Difference between revisions of "Team:EpiphanyNYC/Notebook"

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<p>&nbsp;</p>
 
<p>&nbsp;</p>
 
<div>
 
<div>
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<p><strong>Week 1 (March 19-25)</strong></p>
+
 
−
<ul>
+
      <p class="c2"><span class="c1">Week 1 (March 19-25)</span></p>
−
<li>Consulted with our advisor, Frank, to advise researchers and scientists</li>
+
      <ul class="c3 lst-kix_70t670etpzqj-0 start">
−
<li>Reached out to scientists at Mt Sinai Hospital, Jason Fuller and Russell Hanson</li>
+
        <li class="c0"><span class="c1">Consulted with our advisor, Frank, to advise researchers and scientists</span></li>
−
<li>Met with Jason Fuller and Russell Hanson, scientists at Genetics Department at Mt Sinai Hospital</li>
+
        <li class="c0"><span class="c1">Reached out to scientists at Mt Sinai Hospital, Jason Fuller and Russell Hanson</span></li>
−
<li>Scheduled weekly meetings for the rest of the year</li>
+
        <li class="c0"><span class="c1">Met with Jason Fuller and Russell Hanson, scientists at Genetics Department at Mt Sinai Hospital </span></li>
−
</ul>
+
        <li class="c0"><span class="c1">Scheduled weekly meetings for the rest of the year</span></li>
−
<p><strong>Week 2 (March 26-Apr 1) </strong></p>
+
      </ul>
−
<ul>
+
      <p class="c2"><span class="c1">Week 2 (March 26-Apr 1) </span></p>
−
<li>Brainstormed for a potential topic (Sickle Cell Anemia, Cystic Fibrosis, Huntington’s Disease); decided on HD, proposed by Yeji Cho</li>
+
      <ul class="c3 lst-kix_70t670etpzqj-0">
−
<li>Determined project leaders of the project</li>
+
        <li class="c0"><span class="c1">Brainstormed for a potential topic (Sickle Cell Anemia, Cystic Fibrosis, Huntington&rsquo;s Disease); decided on HD, proposed by Yeji Cho </span></li>
−
<li>Divided into subsections (website, design, fundraising, social media, computational) and assigned roles, such as other team leads</li>
+
        <li class="c0"><span class="c1">Determined project leaders of the project </span></li>
−
</ul>
+
        <li class="c0"><span class="c1">Divided into subsections (website, design, fundraising, social media, computational) and assigned roles, such as other team leads</span></li>
−
<p><strong>Week 3 (Apr 2-8)</strong></p>
+
      </ul>
−
<ul>
+
      <p class="c2"><span class="c1">Week 3 (Apr 2-8)</span></p>
−
<li>Determined possible method to target the disease</li>
+
      <ul class="c3 lst-kix_70t670etpzqj-0">
−
<li>Outlined the abstract for the project</li>
+
        <li class="c0"><span class="c1">Determined possible method to target the disease </span></li>
−
</ul>
+
        <li class="c0"><span class="c1">Outlined the abstract for the project</span></li>
−
<p><strong>Week 4 (Apr 9-15)</strong></p>
+
      </ul>
−
<ul>
+
      <p class="c2"><span class="c1">Week 4 (Apr 9-15)</span></p>
−
<li>Took headshots of members and team pictures to put on website</li>
+
      <ul class="c3 lst-kix_70t670etpzqj-0">
−
<li>Administered Drug Tests for medical clearance</li>
+
        <li class="c0"><span class="c1">Took headshots of members and team pictures to put on website</span></li>
−
<li>HIPAA (Health Insurance Portability and Accountability Act) Training</li>
+
        <li class="c0"><span class="c1">Administered Drug Tests for medical clearance</span></li>
−
<li>Fire Drill Practice</li>
+
        <li class="c0"><span class="c1">HIPAA (Health Insurance Portability and Accountability Act) Training</span></li>
−
</ul>
+
        <li class="c0"><span class="c1">Fire Drill Practice</span></li>
−
<p><strong>Week 5 (Apr 16-22)</strong></p>
+
      </ul>
−
<ul>
+
      <p class="c2"><span class="c1">Week 5 (Apr 16-22)</span></p>
−
<li>Decided to use WordPress as website template</li>
+
      <ul class="c3 lst-kix_jc3tc6twt51v-0 start">
−
<li>Happily received our lab coats and Mount Sinai IDs</li>
+
        <li class="c0"><span class="c1">Decided to use Wordpress as website template </span></li>
−
</ul>
+
        <li class="c0"><span class="c1">Happily received our lab coats and Mount Sinai IDs </span></li>
−
<p><strong>Week 6 (Apr 23-29)</strong></p>
+
      </ul>
−
<ul>
+
      <p class="c2"><span class="c1">Week 6 (Apr 23-29)</span></p>
−
<li>Submitted payment for iGEM group registration</li>
+
      <ul class="c3 lst-kix_vn26atnorkn4-0 start">
−
<li>Signed consent forms</li>
+
        <li class="c0"><span class="c1">Submitted payment for iGEM group registration</span></li>
−
</ul>
+
        <li class="c0"><span class="c1">Signed consent forms</span></li>
−
<p><strong>Week 7 (Apr 30-May 6)</strong></p>
+
      </ul>
−
<ul>
+
      <p class="c2"><span class="c1">Week 7 (Apr 30-May 6)</span></p>
−
<li>Designed graphic of brain and neuron for website</li>
+
      <ul class="c3 lst-kix_jc3tc6twt51v-0">
−
</ul>
+
        <li class="c0"><span class="c1">Designed graphic of brain and neuron for website</span></li>
−
<p><strong>Week 8 (May 7-13)</strong></p>
+
      </ul>
−
<ul>
+
      <p class="c2"><span class="c1">Week 8 (May 7-13)</span></p>
−
<li>Brainstormed for a team name and designed logo; decided on HD Resolution</li>
+
      <ul class="c3 lst-kix_mnv2z11q6een-0 start">
−
</ul>
+
        <li class="c0"><span class="c1">Brainstormed for a team name and designed logo; decided on HD Resolution</span></li>
−
<p><strong>Week 9 (May 14-20)</strong></p>
+
      </ul>
−
<ul>
+
      <p class="c2"><span class="c1">Week 9 (May 14-20)</span></p>
−
<li>Designed T-shirts, hats, and sweaters</li>
+
      <ul class="c3 lst-kix_jc3tc6twt51v-0">
−
<li>Brainstormed for companies/sponsors to partner with us</li>
+
        <li class="c0"><span class="c1">Designed T-shirts, hats, and sweaters </span></li>
−
<li>Received iGEM parts kit</li>
+
        <li class="c0"><span class="c1">Brainstormed for companies/sponsors to partner with us</span></li>
−
</ul>
+
        <li class="c0"><span class="c1">Received iGEM parts kit </span></li>
−
<p><strong>Week 10 (May 21-27)</strong></p>
+
      </ul>
−
<ul>
+
      <p class="c2"><span class="c1">Week 10 (May 21-27)</span></p>
−
<li>Organized home page of website</li>
+
      <ul class="c3 lst-kix_dhoxlsihlhs3-0 start">
−
<li>Created sponsorship/partnership letters for possible companies</li>
+
        <li class="c0"><span class="c1">Organized home page of website</span></li>
−
</ul>
+
        <li class="c0"><span class="c1">Created sponsorship/partnership letters for possible companies </span></li>
−
<p><strong>Week 11 (May 28-Jun 3)</strong></p>
+
      </ul>
−
<ul>
+
      <p class="c2"><span class="c1">Week 11 (May 28-Jun 3)</span></p>
−
<li>Julia, Erik and Stacy organized a table at Peace Angels Benefit Concert at Steinway and Sons Concert Hall in May 28, 2017</li>
+
      <ul class="c3 lst-kix_j0vybz4prg3m-0 start">
−
<li>Mailed out consent forms</li>
+
        <li class="c0"><span class="c1">Julia, Erik and Stacy organized a table at Peace Angels Benefit Concert at Steinway and Sons Concert Hall in May 28, 2017</span></li>
−
<li>Scheduled wetlab training dates for the summer</li>
+
        <li class="c0"><span class="c1">Mailed out consent forms</span></li>
−
</ul>
+
        <li class="c0"><span class="c1">Scheduled wetlab training dates for the summer </span></li>
−
<p><strong>Week 12 (Jun 4-10)</strong></p>
+
      </ul>
−
<ul>
+
      <p class="c2"><span class="c1">Week 12 (Jun 4-10)</span></p>
−
<li>Early Training: lab safety, project overview, practiced lab techniques such as pipetting, centrifugation, PCR, etc.</li>
+
      <ul class="c3 lst-kix_sgmogh7i17px-0 start">
−
</ul>
+
        <li class="c0"><span class="c1">Early Training: lab safety, project overview, practiced lab techniques such as pipetting, centrifugation, PCR, etc.</span></li>
−
<p><strong>Week 13 (Jun 11-17)</strong></p>
+
      </ul>
−
<ul>
+
      <p class="c2"><span class="c1">Week 13 (Jun 11-17)</span></p>
−
<li>Organized team page of website</li>
+
      <ul class="c3 lst-kix_90r8blgf1gec-0 start">
−
<li>Contacted over 50 private and public companies in pharmaceutical and biotech industries, non-profit organizations for sponsorship and support.</li>
+
        <li class="c0"><span class="c1">Organized team page of website</span></li>
−
</ul>
+
        <li class="c0"><span class="c1">Contacted over 50 private and public companies in pharmaceutical and biotech industries, non-profit organizations for sponsorship and support.</span></li>
−
<p><strong>Week 14 (Jun 18-24)</strong></p>
+
      </ul>
−
<ul>
+
      <p class="c2"><span class="c1">Week 14 (Jun 18-24)</span></p>
−
<li>Created Twitter account</li>
+
      <ul class="c3 lst-kix_nwqptpxjthaz-0 start">
−
<li>Christi and Marianne completed safety form 1, 2, 3</li>
+
        <li class="c0"><span class="c1">Created Twitter account</span></li>
−
</ul>
+
        <li class="c0"><span class="c1">Christi and Marianne completed safety form 1, 2, 3</span></li>
−
<p><strong>Week 15 (Jun 25-Jul 1)</strong></p>
+
      </ul>
−
<ul>
+
      <p class="c2"><span class="c1">Week 15 (Jun 25-Jul 1)</span></p>
−
<li>Jason, Justin, Jeewhan, Marianne met up to analyze the data collection</li>
+
      <ul class="c3 lst-kix_306lxmpsexn7-0 start">
−
</ul>
+
        <li class="c0"><span class="c1">Jason, Justin, Jeewhan, Marianne met up to analyze the data collection</span></li>
−
<p><strong>Week 16 (Jul 2-8)</strong></p>
+
      </ul>
−
<ul>
+
      <p class="c2"><span class="c1">Week 18 (Jul 16-22)</span></p>
−
<li><b>Computational Lab Weeks: </b></li>
+
      <ul class="c3 lst-kix_j0vybz4prg3m-0">
−
<li>First weeks  we talked about how to get an application called mFold</li>
+
        <li class="c0"><span class="c1">Christi, Rachel, Esther, and Marianne organized a fundraising and information table at Dominico-American Society of Queens in Flushing Town Hall in Flushing, Queens NY in July 22 2017</span></li>
−
<li>mFold helps to create a simulation of the RNA bonds fusing instead having to actual do it experimentally</li>
+
        <li class="c0"><span class="c1">Created Facebook account</span></li>
−
</ul>
+
        <li class="c0"><span class="c1">Created GoFundMe page</span></li>
−
<p><strong>Week 17 (Jul 9-15)</strong></p>
+
      </ul>
−
<ul>
+
      <p class="c2"><span class="c1">Week 19 (Jul 23-29)</span></p>
−
<li><b>Computational Lab Weeks (continued):</b></li>
+
      <ul class="c3 lst-kix_pbtu2jwv32y-0 start">
−
<li>Looked for Huntington’s mRNA in particular in NCBI</li>
+
        <li class="c0"><span class="c1">Received over 100 likes on Facebook page thanks to our Social Media and Outreach team.</span></li>
−
<li>NM numbers, other prefixes gave types of mRNA</li>
+
      </ul>
−
<li>Found Huntington’s-related sequences in NCBI database  (assumed at least 5-10 sequences)</li>
+
      <p class="c2"><span class="c1">Week 20 (Jul 30-Aug 5)</span></p>
−
</ul>
+
      <ul class="c3 lst-kix_txch3swte3on-0 start">
−
<p><strong>Week 18 (Jul 16-22)</strong></p>
+
        <li class="c0"><span class="c1">Ordered materials (E. coli, etc)</span></li>
−
<ul>
+
      </ul>
−
<li>Christi, Rachel, Esther, and Marianne organized a fundraising and information table at Dominico-American Society of Queens in Flushing Town Hall in Flushing, Queens NY in July 22 2017</li>
+
      <p class="c2"><span class="c1">Week 21 (Aug 6-12)</span></p>
−
<li>Created Facebook account</li>
+
      <ul class="c3 lst-kix_3n5ldna2ea9g-0 start">
−
<li>Created GoFundMe page</li>
+
        <li class="c0"><span class="c1">Wrote letters to respective schools to ask for partnerships</span></li>
−
</ul>
+
      </ul>
−
<p><strong>Week 19 (Jul 23-29)</strong></p>
+
      <p class="c2"><span class="c1">Week 22 (Aug 13-19)</span></p>
−
<ul>
+
      <ul class="c3 lst-kix_c4v382ghf5j3-0 start">
−
<li>Received over 100 likes on Facebook page thanks to our Social Media and Outreach team.</li>
+
        <li class="c0"><span class="c1">Created Instagram account</span></li>
−
<li><strong>Computational Data: </strong></li>
+
        <li class="c0"><span class="c1">Grew cell subcultures of E. Coli for future testing </span></li>
−
<li>We used data we received from [ncbi.com] and used it to compare with the wild type and the infected types.</li>
+
        <li class="c0"><span class="c1">Familiarized ourselves with autoclaving and basic sterilizing processes </span></li>
−
<li>Found 2 accession numbers- NM_002111.8  (mRNA) and NP_002102.4  (protein); however, only one was mRNA and none were disease form</li>
+
        <li class="c0"><span class="c1">Learned how to store E. coli for future experiments</span></li>
−
<li>We then collected the data in our shared folder</li>
+
        <li class="c0"><span class="c1">HDSA agreed to be sponsor</span></li>
−
<li>Looked through other databases for other mutants; found:</li>
+
      </ul>
−
<li>Study with “20 Huntington&#8217;s Disease and 49 neurologically normal control samples from post-mortem human subjects”</li>
+
      <p class="c2"><span class="c1">Week 23 (Aug 20-26)</span></p>
−
<li><a href="http://trace.ddbj.nig.ac.jp/DRASearch/study?acc=SRP051844">http://trace.ddbj.nig.ac.jp/DRASearch/study?acc=SRP051844</a></li>
+
      <ul class="c3 lst-kix_o36rq28sw915-0 start">
−
<li>Results for HTT on DNA Data Bank of Japan (includes above):</li>
+
        <li class="c0"><span class="c1">Learned about transfection and tested cells for competency</span></li>
−
<li><a href="http://trace.ddbj.nig.ac.jp/DRASearch/query?keyword=htt&amp;show=20&amp;fq_rep_name=Homo%20sapiens">http://trace.ddbj.nig.ac.jp/DRASearch/query?keyword=htt&amp;show=20&amp;fq_rep_name=Homo%20sapiens</a></li>
+
        <li class="c0"><span class="c1">Updated team Wiki&rsquo;s page on the iGem website </span></li>
−
<li>Ran wild-type mRNA on mfold (Created a text document with the NM_00211.8 RNA and ran mFold on it <a href="http://unafold.rna.albany.edu/?q=mfold">http://unafold.rna.albany.edu/?q=mfold</a>)</li>
+
        <li class="c0"><span class="c1">Reached out to our respective high schools for sponsorship</span></li>
−
<li>We then collected the data in our shared folder</li>
+
        <li class="c0"><span class="c1">Hereditary Disease Foundation/Huntington&rsquo;s Disease Foundation agreed to be sponsor</span></li>
−
</ul>
+
        <li class="c0"><span class="c1">Spread awareness of our project to Alzheimer&rsquo;s Foundation of America</span></li>
−
<p><strong>Week 20 (Jul 30-Aug 5)</strong></p>
+
      </ul>
−
<ul>
+
      <p class="c2"><span class="c1">Week 24 (Aug 27-Sept 2)</span></p>
−
<li>Ordered materials (E. coli, etc)</li>
+
      <ul class="c3 lst-kix_j0vybz4prg3m-0">
−
<li><strong>Computational Data: </strong></li>
+
        <li class="c0"><span class="c1">Jessica, Erin, Catherine, and Yunsu helped organize a presentation table at Dominico-American Society of Queens Benefit Concert at National Opera America Center in New York NY on August 30 2017 </span></li>
−
<li>Changed programs to ViennaRNA:
+
        <li class="c0"><span class="c1">Reached out to Cerebral Palsy Alliance Research Foundation about partnering and sponsorship and discussed creating a joint campaign CPARF&rsquo;s Steptember campaign to promote 10,000 steps a day goal</span></li>
−
<div></div>
+
        <li class="c0"><span class="c1">Registered team members and created a team roster </span></li>
−
<div>
+
      </ul>
−
<p><span class="m_2664431920058259612gmail-im"><b><img data-attachment-id="1383" data-permalink="https://hdresolutionigem.com/notebook-2/notebook/screen-shot-2017-10-10-at-9-45-23-pm/" data-orig-file="https://i0.wp.com/hdresolutionigem.com/wp-content/uploads/2017/09/Screen-Shot-2017-10-10-at-9.45.23-PM.png?fit=597%2C569&amp;ssl=1" data-orig-size="597,569" data-comments-opened="1" data-image-meta="{&quot;aperture&quot;:&quot;0&quot;,&quot;credit&quot;:&quot;&quot;,&quot;camera&quot;:&quot;&quot;,&quot;caption&quot;:&quot;&quot;,&quot;created_timestamp&quot;:&quot;0&quot;,&quot;copyright&quot;:&quot;&quot;,&quot;focal_length&quot;:&quot;0&quot;,&quot;iso&quot;:&quot;0&quot;,&quot;shutter_speed&quot;:&quot;0&quot;,&quot;title&quot;:&quot;&quot;,&quot;orientation&quot;:&quot;0&quot;}" data-image-title="Screen Shot 2017-10-10 at 9.45.23 PM" data-image-description="" data-medium-file="https://i0.wp.com/hdresolutionigem.com/wp-content/uploads/2017/09/Screen-Shot-2017-10-10-at-9.45.23-PM.png?fit=300%2C286&amp;ssl=1" data-large-file="https://i0.wp.com/hdresolutionigem.com/wp-content/uploads/2017/09/Screen-Shot-2017-10-10-at-9.45.23-PM.png?fit=597%2C569&amp;ssl=1" class="alignnone size-medium wp-image-1383" src="https://i0.wp.com/hdresolutionigem.com/wp-content/uploads/2017/09/Screen-Shot-2017-10-10-at-9.45.23-PM.png?resize=300%2C286&#038;ssl=1" alt="" srcset="https://i0.wp.com/hdresolutionigem.com/wp-content/uploads/2017/09/Screen-Shot-2017-10-10-at-9.45.23-PM.png?resize=300%2C286&amp;ssl=1 300w, https://i0.wp.com/hdresolutionigem.com/wp-content/uploads/2017/09/Screen-Shot-2017-10-10-at-9.45.23-PM.png?w=597&amp;ssl=1 597w" sizes="(max-width: 300px) 100vw, 300px" data-recalc-dims="1" /></b></span></p>
+
      <p class="c2"><span class="c1">Week 25 (Sep 3-9)</span></p>
−
<li>Re-calculated sequences with artificially added CAG repeats (added 42 and 60 repeats)</li>
+
      <ul class="c3 lst-kix_klwsvs4m86wv-0 start">
−
</div>
+
        <li class="c0"><span class="c1">Figured out wet lab schedules and iGEM meetings</span></li>
−
</li>
+
        <li class="c0"><span class="c1">Started introducing each member on Instagram</span></li>
−
</ul>
+
        <li class="c0"><span class="c1">Uploaded sponsors and neuron design on website</span></li>
−
<p><strong>Week 21 (Aug 6-12)</strong></p>
+
      </ul>
−
<ul>
+
      <p class="c2"><span class="c1">Week 26 (Sep 10-16)</span></p>
−
<li>Wrote letters to respective schools to ask for partnerships</li>
+
      <ul class="c3 lst-kix_9ok1theky3vi-0 start">
−
<li>Manually analyzed results of ViennaRNA calculations for chaperone/guideRNA hairpin targets.</li>
+
        <li class="c0"><span class="c1">Arranged tabs on website</span></li>
−
<li>Determined that this process was less reproducible than desired, and it worried the most viable hairpin loop might be buried in the the tertiary structure</li>
+
        <li class="c0"><span class="c1">Sent out emails to organizations for outreach</span></li>
−
<li>Decided to change approach: Use RNAi tools to calculate the toe-hold footing (areas in RNA that can be used to initiate strand displacement and/or break apart mRNA secondary structures).</li>
+
        <li class="c0"><span class="c1">Reached out to organizations on Facebook and iGEM teams for collaboration</span></li>
−
<li>Spent remainder of the day testing the following tools:</li>
+
        <li class="c0"><span class="c1">Decided lab schedules</span></li>
−
<li>We used to addgene.com to find plasmids that had less than 26Q repeats.</li>
+
      </ul>
−
<li>We used a genome to protein sequence converter called [] in order to compare the wild type plasmid and the infected.</li>
+
      <p class="c2"><span class="c1">Week 27 (Sep 17-23)</span></p>
−
<li>At first we thought we had a good candidate with 20Q that was very similar, however, didn’t work because the sequence didn’t completely match up with the wild type.</li>
+
      <ul class="c3 lst-kix_j7kige8g12kr-0 start">
−
<li>Therefore kept looking and decided to use pEGFP-Q23 <a href="https://www.addgene.org/40261/">https://www.addgene.org/40261/</a></li>
+
        <li class="c0"><span class="c1">Reached out to respective high schools for presentation</span></li>
−
</ul>
+
        <li class="c0"><span class="c1">Tested competency of cells</span></li>
−
<p><strong>Week 22 (Aug 13-19)</strong></p>
+
        <li class="c0"><span class="c1">Did miniprep to isolate the Addgene HTT plasmids from E. coli</span></li>
−
<ul>
+
        <li class="c0"><span class="c1">Created reagents to use for separating plasmid DNA</span></li>
−
<li>Created Instagram account</li>
+
      </ul>
−
<li>Grew cell subcultures of E. Coli for future testing</li>
+
      <p class="c2"><span class="c1">Week 28 (Sep 24-30)</span></p>
−
<li>Familiarized ourselves with autoclaving and basic sterilizing processes</li>
+
      <ul class="c3 lst-kix_zag1de21fqov-0 start">
−
<li>Learned how to store E. coli for future experiments</li>
+
        <li class="c0"><span class="c1">Organized project design tab of website</span></li>
−
<li>HDSA agreed to be sponsor</li>
+
        <li class="c0"><span class="c1">Completed Safety Lab forms</span></li>
−
</ul>
+
        <li class="c0"><span class="c1">Ran gel electrophoresis of Addgene Plasmid Digest</span></li>
−
<p><strong>Week 23 (Aug 20-26)</strong></p>
+
      </ul>
−
<ul>
+
      <p class="c2"><span class="c1">Week 29 (Oct 1-Oct 7)</span></p>
−
<li>Learned about transfection and tested cells for competency</li>
+
      <ul class="c3 lst-kix_qxqjqq83enmc-0 start">
−
<li>Updated team Wiki’s page on the iGem website</li>
+
        <li class="c0"><span class="c1">Scheduled collaboration and presentation at Columbia University and Stuyvesant High School, respectively</span></li>
−
<li>Reached out to our respective high schools for sponsorship</li>
+
        <li class="c0"><span class="c1">Cut out Exon1 from Addgene and verified cuts</span></li>
−
<li>Hereditary Disease Foundation/Huntington’s Disease Foundation agreed to be sponsor</li>
+
        <li class="c0"><span class="c5">Did restriction digest on isolated DNA plasmid</span></li>
−
</ul>
+
      </ul>
−
<p><strong>Week 24 (Aug 27-Sept 2)</strong></p>
+
      <p class="c2"><span class="c1">Week 30 (Oct 8-14)</span></p>
−
<ul>
+
      <ul class="c3 lst-kix_iygxz2lsd1yg-0 start">
−
<li>Jessica, Erin, Catherine, and Yunsu helped organize a presentation table at Dominico-American Society of Queens Benefit Concert at National Opera America Center in New York NY on August 30 2017</li>
+
        <li class="c0"><span class="c1">Created banner for Jamboree</span></li>
−
<li>Reached out to Cerebral Palsy Alliance Research Foundation about partnering and sponsorship and discussed creating a joint campaign CPARF’s Steptember campaign to promote 10,000 steps a day goal</li>
+
        <li class="c0"><span class="c1">Started draft for poster</span></li>
−
<li>Registered team members and created a team roster</li>
+
        <li class="c0"><span class="c1">Did gel extraction and PCR purification</span></li>
−
</ul>
+
        <li class="c0"><span class="c1">Purified gel bands from STUL-XBAL digest and amplified isolated DNA fragments</span></li>
 +
      </ul>
 +
      <p class="c2"><span class="c1">Week 31 (Oct 15-21)</span></p>
 +
      <ul class="c3 lst-kix_njukr3be202s-0 start">
 +
        <li class="c0"><span class="c1">Jessica, Sohee, Rachel and Eric presented at Huntington&rsquo;s Disease Society of America headquarters</span></li>
 +
      </ul>
 +
      <ul class="c3 lst-kix_lk0ravwrdi3c-0 start">
 +
        <li class="c0"><span class="c1">Esther, Rachel, Yeji and Eric met with Columbia University Synthetic Biology Initiative and the Columbia iGEM team and presented</span></li>
 +
        <li class="c0"><span class="c1">Verified the PCR amplification from October 14th</span></li>
 +
        <li class="c0"><span class="c1">Digested samples from October 14th with CAC8I</span></li>
 +
        <li class="c0"><span class="c1">Restricted plasmid fragments with CAC8I and ran gel electrophoresis for confirmation</span></li>
 +
        <li class="c0"><span class="c1">Invited Kingsborough Community College iGEM team for collaboration</span></li>
 +
      </ul>
 +
      <p class="c2"><span class="c1">Week 32 (Oct 22-28)</span></p>
 +
      <ul class="c3 lst-kix_jfl286fxvc1w-0 start">
 +
        <li class="c0"><span class="c1">Rachel and Catherine presented the project at Stuyvesant High School</span></li>
 +
      </ul>
 +
      <ul class="c3 lst-kix_hhhwcrx9nwhb-0 start">
 +
        <li class="c0"><span class="c1">Julia, Stacy and Catherine attended the NYC HDSA Family Education Day at Columbia University Medical Center</span></li>
 +
        <li class="c0"><span class="c1">Completed judging forms</span></li>
 +
        <li class="c0"><span class="c1">Worked on transferring from Wordpress website to wiki</span></li>
 +
      </ul>
 +
      <p class="c2"><span class="c1">Week 33 (Oct 29-Nov 1)</span></p>
 +
      <ul class="c3 lst-kix_hhhwcrx9nwhb-0">
 +
        <li class="c0"><span class="c1">Finished transferring Wordpress website content to wiki</span></li>
 +
        <li class="c0"><span class="c1">Organized lab notebook and finalized results</span></li>
 +
        <li class="c0 c6"><span class="c1"></span></li>
 +
      </ul>
 
</div>
 
</div>
 
</section>  
 
</section>  

Revision as of 01:28, 2 November 2017

Notebook

 

Week 1 (March 19-25)

  • Consulted with our advisor, Frank, to advise researchers and scientists
  • Reached out to scientists at Mt Sinai Hospital, Jason Fuller and Russell Hanson
  • Met with Jason Fuller and Russell Hanson, scientists at Genetics Department at Mt Sinai Hospital
  • Scheduled weekly meetings for the rest of the year

Week 2 (March 26-Apr 1)

  • Brainstormed for a potential topic (Sickle Cell Anemia, Cystic Fibrosis, Huntington’s Disease); decided on HD, proposed by Yeji Cho
  • Determined project leaders of the project
  • Divided into subsections (website, design, fundraising, social media, computational) and assigned roles, such as other team leads

Week 3 (Apr 2-8)

  • Determined possible method to target the disease
  • Outlined the abstract for the project

Week 4 (Apr 9-15)

  • Took headshots of members and team pictures to put on website
  • Administered Drug Tests for medical clearance
  • HIPAA (Health Insurance Portability and Accountability Act) Training
  • Fire Drill Practice

Week 5 (Apr 16-22)

  • Decided to use Wordpress as website template
  • Happily received our lab coats and Mount Sinai IDs

Week 6 (Apr 23-29)

  • Submitted payment for iGEM group registration
  • Signed consent forms

Week 7 (Apr 30-May 6)

  • Designed graphic of brain and neuron for website

Week 8 (May 7-13)

  • Brainstormed for a team name and designed logo; decided on HD Resolution

Week 9 (May 14-20)

  • Designed T-shirts, hats, and sweaters
  • Brainstormed for companies/sponsors to partner with us
  • Received iGEM parts kit

Week 10 (May 21-27)

  • Organized home page of website
  • Created sponsorship/partnership letters for possible companies

Week 11 (May 28-Jun 3)

  • Julia, Erik and Stacy organized a table at Peace Angels Benefit Concert at Steinway and Sons Concert Hall in May 28, 2017
  • Mailed out consent forms
  • Scheduled wetlab training dates for the summer

Week 12 (Jun 4-10)

  • Early Training: lab safety, project overview, practiced lab techniques such as pipetting, centrifugation, PCR, etc.

Week 13 (Jun 11-17)

  • Organized team page of website
  • Contacted over 50 private and public companies in pharmaceutical and biotech industries, non-profit organizations for sponsorship and support.

Week 14 (Jun 18-24)

  • Created Twitter account
  • Christi and Marianne completed safety form 1, 2, 3

Week 15 (Jun 25-Jul 1)

  • Jason, Justin, Jeewhan, Marianne met up to analyze the data collection

Week 18 (Jul 16-22)

  • Christi, Rachel, Esther, and Marianne organized a fundraising and information table at Dominico-American Society of Queens in Flushing Town Hall in Flushing, Queens NY in July 22 2017
  • Created Facebook account
  • Created GoFundMe page

Week 19 (Jul 23-29)

  • Received over 100 likes on Facebook page thanks to our Social Media and Outreach team.

Week 20 (Jul 30-Aug 5)

  • Ordered materials (E. coli, etc)

Week 21 (Aug 6-12)

  • Wrote letters to respective schools to ask for partnerships

Week 22 (Aug 13-19)

  • Created Instagram account
  • Grew cell subcultures of E. Coli for future testing
  • Familiarized ourselves with autoclaving and basic sterilizing processes
  • Learned how to store E. coli for future experiments
  • HDSA agreed to be sponsor

Week 23 (Aug 20-26)

  • Learned about transfection and tested cells for competency
  • Updated team Wiki’s page on the iGem website
  • Reached out to our respective high schools for sponsorship
  • Hereditary Disease Foundation/Huntington’s Disease Foundation agreed to be sponsor
  • Spread awareness of our project to Alzheimer’s Foundation of America

Week 24 (Aug 27-Sept 2)

  • Jessica, Erin, Catherine, and Yunsu helped organize a presentation table at Dominico-American Society of Queens Benefit Concert at National Opera America Center in New York NY on August 30 2017
  • Reached out to Cerebral Palsy Alliance Research Foundation about partnering and sponsorship and discussed creating a joint campaign CPARF’s Steptember campaign to promote 10,000 steps a day goal
  • Registered team members and created a team roster

Week 25 (Sep 3-9)

  • Figured out wet lab schedules and iGEM meetings
  • Started introducing each member on Instagram
  • Uploaded sponsors and neuron design on website

Week 26 (Sep 10-16)

  • Arranged tabs on website
  • Sent out emails to organizations for outreach
  • Reached out to organizations on Facebook and iGEM teams for collaboration
  • Decided lab schedules

Week 27 (Sep 17-23)

  • Reached out to respective high schools for presentation
  • Tested competency of cells
  • Did miniprep to isolate the Addgene HTT plasmids from E. coli
  • Created reagents to use for separating plasmid DNA

Week 28 (Sep 24-30)

  • Organized project design tab of website
  • Completed Safety Lab forms
  • Ran gel electrophoresis of Addgene Plasmid Digest

Week 29 (Oct 1-Oct 7)

  • Scheduled collaboration and presentation at Columbia University and Stuyvesant High School, respectively
  • Cut out Exon1 from Addgene and verified cuts
  • Did restriction digest on isolated DNA plasmid

Week 30 (Oct 8-14)

  • Created banner for Jamboree
  • Started draft for poster
  • Did gel extraction and PCR purification
  • Purified gel bands from STUL-XBAL digest and amplified isolated DNA fragments

Week 31 (Oct 15-21)

  • Jessica, Sohee, Rachel and Eric presented at Huntington’s Disease Society of America headquarters
  • Esther, Rachel, Yeji and Eric met with Columbia University Synthetic Biology Initiative and the Columbia iGEM team and presented
  • Verified the PCR amplification from October 14th
  • Digested samples from October 14th with CAC8I
  • Restricted plasmid fragments with CAC8I and ran gel electrophoresis for confirmation
  • Invited Kingsborough Community College iGEM team for collaboration

Week 32 (Oct 22-28)

  • Rachel and Catherine presented the project at Stuyvesant High School
  • Julia, Stacy and Catherine attended the NYC HDSA Family Education Day at Columbia University Medical Center
  • Completed judging forms
  • Worked on transferring from Wordpress website to wiki

Week 33 (Oct 29-Nov 1)

  • Finished transferring Wordpress website content to wiki
  • Organized lab notebook and finalized results
protocol Created with Sketch.

Testing Competency of Cells

Project: HD Resolution iGEM Notebook
Authors: HD Resolution iGEM
Date: 2017-09-19
Tuesday, 9/19/17
1.
Clean working area with 10% ethanol
2.
Throw competent cells on ice
3.
Label one 1.5 mL microcentrifuge tubes for each transformation and pre-chill on ice.
a.
Orange = 50 pg/μl
b.
Yellow = 10 pg/μl
c.
Red = 100 pg/μl
4.
Spin down DNA tubes from competent cell test kit. Quick spin of 20-30 seconds at 8000-10000rpm
5.
Pipet 10μl of DNA into centrifuge tubes. 50μl of competent cells already aliquoted. Flick gentlly
6.
Incubate on ice for 30 min.
7.
Heatshock cells into water bath of 42 degrees Celsius for 45 sec.
8.
Immediately transfer tubes back into ice and incubate on ice for 5 min
9.
Add 950 μl of SOC Media per tube and incubate at 37 degrees Celsius for 1 hour, shaking at 200-300rpm.
10.
Pipet 100μl from each tube onto appropriate plate, spread mixture evenly.
11.
Incubate at 37 degrees Celsius overnight/16 hrs. Place plates with agar side on top and lid on bottom.
protocol Created with Sketch.

Untitled

Project: HD Resolution iGEM Notebook
Authors: HD Resolution iGEM
Date: 2017-09-20
Wednesday, 9/20/17
1.
Innoculated 600 mL LB w/ 1 mL O/N culture
2.
Shake at 37 degrees Celsius, DDC 600 = 0.4, 3 hr
3.
Cooled all materials in ice, including solutions (CaCl3), cells, tubes, & tips
4.
Aliquoted 600 mL of cells into 12 50 mL tubes, 30 mL each
5.
Spin 1.5 k rcf, 5 min
6.
Decant sup, resus, pellets in 1 mL CaCl2 (cold) by gentle rocking, left on ice for 30 min
7.
Spin as above
8.
Discard supernatent
9.
Added fresh cold 100 mM CaCl3
10.
Resuspend as above
11.
Added 10.2 mL 50% glycerol to 50 mL falcon tube (chilled)
12.
Consolidated all resuspended cells into the tube containing 50% glycerol
13.
Did not fully resuspend cells, still looked chunky
14.
Froze in 1 mL aliquots of liiquid nitrogen, left in unmarked box below iGEM box in -80 degrees Celsius freezer
protocol Created with Sketch.

Continuation

Project: HD Resolution iGEM Notebook
Authors: HD Resolution iGEM
Date: 2017-09-22
Friday, 9/22/17
1.
Added 175μl of ice cold Alkaline Lysis Solution III through the viscous bacterial lysate. Inverted tube several times and stored tube on ice for 5 min.
2.
Centrifuged lysate at maximum speed for 5 min at 4 degrees Celsius, and transferrred the supernatant to a new tube
3.
Precipitated the nucleic acids from the supernatant. Added 1 mL of ethanol at room temperature. Mixedsolution by vortexing and allowed mixture to stand for two minutes at room temp.
4.
Collected precipitated nucleic acds by centrifugation at max speed for 5 min at 4 degrees Celsius. Dropped precipitated nucleic aicds. Redid centrifugation at max spped for 5 minutes at 4 degrees Celsius.
5.
Removed supernatant by gentle aspiration, as shown in step 4. Stood tube in an inverted position on a paper towel to allow all the fluid to drain away. Used a kimwipe to remove the drops of fluid adhering to the walls of the tube.
6.
Added 1 mL of 70% ethanol to the pellet and inverted the closed tube several times.
7.
Recovered DNA by centrifugation at max speed for 2 min at 4 degrees Celsius in a microcentrifuge.
protocol Created with Sketch.

Transformation

Project: HD Resolution iGEM Notebook
Authors: HD Resolution iGEM
Date: 2017-09-25
Monday, 9/25/17
1.
Thaw DHS-Alpa Cells on ice
2.
Add 2 mL of DNA to cells (from previously isolated adgene plasmids)
3.
Stand for 34 min on ice
4.
Cells are incubated for 60 sec at 42 degrees Celsius
5.
Put cells on ice for 5 minut s
6.
Add 900μl SOC media
protocol Created with Sketch.

Preparation of LB-Agar Plates

Project: HD Resolution iGEM Notebook
Authors: HD Resolution iGEM
Date: 2017-09-26
Tuesday, 9/26/17
Preparation of LB-Agar Plates
- Adapted from CSH Protocols
LB Agar
- 3.75 gramf of Agar
- 250 mL of 1x LB Medium
1.
Mix
2.
Autoclave on Liquid Cycle for 30 minutes
a.
Do not overfill bottles
I.
250 mL of LB was autoclaved in a 500 mL bottle to prevent it from boiling over.
3.
Add 2.5 mL of Kanamycin Stock (25 mg/mL) to a final concentration of 25ug/mL, once LB-Agar has coded to about 50 degrees Celsius.
4.
Heat on a hot plate to re-melt sugar
protocol Created with Sketch.

Running Electrophoresis Gel of ADDGENE Plasmid Digests

Project: HD Resolution iGEM Notebook
Authors: HD Resolution iGEM
Date: 2017-09-26
Tuesday, 9/26/17
1.
Cut up unused gel and add to remaining unused 2% agarose state.
2.
Warm until gel molds / 2.5 for 5 min
3.
Pour into casting pan

Interlab & Second Competent Cell Test

Project: HD Resolution iGEM Notebook
Authors: HD Resolution iGEM
Date: 2017-09-26
Tuesday, 9/26/17
1.
Clean working area
2.
Thaw competent cells on ice
3.
Label 8 microcentrifuge tubes : 10ug, 50ug, 100ug, 1, 4, 5, 6, Neg.
4.
Spin down DNA from competent cell test kit and interlab DNA
5.
Pipet 1μl of DN from competent cell test kit into tubes labeled 1, 4, 5, 6, neg
6.
Pipet 85μl of competent cells into all tubes, flick gently to mix
7.
Incubate on ice for 30 minutes
8.
Heat shock cells by placing tubes into a hot water bath at 42 degrees Celsius for 45 seconds
9.
Transfer back and incubate on ice for 5 min
10.
Add 45μl of SOC Media into eah cand incubate at 37 degrees for 1 hour shaking at 200-300 rpm
11.
Warm up agar plates, label
12.
Pipet 100μl from each tube onto appropriate plate, spread mixture evenly across plates via sterile glass beads.
13.
Close with parafilm
14.
Incubate at 37 degrees Celsius overnight w/ plates positioned w/ agar side at top and lid at bottom

Interlab

Project: HD Resolution iGEM Notebook
Authors: HD Resolution iGEM
Date: 2017-09-27
Wednesday, 9/27/17
1.
Clean working area
2.
Pipette 1μl of 1, 4, 5, 6, neg - and control each with their respective cell tube
3.
INcubate for 30 min (until 7:02)
4.
Pipette 100μl of LUDOX into wells A1, B1, C1. D1 each
5.
Pipette 100μl of H2O each into wells A2, B2, C2, D2
6.
Spin down fluorescein tube
7.
Resuspend 100μM florescein solution in 1 mL 1xPBS to make 2x solution
8.
Dilute to 1x.
9.
Perform serial dilution with 10 columns of 1x PBS (leave an additional column as regular PBS)
protocol Created with Sketch.

Restriction Digest on Previously Isolated DNA Plasmid

Project: HD Resolution iGEM Notebook
Authors: HD Resolution iGEM
Date: 2017-10-06
Friday, 10/6/17
1.
Placed previously isolated DNA plasmid on ie (61 and 62)
2.
Measured the concentration of the DNA Plasmids with the nanodrop (2μl)
a.
DNA plasmid 40261 : 356.2 ng/μl
b.
DNA plasmid 40262 : 300.5 ng/μl
3.
Diluted DNA plasmid 1 : 3 to approximate 100 ng/μl and measured with nanodrop
a.
DNA plasmid 40261 : 117.9 ng/μl
b.
DNA plasmid 40262 : 106.1 ng/μl
4.
Labeled fresh 0.5 mL tubes 61 and 62
5.
For each tube, transferred 5 ul of 10x NEBuffer, 1 ul of Stul, 1 mL of cac 81, and 33 ul of dH2O
6.
Transferred 10μl DNA Plasmid 40261 to 0.5 mL tube 61 and 10μl of DNA plasmid 40262 to 0.5 mL tube 62
7.
Incubated 0.5 mL tubes at 37 degrees Celsius for fifteen minutes
8.
Added 10μl purple 6x loading dye to each of the 0.5 mL tube and mixed
9.
Gel was loaded as follows:
A
B
C
D
E
F
G
1
 Cane 1 2 3 4 5 6
2
 10μl of 2 log ladder 10μl of digested ladder 10μl of digested 40262 15μl of digested 40262 15μl of digested 40261 15μl of digested 40261 10μl of 2 log ladder
Table1
protocol Created with Sketch.

Re-Running Gel Electrophoresis

Project: HD Resolution iGEM Notebook
Authors: HD Resolution iGEM
Date: 2017-10-10
Tuesday, 10/10/17
1.
Repeat of 10/6/17 procedure from Step 5
2.
Placed unused gel in gel box for 30 min
3.
Pipetted 10μl in 1st and last lane (purple ladder)
4.
Pipetted 15μl in 2nd and 2nd to last lane (3x concentration ol-digest)
5.
Pipetted 30μl (double bond) in outer lanes
6.
Make ETBR solution and place gel in there and on SHAKER (5 min)
7.
Image gel and results: Showed that plasmids were restricted at least once, but 100 bp fragmentts were not seen
8.
Conclusion: Restriction enzymes are to be checked for cut/not.
protocol Created with Sketch.

Re-Run Restrictions on 3x oncnerated samples for 1 hour

Project: HD Resolution iGEM Notebook
Authors: HD Resolution iGEM
Date: 2017-10-12
Thursday, 10/12/17
1.
Labeled 0.5 mL tubes "61" and "62"
2.
Transferred 5μl of 10x NEBuffer, 1μl of StuI, 1μl of Cac 81, and 33μl H2O
3.
Transferred 10μl of DNA plasmid 40261 to 0.5 mL tube "62"
4.
Incubated tubes at 37 degrees Celsius for 1 hour
5.
Added 10μl 6x purple loading dye to each tube and mixed
6.
Gel was labeled as follows
A
B
C
D
E
F
G
H
1
 Lane 1 2 3 4 5 6 7
2
 empty 10μl 2 log buffer 15μl digested 40261 26μl digested 40261 26μl digested 40262 15μl digested 40262 10μl 2 log buffer
3
Table1
protocol Created with Sketch.

Test of DH5Alfa Souls

Project: HD Resolution iGEM Notebook
Authors: HD Resolution iGEM
Date: 2017-10-12
Thursday, 10/12/17
protocol Created with Sketch.

Gel Extraction and PCR Purification

Project: HD Resolution iGEM Notebook
Authors: HD Resolution iGEM
Date: 2017-10-13
Friday, 10/13/17
1.
Weighed gel slices containing DNA fragment
a.
61a)0.3620g b)0.3895g
b.
62a) 0.2865g b)0.3877g
2.
Added gel solubilization buffer L3 in 3:1 ratio to gel in a 1.7 mL polypropylene tube
3.
Incubated tubes at 50 degrees Celsius for 15 min and inverted tubes every 3 min
4.
Incubated the tubes for an additional 5 min when gel slice appeared dissolved
5.
Pipetted and dissolved gel piece onto a column at 12000 x g for 1 minute. Discarded flow-through and placed column into wash tube
6.
Added 500μl wash buffer containing ethanol to the column. Centrifuged column at >12000 x g for one minute. DIscarded flow thorugh and placed column into wash tube. Centrifuged column at max speed for 2-3 min. Discarded flow-through.
7.
Placed column into recovery tube. Added 50 mL elution buffer (E1) to the column. INcubated tube for a minute at room temperature. Centrifuged tube > 12000 x g/minute.
8.
Stored, purified DNA at -20 degrees Celsius for immediate use
protocol Created with Sketch.

Purifying Gel Bonds from STUL-XBAL Digest

Project: HD Resolution iGEM Notebook
Authors: HD Resolution iGEM
Date: 2017-10-14
Saturday, 10/14/17
1.
weigh gel slices containing dna fragments
a.
61 > 0.2100 g
b.
62 > 0.2230 g
2.
Add Gel solubilization buffer L3 to excised gels in the tubes in a 3:1 ratop
a.
61 > 0. 63 g = 630μl
b.
62 > 0.669 g = 669μl
3.
Incubate at 50 degrees Celsius for 10 minutes ad invert tube every 3 minutes. After the gel slie appears dissolves, incubate tube for an additional 3 min
4.
Add 1 gel volume of isporopanol to dissolved gel slices. Mix well
a.
61 > 210μl
b.
62 > 223μl
5.
Pipet 850μl of dissolved gel pieces into wash tubes. Centrifuge for
sequence Created with Sketch.

How to Assemble the Parts we currently have

Project: HD Resolution Shared Project
Authors: jason fuller
Date: 2017-10-18
Wednesday, 10/18/17
Assuming Exon 1 Flanks have been properly assembled, the remaing ligations could proceed as follows:
Pair and join every 2 G-Blocks, then PCR amplify (IDT doesn't reccomend PCR amplifying parts greater than 1000 bases)
- Start working backwards (GB15 + GB14, GB13+GB12, etc...)
- Pair the Flanked Exon1 part with GB1
Should work as you are only requring polymerase to amplify 500kb or less
OR
LOW EFFICIENCY - extra work, but probably will work!
Pair 3 sets of 5 G-Blocks using mastermix
Blunt cut a plasmid with a restriction enzyme (peferably one with other restriction sites near by - or where a restriction site will be made where the insert is) CUT in the middle of the GFP!!!!!!!
Blunt assemble
transform
colony pick many colonies (THAT DON'T GLOW)
miniprep separtate colonies
- run gel to look for plasmid of correct size. (on side)
Run Gibson assembly.....
OR
remove phosphates from above plasmid cuts via pcr amplification
add phosphates to gibson assembled g-blocks* (how? / how to separate from other smaller fragments?)
Gibson assemble the 8 parts into 2 separate plasmids (could try 1 but success seesm unlikely):
- prepare a unphosphorlated vector backbone (via reverse amplification PCR is best)
- after mastermix step, phosphoralate the DNA
- Vector insert as shown here: https://youtu.be/-Vh-LbJAJpk?t=19m52s
Need to order?: phosphoralase, dephosphoralase (or primers for the backbone), blunt-end lligase, ligase
Questions to answer ASAP:
Do we have Exon1 properly assmebled?
Do we have the primers needed so we don't have to dephosphoralate?
sequence Created with Sketch.

ToeHold Exchange Questions

Project: HD Resolution Shared Project
Authors: jason fuller
Date: 2017-10-18
Wednesday, 10/18/17
Do we need to assemble better Kozak sequences for our syntheitc mRNA replacement?
WT-Mutated = exact gene
chaperone target footing = fagments of exact gene starting at posion 7 up though position 47
- seems like we shouldn't start later than 30 or sooner than 15
- 22 to
image.png
thumbnail
Kozak Sequences should be evaluated and/or Alternative codon used from positions 7 though 47
Chaperone extension though the start codon could therotically go up though the BbCI cut site at position 209 but is perhaps better to keep short and extend no further than the StuI cut site at positon 11O
image.png
thumbnail
Kozak Sequence is imperfect in WT HTT, syntetic form should be changed to contain the GCCG portion:
image.png
thumbnail
Everything before this sequence doesn't really matter and could use alternative splicing.
Perhaps the sequence can just be trimed before this and the guid strand be the hairpin target overhang?
Else it might be nice for it to be D
Wild Type Huntinton mRNA - GBlock Aligned
sequence Created with Sketch.

What did our PCR actually amplify?

Project: HD Resolution Shared Project
Authors: jason fuller
Date: 2017-10-18
Wednesday, 10/18/17
According to PCR theroy the "short" amplicons swamp out all others during an aplification:
http://www.cybertory.org/exercises/primerDesign/#Background (about 1/2 way down)
Thus, we don't actually need perfect cut sites if we are getting enough sticky ends to start an amplification....
From:
Wild Type Huntinton mRNA - GBlock Aligned
image.png
thumbnail
image.png
thumbnail
sequence Created with Sketch.

NEBuilder HiFi DNA Assembly Reaction Protocol

Project: HD Resolution Shared Project
Authors: Sohee
Date: 2017-10-20
Friday, 10/20/17
Optimal Quantities
NEB recommends a total of 0.03–0.2 pmols of DNA fragments when 1 or 2 fragments are being assembled into a vector, and 0.2–0.5 pmols of DNA fragments when 4–6 fragments are being assembled. Efficiency of assembly decreases as the number or length of fragments increases. To calculate the number of pmols of each fragment for optimal assembly, based on fragment length and weight, we recommend the following formula, or using the tool, NEBiocalculator.
pmols = (weight in ng) x 1,000 / (base pairs x 650 daltons)
50 ng of 5000 bp dsDNA is about 0.015 pmols
50 ng of 500 bp dsDNA is about 0.15 pmols
The mass of each fragment can be measured using the NanoDrop instrument, absorbance at 260 nm or estimated from agarose gel electrophoresis followed by ethidium bromide staining.
HiFi DNA Assembly Protocol
1.
Set up the following reaction on ice:
Table 1: Recommended Amount of Fragments Used for Assembly
A
B
C
D
1
 2–3 Fragment Assembly* 4–6 Fragment Assembly** Positive Control✝
2
 Recommended DNA Molar Ratio vector:insert = 1:2 vector:insert = 1:1
3
 Total Amount of Fragments 0.03–0.2 pmols*X μl 0.2–0.5 pmols**X μl 10 μl
4
 NEBuilderHiFi DNA Assembly Master Mix 10 μl 10 μl 10 μl
5
 Deionized H2O 10-X μl 10-X μl 0
6
 Total Volume 20 μl✝✝ 20 μl✝✝ 20 μl
Table2
* Optimized cloning efficiency is 50–100 ng of vector with 2-fold excess of inserts.
Use 5 times more insert if size is less than 200 bp. Total volume of unpurified PCR fragments in the assembly reaction should not exceed 20%.
** To achieve optimal assembly efficiency, design ≥ 20 bp overlap regions between each fragment with equimolarity (suggested: 0.05 pmol each).
† Control reagents are provided for 5 experiments.
†† If greater numbers of fragments are assembled, increase the volume of the reaction, and use additional NEBuilder HiFi DNA Assembly Master Mix.
2. Incubate samples in a thermocycler at 50°C for 15 minutes (when 2 or 3 fragments are being assembled) or 60 minutes (when 4–6 fragments are being assembled). Following incubation, store samples on ice or at –20°C for subsequent transformation.
Note: Extended incubation up to 60 minutes may help to improve assembly efficiency in some cases (for further details see FAQ section).
3. Transform NEB 5-alpha or 10-beta Competent E. coli cells (provided in the cloning kit, bundle or purchased separately from NEB) with 2 μ l of the assembled product, following the transformation protocol.
sequence Created with Sketch.

iGEM Exon 1 Assembly

Project: HD Resolution Shared Project
Authors: Sohee
Date: 2017-10-20
Friday, 10/20/17
HiFi DNA Assembly Protocol
1.
Set up the following reaction on ice:
Table 1: Recommended Amount of Fragments Used for Assembly
A
B
C
D
1
 2–3 Fragment Assembly* 4–6 Fragment Assembly** Positive Control✝
2
 Recommended DNA Molar Ratio vector:insert = 1:2 vector:insert = 1:1
3
 Total Number of Fragments*** 0.03–0.2 pmols*X μl 0.2–0.5 pmols**X μl 10 μl
4
 NEBuilderHiFi DNA Assembly Master Mix 10 μl 10 μl 10 μl
5
 Deionized H2O 10-X μl 10-X μl 0
6
 Total Volume 20 μl✝✝ 20 μl✝✝ 20 μl
Table2
* Optimized cloning efficiency is 50–100 ng of vector with 2-fold excess of inserts.
Use 5 times more insert if size is less than 200 bp. Total volume of unpurified PCR fragments in the assembly reaction should not exceed 20%.
** To achieve optimal assembly efficiency, design ≥ 20 bp overlap regions between each fragment with equimolarity (suggested: 0.05 pmol each).
*** When attempting to add flanking reagents, do not add flanking DNA until the reaction mixture has been heated until 50˚C has been reached.
† Control reagents are provided for 5 experiments.
†† If greater numbers of fragments are assembled, increase the volume of the reaction, and use additional NEBuilder HiFi DNA Assembly Master Mix.
2. Incubate samples in a thermocycler at 50°C for 15 minutes (when 2 or 3 fragments are being assembled) or 60 minutes (when 4–6 fragments are being assembled). Following incubation, store samples on ice or at –20°C for subsequent transformation.
Note: Extended incubation up to 60 minutes may help to improve assembly efficiency in some cases (for further details see FAQ section).
3. Transform NEB 5-alpha or 10-beta Competent E. coli cells (provided in the cloning kit, bundle or purchased separately from NEB) with 2 μ l of the assembled product, following the transformation protocol.
sequence Created with Sketch.

G-block Assembly

Project: HD Resolution Shared Project
Authors: HD Resolution iGEM
Date: 2017-10-20
Friday, 10/20/17
Purpose: Begin gibson assembly so that we can PCR once primers are in.
Materials:
IDT G-blocks (15)
NEB Hifi Builder Kit
Nuclease free water
A
B
C
D
1
 Concentration Range of DNA fragments 02 - .5 pmols* .2 - 1.0 pmols* 0 pmols
2
 Total Volume of Fragments (μl) 10
3
 Gibson Assembly Master Mix (2x) (μl) 10 10 10
4
 Deionized Water (μl) 10 10 0
5
 Total Volume (μl) *** 20 20 20
Table1
1.
Normalized concentrations of all G-blocks to 10 fmol per microliter.
2.
Take 5 microliters of each G-block sample and put into a new tube of each paired G-block sample.
3.
Then add 10 microliters of Gibson assembly master mix to each tube.
4.
Incubate for 15 minutes at 50 C in the thermocycler.

Gibson Assembly® Protocol (E5510)

Introduction

This is the protocol for Gibson Assembly using the Gibson Assembly® Cloning Kit (E5510). More information from NEB can be found here.

Materials

Procedure

  • Set up the following reaction on ice:
  1. Reaction volumes: Use this table to calculate reaction volumes and set up the reaction. Remember to input your total DNA fragment volume in cells B3 and C3 for assemblies with 2-3 fragments and 4-6 fragments, respectively.
  • - NEB recommends a total of 0.02–0.5 pmols of DNA fragments when 1 or 2 fragments are being assembled into a vector and 0.2–1.0 pmoles of DNA fragments when 4–6 fragments are being assembled. Efficiency of assembly decreases as the number or length of fragments increases. To calculate the number of pmols of each fragment for optimal assembly, based on fragment length and weight, we recommend using NEB's online tool, NEBioCalculator.
  • - The mass of each fragment can be measured using the NanoDrop instrument, absorbance at 260 nm, or estimated from agarose gel electrophoresis followed by ethidium bromide staining.
A
B
C
D
1
 2-3 Fragments Assembly 4-6 Fragments Assembly Positive Control **
2
 Concentration Range of DNA fragments 02 - .5 pmols* .2 - 1.0 pmols* 0 pmols
3
 Total Volume of Fragments (μl) 10
4
 Gibson Assembly Master Mix (2x) (μl) 10 10 10
5
 Deionized Water (μl) 10 10 0
6
 Total Volume (μl) *** 20 20 20
Table1
  • *Optimized cloning efficiency is 50–100 ng of vectors with 2–3 fold of excess inserts. Use 5 times more of inserts if size is less than 200 bps. Total volume of unpurified PCR fragments in Gibson Assembly reaction should not exceed 20%.
  • ** Control reagants are provided for 5 experiments with the Gibson Asembly Kit.
  • *** If greater numbers of fragments are assembled, additional Gibson Assembly Master Mix may be required.
  1. Incubate samples in a thermocycler at 50°C for 15 minutes when 2 or 3 fragments are being assembled or 60 minutes when 4-6 fragments are being assembled.
  • Note: Extended incubation up to 60 minutes may help to improve assembly efficiency in some cases (for further details see FAQ section).
  • *Select the pencil icon and change the timer to 60 minutes if working with 4-6 fragments
00:15:00
  1. Store samples on ice or at –20°C for subsequent transformation.
  1. Transform NEB 5-alpha Competent E. coli cells (provided with the kit) with 2 μl of the assembly reaction, following the chemcial transformation protocol or electro competent cells transformation protocol
protocol Created with Sketch.

Finalize Exon 1 Assembly

Project: HD Resolution iGEM Notebook
Authors: HD Resolution iGEM
Date: 2017-10-20
Friday, 10/20/17
Purpose: To add the prefix and suffix fragments to the addgene insert to complete exon 1 assembly.
Materials:
NEB Hifi Assembly Kit
Addgene Insert with adapter overhang (previously PCRed)
IDT exon 1 flanks (previously restricted)
Water
A
B
C
1
 Concentration Volume
2
 Prefix Fragment
3
 Suffix Fragment
4
 Addgene Insert
5
 Total Volume of Fragments
6
 Gibson Assembly Master Mix (2x) (μl)
7
 Deionized Water (μl)
8
 Total Volume (μl) ***
Table1
*Note: Experiment postponed bc previous digested exon flank was missing upon review of the digestion protocol it seems it was the restriction enzymes chosen were wrong. As StuI cuts exon flank in two places.

Gibson Assembly® Protocol (E5510)

Introduction

This is the protocol for Gibson Assembly using the Gibson Assembly® Cloning Kit (E5510). More information from NEB can be found here.

Materials

Procedure

  • Set up the following reaction on ice:
  1. Reaction volumes: Use this table to calculate reaction volumes and set up the reaction. Remember to input your total DNA fragment volume in cells B3 and C3 for assemblies with 2-3 fragments and 4-6 fragments, respectively.
  • - NEB recommends a total of 0.02–0.5 pmols of DNA fragments when 1 or 2 fragments are being assembled into a vector and 0.2–1.0 pmoles of DNA fragments when 4–6 fragments are being assembled. Efficiency of assembly decreases as the number or length of fragments increases. To calculate the number of pmols of each fragment for optimal assembly, based on fragment length and weight, we recommend using NEB's online tool, NEBioCalculator.
  • - The mass of each fragment can be measured using the NanoDrop instrument, absorbance at 260 nm, or estimated from agarose gel electrophoresis followed by ethidium bromide staining.
A
B
C
D
1
 2-3 Fragments Assembly 4-6 Fragments Assembly Positive Control **
2
 Concentration Range of DNA fragments 02 - .5 pmols* .2 - 1.0 pmols* 0 pmols
3
 Total Volume of Fragments (μl) 10
4
 Gibson Assembly Master Mix (2x) (μl) 10 10 10
5
 Deionized Water (μl) 10 10 0
6
 Total Volume (μl) *** 20 20 20
Table1
  • *Optimized cloning efficiency is 50–100 ng of vectors with 2–3 fold of excess inserts. Use 5 times more of inserts if size is less than 200 bps. Total volume of unpurified PCR fragments in Gibson Assembly reaction should not exceed 20%.
  • ** Control reagants are provided for 5 experiments with the Gibson Asembly Kit.
  • *** If greater numbers of fragments are assembled, additional Gibson Assembly Master Mix may be required.
  1. Incubate samples in a thermocycler at 50°C for 15 minutes when 2 or 3 fragments are being assembled or 60 minutes when 4-6 fragments are being assembled.
  • Note: Extended incubation up to 60 minutes may help to improve assembly efficiency in some cases (for further details see FAQ section).
  • *Select the pencil icon and change the timer to 60 minutes if working with 4-6 fragments
00:15:00
  1. Store samples on ice or at –20°C for subsequent transformation.
  1. Transform NEB 5-alpha Competent E. coli cells (provided with the kit) with 2 μl of the assembly reaction, following the chemcial transformation protocol or electro competent cells transformation protocol
protocol Created with Sketch.

Mini Prep Add gene HTT plasmids from E Coli

Project: HD Resolution iGEM Notebook
Authors: HD Resolution iGEM
Date: 2017-10-20
Friday, 10/20/17
1.
Prepared lysis buffer (calculated amount needed)
2.
Poured 1.5 mL of the culture into a microcentrifuge tube.
3.
Spun in microcentrifuge tube for 3 min at room temp. Placed in 4 degrees Celsius - store unused portion of the original culture at 4 degreees Celsuis
4.
Removed media w/ aspiration, leaving bacterial pellets as dry as possible
5.
Resuspended bacterial pellet in 200 mL of ice cole alkaline lysis solution 1 with vigorous vortexing (by hand)
6.
Added 250μl of freshly prepared alkaline lysis solution II to each bacterial suspension. Closed the tube tightly and mixed the contents by inverting the tube rapidly five times. Did not vortex - place the tube on ice.
sequence Created with Sketch. protocol Created with Sketch.

Creating Exon 1 Fragment via PCR

Project: Lab Notebook
Authors: jason fuller
Date: 2017-10-23
Monday, 10/23/17
Purpose:
To couple IDT flanking sequences to AddGene HTT sequences.
Materials
See proceedure for PCR specific reagents
Template DNA:
PCRed out and Adapted AddGene Sequence
Exon1_flank_XmnI-Prefix
Exon1_flank-Cac8I-Adapter
Primers:
HTT-Assembly-Fwd
HTT-Assembly-REV
Procedure:
Follow protocol below
Flanking Region PCR Reaction
Calculations:
Previous gel of AddGene HTT sequences were compared to the NEB 2 log ladder.
●
All amplified parts appeared to coorespond to approximately 100 ng on gel. Gel lanes were loaded with 10ul of sample, thus 10 ul of each sample = ~100ng.
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Flanking region DNA total = 1 pg in 50 ul
●
Therefore, 5 ul = 100 ng
Primer Dilution:
●
Starting quantity = 35 nM diluted into 100 ul
○
350 uM/L = stock primer concentration
●
1 ul of stock primer into 34 ul of H2O = 10 uM
○
1.25 ul of diluted stock = 0.5uM when diluted into a 25ul total reaction volume.
dNTP calculations
●
10 mM starting Conc.
○
dilute 1:2 to get 5mM
Taq Pol Calculations
●
5 U/uL starting Conc.
○
dilute 1:10 to get 0.5 U/uL
Anneling tempature calculations:
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Elongation Calculations (1min / 1Kb)
●
Size of AddGene insert + AddGene Primer overlap =
Records of Quantities Used:
A
B
C
1
 Componet Volume (uL) Final Conc
2
 AddGene HTT DNA 9 ~ 90 ng
3
 cut IDT Flaking DNA 9 90 ng
4
 10x PCR buffer 2.5 1 X
5
 5mM dNTP Mix 1 0.2 mM (each)
6
 10 uM forward primer 1.25 0.5 uM
7
 10 uM reverse primer 1.25 0.5 uM
8
 0.5 U/uL Taq Polymerase * 1 2.5 U / rxn
9
 Water 0 n/a
Table1
*Add TaqPol after intial denatuartion at 95˚C for 10 min
PCR Protocol As Implemented:
1.
Intially denature dna at 95˚C for 10 min (with no polymerase)
2.
Add Polymerase
3.
Cycle 20x as follows
A
B
C
1
 Tempature Time Cycle name
2
 95˚C 45 s Melt
3
 65˚C 45 s Anneal
4
 72˚C 1 min / kb Elongate
Table2
●
Cycle Name: JHF
3. End cycle as follows
A
B
C
1
 72˚C 10 min Final Elongation
2
 4˚C Indefinitly Hold Chilled
PCR Finalization Parameters
●
Cycle Name: 95C-10M
4. Store DNA at -20˚C
A
B
C
1
 Tempature Time Cycle name
2
 95˚C 45 s Melt
3
 Tm - 5˚C (https://tmcalculator.neb.com/) 45 s Anneal
4
 72˚C 1 min / kb Elongate
Post Hot start cycle values
●
Cycle Name: JHF
3. End cycle as follows
A
B
C
1
 72˚C 10 min Final Elongation
2
 4˚C Indefinitly Hold Chilled
Final PCR steps
●
Cycle Name: 95C-10M
4. Store DNA at -20˚C
Expected Product (with varied CAG repeats):
HTT WildType Exon1 - Full

DNA Ladders

Project: HD Resolution iGEM Notebook
Authors: HD Resolution iGEM
Date: 2017-10-24
Tuesday, 10/24/17
NEB Quick Load Purple 2-Log Ladder
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Thermoscientific Gene Ruler 1kb DNA Ladder
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Invitrogen 10bp DNA Ladder
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protocol Created with Sketch.

Amplification of Gibson Assembly Fragments

Project: HD Resolution iGEM Notebook
Authors: jason fuller
Date: 2017-10-24
Tuesday, 10/24/17
Purpose:
To amplify paired G-blocks (Blocks 1-3, 4-5, 6-7, 8-9, 10-11, 12-13, 14-15) from the Friday's lab.
Protocol:
The following protocol was followed:
Q5 Hi Fi 2X Master Mix Protocol
Additionally:
1.
All dry primer stocks were diluted to 500uM (0.5 nMoles/ µL).
2.
Diluted primer stocks to a 'working primer solution' with conenctration of 12.5 uM (1ul stock + 39 water)
3.
New PCR tubes were labled for each previous assembly.
4.
A 25 ul Reaction volume was chosen:
5.
Reagents were added as follows
A
B
1
 Component 25 μL Reaction
2
 Q5 High-Fidelity 2X Master Mix 12.5 μL
3
 10 μM Forward Primer 1 μL
4
 10 μM Reverse Primer 1 μL
5
 Template DNA 5 ul + 5 ul
6
 Nuclease-Free Water 5.5 ul *
Table1
* this volume was reduced by 2ul for the 1-3 reaction as 2 more primers were added.
Calculations
Primer dilution:
We need a final concentration of 0.5 uM in 25 uL. The minimum we can add with our pipets is 1 ul.
Thus we need a working solution containing 25x the concentration of 0.5 uM, or a 12.5 uM solution.
To get a 12.5uM solution from a 500uM stock, we need to dilute by a factor of 40. (500/12.5)
Thus we need 1ul of stock in 39 of water.
Primers Tms
2 Fwd & 3 Rev
image.png
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2 Rev & 3 Fwd
image.png
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4 Fwd & Rev
image.png
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5 Fwd & Rev
image.png
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6 Fwd & Rev
image.png
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7 Fwd & Rev
image.png
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8 Fwd & Rev
image.png
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9 Fwd & Rev
image.png
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All but 9 seem to anneal at 72, so we will use 72.
protocol Created with Sketch.

Visualization of PCR Exon 1 Fragments

Project: HD Resolution iGEM Notebook
Authors: jason fuller
Date: 2017-10-24
Tuesday, 10/24/17
Purpose: To run a gel electrophoresis to visualize the PCR amplicons of the full Exon 1 Assembly
1.
Split 10 lanes from precast 2% agarose gel
2.
Transfer 10ul from PCR tube to reaction tube
3.
Add 6x purple loading dye to each sample (2ul)
4.
Load 2% gel as follows:
A
B
C
D
E
F
G
H
I
J
K
1
 Lane # 1 2 3 4 5 6 7 8 9 10
2
 Sample 2 log ladder 1: MP Taq 24061 2: MP Taq 24061 3: Sat 24061 4: Invitrogen 24062 J1: MP Taq 24061 J2: MP Taq 24061 J3: Sat 24061 J4: Invitrogen 24062 2 log ladder
Table1
2 log ladder will resolve as follows:
image.png
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4. Run gel at 100 volts for 30 min
5. Stain with ETBR with GENTLE shaking
6. Image using gel imager
RESULTS
sequence Created with Sketch.

Final days time calculaton: How Much Time do we need?

Project: HD Resolution Shared Project
Authors: jason fuller
Date: 2017-10-25
Wednesday, 10/25/17
Working Backwards:
Shipping the part: (7 hrs)
MiniPrep the Plasmid out of the cells = 1 hr
Dry the DNA (in 2 fractions) = 30 min
Reconstitute 1 of 2 fractions in water and take OD reading 10 min
Reconsititue remaning fration in ethanol to a concentration of 25ng/ul and transfer 10ul to shipping plate, then drying = 4-6 hrs
(I think we can do this in 1)
Add foil seal, pack, and get to a shipper 20 min
Unplanned Gotchas:
Guessing the pakage weight and getting and printing a shipping label with tracking number
People using the nanodrop when we go to take OD readings
Genewiz shipping number and sample tracking info needed to be submitted along with part before this point!
Cell Culture outgrowth: (19hrs) - 26
Pick colonies (that glow? that don't glow? how can we ensure we have the right plasmid?): 1 hr
Grow cultures over night: 16- 20 hrs
Unplanned Gotchas:
Not having sterlilized Chloramphenical containing media
Colonys too small
Colonies hard to differentatiate (no flourescense? - add 2 hrs?)
Transform and Grow Colonies (19 hrs) - 45
Prep and Transform colonies with Plasmid containing our Part: 1 hr
Grow colonies over night: 16- 20 hr
Ligate part into backbone and isolate
Order Oligos
Arrive noon tomorrow: 28 HRs 28 +45 = 6
Notes:
Separating supercoild plasmid dna and nicked plasmids from linear can be done using λ endonuclease (it can't restrict nicked plasmids) and RecJf https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2832929/
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protocol Created with Sketch.

G-block 5-way Assembly

Project: HD Resolution iGEM Notebook
Authors: jason fuller
Date: 2017-10-25
Wednesday, 10/25/17
Purpose: Begin gibson assembly so that we can PCR once primers are in.
Materials:
IDT G-blocks (15)
NEB Hifi Builder Kit
Nuclease free water
A
B
C
D
1
 Concentration Range of DNA fragments 02 - .5 pmols* .2 - 1.0 pmols* 0 pmols
2
 Total Volume of Fragments (μl) 10
3
 Gibson Assembly Master Mix (2x) (μl) 10 10 10
4
 Deionized Water (μl) 10 10 0
5
 Total Volume (μl) *** 20 20 20
Table1
1.
Normalized concentrations of all G-blocks to 10 fmol per microliter.
2.
Take 5 microliters of each G-block sample and put into a new tube of each paired G-block sample.
3.
Then add 10 microliters of Gibson assembly master mix to each tube.
4.
Incubate for 15 minutes at 50 C in the thermocycler.

Gibson Assembly® Protocol (E5510)

Introduction

This is the protocol for Gibson Assembly using the Gibson Assembly® Cloning Kit (E5510). More information from NEB can be found here.

Materials

Procedure

  • Set up the following reaction on ice:
  1. Reaction volumes: Use this table to calculate reaction volumes and set up the reaction. Remember to input your total DNA fragment volume in cells B3 and C3 for assemblies with 2-3 fragments and 4-6 fragments, respectively.
  • - NEB recommends a total of 0.02–0.5 pmols of DNA fragments when 1 or 2 fragments are being assembled into a vector and 0.2–1.0 pmoles of DNA fragments when 4–6 fragments are being assembled. Efficiency of assembly decreases as the number or length of fragments increases. To calculate the number of pmols of each fragment for optimal assembly, based on fragment length and weight, we recommend using NEB's online tool, NEBioCalculator.
  • - The mass of each fragment can be measured using the NanoDrop instrument, absorbance at 260 nm, or estimated from agarose gel electrophoresis followed by ethidium bromide staining.
A
B
C
D
1
 2-3 Fragments Assembly 4-6 Fragments Assembly Positive Control **
2
 Concentration Range of DNA fragments 02 - .5 pmols* .2 - 1.0 pmols* 0 pmols
3
 Total Volume of Fragments (μl) 10
4
 Gibson Assembly Master Mix (2x) (μl) 10 10 10
5
 Deionized Water (μl) 10 10 0
6
 Total Volume (μl) *** 20 20 20
Table1
  • *Optimized cloning efficiency is 50–100 ng of vectors with 2–3 fold of excess inserts. Use 5 times more of inserts if size is less than 200 bps. Total volume of unpurified PCR fragments in Gibson Assembly reaction should not exceed 20%.
  • ** Control reagants are provided for 5 experiments with the Gibson Asembly Kit.
  • *** If greater numbers of fragments are assembled, additional Gibson Assembly Master Mix may be required.
  1. Incubate samples in a thermocycler at 50°C for 15 minutes when 2 or 3 fragments are being assembled or 60 minutes when 4-6 fragments are being assembled.
  • Note: Extended incubation up to 60 minutes may help to improve assembly efficiency in some cases (for further details see FAQ section).
  • *Select the pencil icon and change the timer to 60 minutes if working with 4-6 fragments
00:15:00
  1. Store samples on ice or at –20°C for subsequent transformation.
  1. Transform NEB 5-alpha Competent E. coli cells (provided with the kit) with 2 μl of the assembly reaction, following the chemcial transformation protocol or electro competent cells transformation protocol
protocol Created with Sketch.

Gel Images

Project: HD Resolution iGEM Notebook
Authors: HD Resolution iGEM
Date: 2017-10-25
Wednesday, 10/25/17
IMG_4006.JPG
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protocol Created with Sketch.

PCR Amplification of New Restriction Part

Project: HD Resolution iGEM Notebook
Authors: HD Resolution iGEM
Date: 2017-10-25
Wednesday, 10/25/17
Additionally:
1.
All dry primer stocks were diluted to 500uM (0.5 nMoles/ µL).
2.
Diluted primer stocks to a 'working primer solution' with conenctration of 12.5 uM (1ul stock + 39 water)
3.
New PCR tubes were labled for each previous assembly.
4.
A 25 ul Reaction volume was chosen:
5.
Reagents were added as follows
HTT AddGene
(FWD)
GAC CCT GGA AAA GCT GAT GAA GGC CTT CGA GTC CCT CA
(REV)
GAG GCA GCA GCG GCT GCT GTG CCT GCG GCG GCG GCT GAG GAA GCT
IMG_4007.JPG
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Part Assembly with EcoR1 Restriction Sites

Project: HD Resolution iGEM Notebook
Authors: HD Resolution iGEM
Date: 2017-10-25
Wednesday, 10/25/17
Purpose: Reassemble and cut existing Exon 1 parts such that there is a definite Eco R1 restriction site so that they can be added into a pSB1C3 backbone
1.
Placed previously isolated DNA plasmid on ie (61 and 62)
2.
Measured the concentration of the DNA Plasmids with the nanodrop (2μl)
a.
DNA plasmid 40261 : 356.2 ng/μl
b.
DNA plasmid 40262 : 300.5 ng/μl
3.
Diluted DNA plasmid 1 : 3 to approximate 100 ng/μl and measured with nanodrop
a.
DNA plasmid 40261 : 117.9 ng/μl
b.
DNA plasmid 40262 : 106.1 ng/μl
4.
Labeled fresh 0.5 mL tubes 61 and 62
5.
For each tube, transferred 4 ul of 10x NEBuffer, 1 ul of Eco R1, 1 ul of Xmn 1, and 25.6 ul of dH2O*
6.
Transferred 10μl DNA Plasmid 40261 to 0.5 mL tube 61 and 10μl of DNA plasmid 40262 to 0.5 mL tube 62
7.
Incubated 0.5 mL tubes at 37 degrees Celsius for fifteen minutes
8.
Added 10μl purple 6x loading dye to each of the 0.5 mL tube and mixed
9.
Gel was loaded as follows:
A
B
C
D
E
F
G
1
 Cane 1 2 3 4 5 6
2
 10μl of 2 log ladder 10μl of digested ladder 10μl of digested 40262 15μl of digested 40262 15μl of digested 40261 15μl of digested 40261 10μl of 2 log ladder
Table1
*NOTE: NEBuffer and H2O was accidentally pippeted into the DNA Plasmids (tubes 61 and 62)
- So 18 ul of each 61 and 62 were pipetted into corresponding 61 and 62 tubes (the other ones)

 

 

HD RESOLUTION

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