes</li>
 +
<li>Aliquot 750 uL each into pre-chilled microcentrifuge tubes</li>
 +
<li>Store at -80C until use</li>
 +
</ol>
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<p>Note: we never refreeze competent cells once thawed. Any unused cells in an aliquot are discarded.</p>
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</ol>
</div>
</div>
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</div>
 
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<div id="undergrad">
 
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<h1> Undergrads </h1>
 
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<div class="bio">
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<div class="pro">
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<div id="ciesla">
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<a id="transform"><h2> Transformation </h2></a>
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<h2> TJ Ciesla </h2>
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This is our standard heat-shock transformation protocol for E. coli cells. We use it for ligations and other assemblies as well as transformation of intact plasmids (i.e. stocks from the distribution kit).
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<img src="https://static.igem.org/mediawiki/2014/3/31/Imgcomingsoon.jpg" />
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<ol>
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<!-- image should be 200px by 300px, I think. Height of the bio class should be bigger than the height of the picture.-->
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<li>Thaw chemically competent cells on ice</li>
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TJ is a Duke junior majoring in biomedical engineering with a certificate in genome science and policy from Tampa, FL. While not iGEMing, he dances on the Duke Raas team, plays the ukulele and optimizes his calorie to vegetable ratio. He likes his coffee black.
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<li>Add 50 uL cells to 10 uL DNA (plasmid or assembly reaction)</li>
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</div>
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<li>Incubate on ice for 30 minutes</li>
 +
<li>Heat shock for 45 seconds at 42C</li>
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<li>Return to ice, incubate for 2 minutes</li>
 +
<li>Transfer cells into 1 mL SOC medium and shake at 37C for 1 hour</li>
 +
<li>Pellet cells at 4500 RPM for 3 minutes and pour off supernatant</li>
 +
<li>Vortex to resuspend and spread on LB+antibiotic agar plates</li>
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<li>Incubate overnight at 37C</li>
 +
</ol>
</div>
</div>
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<div class="bio">
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<div class="pro">
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<div id="farnitano">
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<a id="ligate"><h2> Ligation </h2></a>
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<h2> Matthew Farnitano </h2>
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We had some trouble with ligations earlier in the year, and our protocol has undergone changes in an attempt to improve our ligation success. This protocol represents the most recent (and most successful) version of our ligation protocol.
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<img src="https://static.igem.org/mediawiki/2014/3/31/Imgcomingsoon.jpg" />
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<ol>
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Matthew Farnitano is a junior at Duke University majoring in Biology with minors in chemistry and music. This is his second year as a member of Duke’s iGEM team. In addition to his lab life, Matt plays French horn with the Duke Wind Symphony, and has served as piano accompanist and pit orchestra member with Duke’s Hoof’n’horn musical theatre troupe. He grew up in the San Francisco Bay Area and loves travelling, having visited 49 US states (and counting). He is spending the fall semester in South Africa studying wildlife ecology and conservation with the Organization for Tropical Studies.  
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<li> Restriction digestion
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</div>
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<ul>
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<li>To insert a single part into a desired backbone, digest both the insert and backbone with EcoRI and SpeI</li>
 +
<li>To insert a new part downstream of an existing part, digest the backbone with PstI and XbaI, and the insert with PstI and SpeI</li>
 +
<li>Digest four individual tubes of each mini prepped plasmid</li>
 +
<li>Add 10 uL Cutsmart buffer and 5 uL total enzyme, along with dH20 to a final volume of 100 uL for each mini prep</li>
 +
<li>Incubate at 37C for 3+ hrs</li>
 +
</ul></li>
 +
<li> Gel extraction </li>
 +
<ul>
 +
<li>Load 200 uL (two digestion tubes) plus 20 uL loading dye into each lane of a 0.8% agarose/TAE gel
 +
<li>Run gel electrophoresis at 100 volts for 18 minutes
 +
<li>Cut desired band from gel
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<li>Extract DNA from gel band using <a href = http://www.zymoresearch.com/dna/dna-clean-up/gel-dna-recovery/zymoclean-gel-dna-recovery-kit>Zymoclean gel DNA recovery kit</a>
 +
</ul></li>
 +
<li> Antarctic Phosphatase treatment
 +
<ul>
 +
<li> Treat only the backbone with phosphatase</li>
 +
<li> We used Calf Intestinal Phosphatase earlier in the year, but seemed to have better results with Antarctic Phosphatase</li>
 +
<li>Combine two samples of gel extracted DNA (60 uL) with 10 uL Antarctic Phosphatase buffer, 2.5 uL phosphatase, and dH20 to a final volume of 100 uL</li>
 +
<li>Incubate for 1-2 hours at 37C</li>
 +
<li>Clean up sample by running our PCR cleanup protocol using the Qiagen miniprep kit</li>
 +
<li>Typical yield from minipreps to cleaned, processed DNA is about 10-25%, which is why we start with 4 minipreps to obtain one tube of product.</li>
 +
</ul></li>
 +
<li> Ligation
 +
<ul>
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<li>Combine the following in a 0.5 mL microcentrifuge tube:
 +
<ul>
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<li>100 ng backbone DNA</li>
 +
<li>3x molar excess of insert DNA (ex. 150 ng for an insert half the size of the backbone)</li>
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<li>1 uL T4 Ligase buffer </li>
 +
<li>0.5 uL T4 Ligase </li>
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<li>dH20 to a final volume of 10 uL</li>
 +
</ul></li>
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<li>For each reaction, make a backbone-only control and an insert-only control, with an equal volume of dH20 replacing the missing DNA</li>
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<li>Incubate for 1 hour at room temperature (shorter incubation times do not seem to affect efficiency, but this was our default)</li>
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<li>Transform into chemically competent E. coli</li>
 +
</ul></li>
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</ol>
</div>
</div>
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<div class="bio">
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<div class="pro">
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<div id="faw">
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<a id="pcrprep"><h2> PCR preparation of inserts </h2></a>
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<h2> Matthew Faw </h2>
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This protocol was used to isolate PCR products for insertion into BioBrick backbones. It is derived from the <a href="https://www.neb.com/protocols/2013/12/13/pcr-using-q5-high-fidelity-dna-polymerase-m0491">Q5 polymerase PCR protocol</a> by New England Biolabs.
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<img src="https://static.igem.org/mediawiki/2014/3/31/Imgcomingsoon.jpg" />
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<ol>
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Matthew Faw still believes in Santa. Nobody ruin it for him.
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<li>Combine the following in individual strip-capped tubes on ice:
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</div>
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<ul>
 +
<li>10 uL 5x Q5 Reaction buffer</li>
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<li>1 uL dNTPs</li>
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<li>0.25 uL each primer (100uM)</li>
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<li>0-1 ng template DNA</li>
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<li>0.5 uL Q5 DNA polymerase</li>
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<li>dH20 to 50 uL final volume</li>
 +
</ul></li>
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<li>Run thermocycler protocol:
 +
<ol>
 +
<li>30 seconds at 98C</li>
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<li>35 cycles:
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<ul>
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<li>10 seconds at 98C</li>
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<li>30 seconds annealing at 2-3C above the lower Tm of the two primers</li>
 +
<li>20-30 seconds per kilobase extension at 72C</li>
 +
</ul></li>
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<li>10 minutes at 72C</li>
 +
<li>hold at 4C</li>
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</ol></li>
 +
<li>Run 2 uL per tube on a 0.8% agarose/TAE gel for 20 minutes to confirm reaction</li>
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<li>Combine tubes and clean up using PCR cleanup protocol</li>
 +
</ol>
</div>
</div>
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<div class="bio">
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<div class="pro">
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<div id="ghoshal">
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<a id="pcrclean"><h2> PCR cleanup </h2></a>
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<h2> Delta Ghoshal </h2>
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We used this protocol to clean up DNA after PCRs, digestions, and other enzyme treatments. It is derived from, and uses materials from, the protocol for the <a href="http://www.qiagen.com/products/catalog/sample-technologies/dna-sample-technologies/plasmid-dna/qiaprep-spin-miniprep-kit">Qiagen spin miniprep kit.</a>
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<img src="https://static.igem.org/mediawiki/2014/3/31/Imgcomingsoon.jpg" />
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<ol>
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Delta is a sophomore majoring in Biomedical Engineering, with a certificate in Genome Sciences and Policy. She sings in the Duke Chorale and likes science a lot. She also enjoys eating, sleeping, reading, knitting, jokes concerning her name, and dancing, <em>sometimes all at the same time</em>.
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<li>Add 5x volume of Buffer PB to sample, mix and transfer to a spin column.</li>
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</div>
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<li>Spin at 13,000 rpm for 30 seconds. Pour the flow-through back into the column and spin again. Then discard the flow-through.</li>
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<li>Add 750 uL of Buffer PE to the column. Spin for 30 seconds, discard the flow-through, then spin again for 1 minute to remove residual buffer.</li>
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<li>Transfer the spin column to a labelled microcentrifuge tube. Add 30 uL of Buffer EB, let stand for 3-5 minutes, then spin for 1 minute to elute DNA.</li>
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</ol>
</div>
</div>
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<div class="bio">
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<div class="pro">
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<div id="zhu">
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<a id="colpcr"><h2> Colony PCR </h2></a>
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<h2> Mike Zhu </h2>
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This is a screening method for identifying transformants after a ligation or other assembly. We used our oligos SB1C3-up and SB1C3-dn with this protocol to screen BioBricks in the standard backbone pSB1C3. We also used our oligos pdCas9-up and pdCas9-dn with this protocol to screen for the addition of crRNA sequences into our pdCas9 plasmid.
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<img src="https://static.igem.org/mediawiki/2014/3/31/Imgcomingsoon.jpg" />
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<ol>
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Mike is a junior studying Biomedical Engineering and Computer Science. Outside of iGEM, Mike conducts research with Dr. John Reif on DNA nanotechnology and is involved with the Chinese Dance team. He enjoys spending his free time cooking, eating, being Canadian, and falling asleep.
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<li>Combine the following in individual strip-capped tubes on ice:
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<ul>
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<li>5 uL Taq buffer</li>
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<li>1 uL dNTPs</li>
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<li>0.25 uL each primer (100 uM)</li>
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<li>0.25 uL Taq polymerase</li>
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<li>dH20 to 50 uL final volume</li>
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</ul></li>
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<li>Prepare a 50 uL SOC tube parallel to each PCR tube</li>
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<li>Inoculate each colony from the transformation plate into a PCR tube, then into its parallel SOC tube</li>
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<li>Run PCR tubes in thermocycler protocol</li>
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<li>Run 3 uL from each PCR tube on a 0.8% agarose/TAE gel to identify successful amplicons</li>
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<li>Add SOC copies of successful amplicons to 5 mL LB+antibiotic medium and grow overnight at 37C</li>
 +
</ol>
 +
PCR Program:
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<ul>
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<li>95°C 5min</li>
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<li>Repeat the following three steps 30 times:</li>
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<li>94°C 45s</li>
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<li>50°C 1min</li>
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<li>68°C 1min 30s</li>
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<li>72°C 10min</li>
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<li>4°C, indefinitely (try to be max 1 hour)</li>
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</ul>
</div>
</div>
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<div id="hischool">
 
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<h1> High School Students </h1>
 
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<div class="bio">
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<div class="pro">
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<div id="tomar">
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<a id="flow"><h2> Flow Cytometry </h2></a>
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<h2> Garima Tomar </h2>
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We used Flow Cytometry to view the fluorescence of our cells at the individual level. This is the protocol we used when running flow samples.
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<img src="http://upload.wikimedia.org/wikipedia/en/thumb/b/b7/Stanford_University_seal_2003.svg/1024px-Stanford_University_seal_2003.svg.png" />
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<ol>
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Garima is a senior at the North Carolina School of Science and Mathematics. In her free time, she enjoys playing tennis, spending time with her sister, and learning cool things about math. She also dances, and is one of the captains of NCSSM Bhangra. Garima is really excited to be a part of the Duke iGEM team this year, as she is interested in both applying to Duke and majoring in biomedical engineering!</div>
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<li>Culture cells
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</div>
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<ul>
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<li>Inoculate colonies of desired strains into 5 mL each of LB+antibiotic</li>
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<li>If testing induction with aTc:
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<ul>
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<li>Inoculate colonies into 50 uL SOC and mix</li>
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<li>Add 25 uL of SOC mix into medium with aTc/EtOH added</li>
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<li>Add 25 uL of SOC mix into medium with 100% EtOH added (no aTc)</li>
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</ul></li>
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<li>Grow to stationary phase (~18 hrs) at 37C
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</ul></li>
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<li>Dilute cultures 1/1000
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<ul>
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<li>Add 5 uL culture to 5 mL equivalent medium</li>
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<li>Grow to mid-log phase for 4.5 hours at 37C</li>
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</ul></li>
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<li>Prepare samples for flow
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<ul>
 +
<li>Add 20 uL culture to 1 mL PBS (1/50 dilution)</li>
 +
<li>Place on ice until sample is run</li>
 +
</ul></li>
 +
<li>Run samples with the following parameters:
 +
<ul>
 +
<li>FSC: log3 scale, 350 Volts</li>
 +
<li>SSC: log3 scale, 350 Volts</li>
 +
<li>B1 (GFP/FITC): log5 scale, 350 Volts</li>
 +
<li>FSC trigger 10.0</li>
 +
<li>10,000 events per sample</li>
 +
</ul></li>
 +
</ol>
</div>
</div>
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<div id="grad">
 
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<h1> Graduate Students </h1>
 
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<div class="bio">
 
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<div id="cooper">
 
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<h2> Charlie Cooper </h2>
 
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<img src="https://static.igem.org/mediawiki/2014/3/31/Imgcomingsoon.jpg" />
 
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Charlie is a PhD candidate in Nick Buchler's lab. He is currently working on developing new tools to control biological processes.
 
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<h1> Instructors </h1>
 
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<div class="bio">
 
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<div id="buchler">
 
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<h2> Dr. Nicolas Buchler </h2>
 
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<img src= "https://static.igem.org/mediawiki/2014/3/31/Imgcomingsoon.jpg">
 
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Nick really likes oscillators, such as the cell cycle, metabolic rhythms, circadian clocks, and synthetic oscillators.  Folks in his lab use a combination of synthetic biology, time lapse microscopy, microfluidics, comparative genomics, mathematical modelling, and molecular genetics to understand biological oscillation.  When he’s not in lab, he’s at home on 5 acres in the Duke forest with his family.  The summer fireflies are very satisfying to watch.
 
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<div class="bio">
 
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<div id="gersbach"><h2> Dr. Charlie Gersbach </h2>
 
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<img src= "https://static.igem.org/mediawiki/2014/9/9a/073911_gersbach001.jpg">
 
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Dr. Charles A. Gersbach is an Assistant Professor in the Department of Biomedical Engineering and Center for Genomic and Computational Biology at Duke University.  He has research interests in gene therapy, regenerative medicine, biomolecular and cellular engineering, synthetic biology, and genomics.  Dr. Gersbach received his Bachelor's degree in Chemical Engineering from the Georgia Institute of Technology and Ph.D. in Biomedical Engineering from the Georgia Institute of Technology and Emory University School of Medicine focusing on the genetic reprogramming of adult stem cells for musculoskeletal tissue regeneration.  Dr. Gersbach completed his postdoctoral training at The Scripps Research Institute in molecular biology and biochemistry.    Dr. Gersbach's laboratory at Duke University is focused on applying molecular and cellular engineering to applications in gene therapy, regenerative medicine, and basic science.  Examples of technologies used in his research include genome engineering, protein engineering, directed evolution, genetic reprogramming, gene delivery, and optogenetics. 
 
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Latest revision as of 03:56, 18 October 2014

Protocols

Preparing Chemically Competent Cells

This protocol is for making chemically competent E. coli for transformations. It is derived from the official iGEM protocol, which can be found here
  1. Making CCMB 80 Buffer
    • 10 mM KOAc pH 7.0 (10 ml of a 1M stock/L)
    • 80 mM CaCl2.2H2O (11.8 g/L)
    • 20 mM MnCl2.4H2O (4.0 g/L)
    • 10 mM MgCl2.6H2O (2.0 g/L)
    • 10% glycerol (100 ml/L)
    • adjust pH DOWN to 6.4 with 0.1M HCl if necessary
  2. Culturing Cells
    1. Scrape cells from a colony or frozen stock of the desired strain
    2. Inoculate into 5 mL SOC (or LB+Antibiotic for plasmid-containing strains)
    3. Grow for ~8 hrs (morning to late afternoon) in 37C shaker
    4. Add 5 mL of culture to 250 mL SOC (or LB+Antibiotic) in a shaker flask
      • 250 mL culture will yield approximately 50 chemically competent samples. For smaller batches, we add 1 mL into 50 mL.
    5. Grow overnight for ~16 hrs in a shaker at room temperature
  3. Treating cells
    1. Transfer culture into 50 mL centrifuge tubes
    2. Pellet cells at 4500 RPM for 10 mins
    3. Pour off supernatant and resuspend cells in 40 mL CCMB 80 Buffer
    4. Incubate on ice for 20 minutes
    5. Pellet cells at 4500 RPM for 10 mins
    6. Pour off supernatant and resuspend cells in 5 mL CCMB 80 Buffer
    7. Incubate on ice for 20 minutes
    8. Aliquot 750 uL each into pre-chilled microcentrifuge tubes
    9. Store at -80C until use

    Note: we never refreeze competent cells once thawed. Any unused cells in an aliquot are discarded.

Transformation

This is our standard heat-shock transformation protocol for E. coli cells. We use it for ligations and other assemblies as well as transformation of intact plasmids (i.e. stocks from the distribution kit).
  1. Thaw chemically competent cells on ice
  2. Add 50 uL cells to 10 uL DNA (plasmid or assembly reaction)
  3. Incubate on ice for 30 minutes
  4. Heat shock for 45 seconds at 42C
  5. Return to ice, incubate for 2 minutes
  6. Transfer cells into 1 mL SOC medium and shake at 37C for 1 hour
  7. Pellet cells at 4500 RPM for 3 minutes and pour off supernatant
  8. Vortex to resuspend and spread on LB+antibiotic agar plates
  9. Incubate overnight at 37C

Ligation

We had some trouble with ligations earlier in the year, and our protocol has undergone changes in an attempt to improve our ligation success. This protocol represents the most recent (and most successful) version of our ligation protocol.
  1. Restriction digestion
    • To insert a single part into a desired backbone, digest both the insert and backbone with EcoRI and SpeI
    • To insert a new part downstream of an existing part, digest the backbone with PstI and XbaI, and the insert with PstI and SpeI
    • Digest four individual tubes of each mini prepped plasmid
    • Add 10 uL Cutsmart buffer and 5 uL total enzyme, along with dH20 to a final volume of 100 uL for each mini prep
    • Incubate at 37C for 3+ hrs
  2. Gel extraction
    • Load 200 uL (two digestion tubes) plus 20 uL loading dye into each lane of a 0.8% agarose/TAE gel
    • Run gel electrophoresis at 100 volts for 18 minutes
    • Cut desired band from gel
    • Extract DNA from gel band using Zymoclean gel DNA recovery kit
  3. Antarctic Phosphatase treatment
    • Treat only the backbone with phosphatase
    • We used Calf Intestinal Phosphatase earlier in the year, but seemed to have better results with Antarctic Phosphatase
    • Combine two samples of gel extracted DNA (60 uL) with 10 uL Antarctic Phosphatase buffer, 2.5 uL phosphatase, and dH20 to a final volume of 100 uL
    • Incubate for 1-2 hours at 37C
    • Clean up sample by running our PCR cleanup protocol using the Qiagen miniprep kit
    • Typical yield from minipreps to cleaned, processed DNA is about 10-25%, which is why we start with 4 minipreps to obtain one tube of product.
  4. Ligation
    • Combine the following in a 0.5 mL microcentrifuge tube:
      • 100 ng backbone DNA
      • 3x molar excess of insert DNA (ex. 150 ng for an insert half the size of the backbone)
      • 1 uL T4 Ligase buffer
      • 0.5 uL T4 Ligase
      • dH20 to a final volume of 10 uL
    • For each reaction, make a backbone-only control and an insert-only control, with an equal volume of dH20 replacing the missing DNA
    • Incubate for 1 hour at room temperature (shorter incubation times do not seem to affect efficiency, but this was our default)
    • Transform into chemically competent E. coli

PCR preparation of inserts

This protocol was used to isolate PCR products for insertion into BioBrick backbones. It is derived from the Q5 polymerase PCR protocol by New England Biolabs.
  1. Combine the following in individual strip-capped tubes on ice:
    • 10 uL 5x Q5 Reaction buffer
    • 1 uL dNTPs
    • 0.25 uL each primer (100uM)
    • 0-1 ng template DNA
    • 0.5 uL Q5 DNA polymerase
    • dH20 to 50 uL final volume
  2. Run thermocycler protocol:
    1. 30 seconds at 98C
    2. 35 cycles:
      • 10 seconds at 98C
      • 30 seconds annealing at 2-3C above the lower Tm of the two primers
      • 20-30 seconds per kilobase extension at 72C
    3. 10 minutes at 72C
    4. hold at 4C
  3. Run 2 uL per tube on a 0.8% agarose/TAE gel for 20 minutes to confirm reaction
  4. Combine tubes and clean up using PCR cleanup protocol

PCR cleanup

We used this protocol to clean up DNA after PCRs, digestions, and other enzyme treatments. It is derived from, and uses materials from, the protocol for the Qiagen spin miniprep kit.
  1. Add 5x volume of Buffer PB to sample, mix and transfer to a spin column.
  2. Spin at 13,000 rpm for 30 seconds. Pour the flow-through back into the column and spin again. Then discard the flow-through.
  3. Add 750 uL of Buffer PE to the column. Spin for 30 seconds, discard the flow-through, then spin again for 1 minute to remove residual buffer.
  4. Transfer the spin column to a labelled microcentrifuge tube. Add 30 uL of Buffer EB, let stand for 3-5 minutes, then spin for 1 minute to elute DNA.

Colony PCR

This is a screening method for identifying transformants after a ligation or other assembly. We used our oligos SB1C3-up and SB1C3-dn with this protocol to screen BioBricks in the standard backbone pSB1C3. We also used our oligos pdCas9-up and pdCas9-dn with this protocol to screen for the addition of crRNA sequences into our pdCas9 plasmid.
  1. Combine the following in individual strip-capped tubes on ice:
    • 5 uL Taq buffer
    • 1 uL dNTPs
    • 0.25 uL each primer (100 uM)
    • 0.25 uL Taq polymerase
    • dH20 to 50 uL final volume
  2. Prepare a 50 uL SOC tube parallel to each PCR tube
  3. Inoculate each colony from the transformation plate into a PCR tube, then into its parallel SOC tube
  4. Run PCR tubes in thermocycler protocol
  5. Run 3 uL from each PCR tube on a 0.8% agarose/TAE gel to identify successful amplicons
  6. Add SOC copies of successful amplicons to 5 mL LB+antibiotic medium and grow overnight at 37C
PCR Program:
  • 95°C 5min
  • Repeat the following three steps 30 times:
  • 94°C 45s
  • 50°C 1min
  • 68°C 1min 30s
  • 72°C 10min
  • 4°C, indefinitely (try to be max 1 hour)

Flow Cytometry

We used Flow Cytometry to view the fluorescence of our cells at the individual level. This is the protocol we used when running flow samples.
  1. Culture cells
    • Inoculate colonies of desired strains into 5 mL each of LB+antibiotic
    • If testing induction with aTc:
      • Inoculate colonies into 50 uL SOC and mix
      • Add 25 uL of SOC mix into medium with aTc/EtOH added
      • Add 25 uL of SOC mix into medium with 100% EtOH added (no aTc)
    • Grow to stationary phase (~18 hrs) at 37C
  2. Dilute cultures 1/1000
    • Add 5 uL culture to 5 mL equivalent medium
    • Grow to mid-log phase for 4.5 hours at 37C
  3. Prepare samples for flow
    • Add 20 uL culture to 1 mL PBS (1/50 dilution)
    • Place on ice until sample is run
  4. Run samples with the following parameters:
    • FSC: log3 scale, 350 Volts
    • SSC: log3 scale, 350 Volts
    • B1 (GFP/FITC): log5 scale, 350 Volts
    • FSC trigger 10.0
    • 10,000 events per sample

Retrieved from "http://2014.igem.org/Team:Duke/Notebook/Protocols"