Difference between revisions of "Team:Groningen/test"

 
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{{Groningen}}
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{{groningen}}
 
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<nav id="myScrollspy">
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/*div.bar li a {display: block;}*/
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  <ul class="nav">
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div.bar li.icon {display: none;}
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    <li class="active"><a href="#nav1">Restriction digestion</a></li>
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−
    <li><a href="#nav2">Ligation</a></li>
+
−
    <li><a href="#nav3">Gibson assembly</a></li>
+
−
    <li><a href="#nav4">Preparing competent <i>E. coli</i></a></li>
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−
    <li><a href="#nav5">Preparing competent <i>L. lactis</i></a></li>
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−
    <li><a href="#nav6">Transformation <i>E. coli</i></a></li>
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−
    <li><a href="#nav7">Electrotransformation <i>L. lactis</i></a></li>
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    <li><a href="#nav8">Colony PCR</a></li>
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−
    <li><a href="#nav9">Quickchange PCR</a></li>
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−
    <li><a href="#nav10">Taq PCR</a></li>
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−
    <li><a href="#nav11">Phusion PCR</a></li>
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    <li><a href="#nav12">PCR cleanup</a></li>
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−
    <li><a href="#nav13">Media</a></li>
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    <li><a href="#nav14">Antibiotic</a></li>
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  </ul>
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</nav>
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<div style="margin-left:25%;">
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<div class="main-col">
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@media screen and (max-width:800px) {
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<h1> Protocols</h1>
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<br>
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ul.menu.res li {float: left;}
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<ol>
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<li id="nav1"><b>Restriction Digestion</b>
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  <ol type=a>
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</style>
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   <li>Materials
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   <ol type=i>
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     <li>(1) 8-tube strip, or (3) 0.6ml thin-walled tubes</li>
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  <section>
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    <li>BioBrick Part in BioBrick plasmid (Purified DNA, > 16ng/ul)</li>
+
  <div class="bar">  
−
    <li>dH2O</li>
+
<!-- Begin content block -->
−
    <li>NEB Buffer 2</li>
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   <!--<ul id="menu"> -->
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    <li>BSA</li>
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   <ul class="menu">
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    <li>Restriction Enzymes: EcoRI, SpeI, XbaI, PstI</li>
+
     <li class="icon"> <a href="javascript:void(0);" onclick="myFunction()">≡ Menu</a></li>
−
  </ol>
+
  <li><a href="?page=home">Home</a></li>
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   <li>Procedure
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   <li><a href="#">Symposium</a>
−
    <ol type=i>
+
    <ul>
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    <li>Add 250ng of DNA to be digested, and adjust with dH20 for a total volume of 16ul</li>
+
     <li><a href="?page=program" onclick="">Program</a></li>
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    <li>Add 2.5ul of NEBuffer 2.1</li>
+
     <li><a href="?page=daychair" onclick="">Day chair</a></li>
−
     <li>Check here for buffer selection (depending on the enzyme)</li>
+
     <li><a href="?page=speakers" onclick="">Speakers</a></li>
−
    <li>Add 0.5ul of BSA</li>
+
    <li><a href="?page=guestspeaker" onclick="">Guest speaker</a></li>
−
     <li>Add 0.5ul of EcoRI</li>
+
     <li><a href="?page=location" onclick="">Location</a></li>
−
    <li>Add 0.5ul of PstI</li>
+
     </ul>
−
    <li>There should be a total volume of 20ul. Mix well and spin down briefly</li>
+
−
     <li>Incubate the restriction digest at 37C for 30min, and then 80C for 20min to heat kill the enzymes. <i>We incubate in a thermal cycler with a heated lid</i></li>
+
−
    <li>Run a portion of the digest on a gel (8ul, 100ng), to check that both plasmid backbone and part length are accurate.</li>
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    </ol>
+
−
  </li>
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−
    <li>Source
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    <ol type=i>
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      <li>iGEM http://parts.igem.org/Help:Protocols/Restriction_Digest</li>
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     </ol>
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−
    </li>
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−
  </li>
+
−
  </ol>
+
−
</li>
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−
<br>
+
−
<li id="nav2"><b>Ligation</b>
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−
  <ol type=a>
+
−
  <li>Materials
+
−
    <ol type=i>
+
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    <li>Digested backbone & inserts</li>
+
−
    <li>T4 DNA ligase</li>
+
−
    <li>T4 DNA ligase buffer</li>
+
−
     </ol>
+
 
   </li>
 
   </li>
−
  <li>Procedure
+
<!--  <li><a href="?page=gallery">Gallery</a></li>-->
−
    <ol type=i>
+
  <li><a href="?page=sponsors">Sponsors</a></li>
−
    <li>Add 2ul of digested plasmid backbone (25 ng)</li>
+
  <li><a href="#">About us</a>
−
    <li>Add equimolar amount of EcoRI-HF SpeI digested fragment (< 3 ul)</li>
+
    <ul>
−
    <li>Add equimolar amount of XbaI PstI digested fragment (< 3 ul)</li>
+
     <li><a href="?page=nanoscience" onclick="">Nanoscience</a></li>
−
     <li>Molar ratios of 1:1, 1:10, 1:20 are recommended</li>
+
     <li><a href="?page=topmaster" onclick="">Top Master</a></li>
−
    <li>Add 1 ul T4 DNA ligase buffer. Note: Do not use quick ligase</li>
+
     <li><a href="?page=recom" onclick="">Recommendations</a></li>
−
     <li>Add 0.5 ul T4 DNA ligase</li>
+
     </ul>
−
    <li>Add water to 10 ul</li>
+
−
     <li>Ligate 16C/30 min, heat kill 80C/20 min</li>
+
−
    <li>Transform with 1-2 ul of product</li>
+
−
     </ol>
+
 
   </li>
 
   </li>
−
   <li>Source
+
   <li><a href="?page=contact">Contact</a></li>
−
    <ol type=i>
+
   <li class="lilast"><a href="?page=registration">Registration</a></li>
−
    <li>iGEM http://parts.igem.org/Help:Protocols/Ligation</li>
+
  </ul>
−
    </ol>
+
</div>
−
   </li>
+
</section>
−
  </ol>
+
 
−
</li>
+
<script>
−
<br>
+
function myFunction() {
−
<li id="nav3"><b>Gibson assembly</b>
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     document.getElementsByClassName("bar")[0].classList.toggle("res");
−
  <ol type=a>
+
    document.getElementsByClassName("menu")[0].classList.toggle("res");
−
  <li>Materials
+
}
−
    <ol type=i>
+
</script>
−
    <li>Compatible Fragments</li>
+
 
−
    <li>Gibson Assembly Master Mix 2x</li>
+
<div class="main-col">
−
    <li>Positive control (NEB)</li>
+
 
−
    </ol>
+
<h2><a href="https://2017.igem.org/Team:Groningen/test1">back to test 1</a></h2>
−
  </li>
+
 
−
  <li>Procedure
+
<div>hicula risus et, congue mi. Praesent quis auctor enim. Aenean gravida, libero ut dignissim porta, elit nisi faucibus nisi, vitae faucibus lorem eros in augue. Pellentesque habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas. In ac varius diam. Mauris consectetur sed erat at dapibus. Fusce pulvinar est eu orci lacinia egestas.
−
     <table style="width:100%">
+
 
−
    <tr>
+
Phasellus facilisis nulla quis tortor adipiscing, eu blandit tortor pulvinar. In lacus purus, aliquet eu ante ut, tempor ultricies diam. Donec cursus neque neque, eget vehicula nisl commodo at. Fusce eu urna tempus, egestas ante id, facilisis dolor. Pellentesque venenatis ante convallis, ullamcorper erat at, malesuada elit. Interdum et malesuada fames ac ante ipsum primis in faucibus. Aliquam commodo lectus ut luctus malesuada. Donec leo nisl, dignissim et ullamcorper ut, porta id ante. Pellentesque habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas. Vestibulum eros felis, consequat sed augue non, semper eleifend nisi.
−
      <th> </th>
+
 
−
      <th>2-3 Fragment Assembly</th>
+
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−
      <th>4-6 Fragment Assembly</th>
+
Sub heading style
−
      <th>Positive Control**</th>
+
 
−
    </tr>
+
Nunc ut ornare odio. Proin massa turpis, porta at dolor non, tempus sagittis risus. Maecenas ornare, eros eget commodo sagittis, neque orci consectetur risus, sed convallis purus
−
    <tr>
+
# First Name Last Name Username
−
      <td>Total Amount of Fragments</td>
+
1 Mark Otto @mdo
−
      <td>0.02–0.5 pmols* X μl</td>
+
2 Jacob Thornton @fat
−
      <td>0.2–1 pmols* X μl</td>
+
3 Larry the Bird @twitter
−
      <td>10 μl</td>
+
The Route
−
    </tr>
+
 
−
    <tr>
+
Lorem ipsum dolor sit amet, consectetur adipiscing elit. Ut egestas consectetur ante a rhoncus. Praesent a sapien vulputate, vehicula risus et, congue mi. Praesent quis auctor enim. Aenean gravida, libero ut dignissim porta, elit nisi faucibus nisi, vitae faucibus lorem eros in augue. Pellentesque habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas. In ac varius diam. Mauris consectetur sed erat at dapibus. Fusce pulvinar est eu orci lacinia egestas.
−
      <td>Gibson Assembly Master Mix (2X)</td>
+
Hotel.
−
      <td>10 μl</td>
+
795 Folsom Ave, Suite 600
−
      <td>10 μl</td>
+
San Francisco, CA 94107
−
      <td>10 μl</td>
+
P: (123) 456-7890
−
    </tr>
+
Full Name
−
    <tr>
+
first.last@example.com
−
      <td>Deionized H2O</td>
+
 
−
      <td>10-X μl</td>  
+
Phasellus facilisis nulla quis tortor adipiscing, eu blandit tortor pulvinar. In lacus purus, aliquet eu ante ut, tempor ultricies diam. Donec cursus neque neque, eget vehicula nisl commodo at. Fusce eu urna tempus, egestas ante id, facilisis dolor. Pellentesque venenatis ante convallis, ullamcorper erat at, malesuada elit. Interdum et malesuada fames ac ante ipsum primis in faucibus. Aliquam commodo lectus ut luctus malesuada. Donec leo nisl, dignissim et ullamcorper ut, porta id ante. Pellentesque habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas. Vestibulum eros felis, consequat sed augue non, semper eleifend nisi.
−
      <td>10-X μl</td>
+
 
−
      <td>0</td>
+
Maecenas tincidunt, ante in ultricies porta, magna ipsum convallis augue, non ornare turpis orci ac risus. Duis non enim sit amet libero ultricies egestas. Duis bibendum felis est, nec feugiat erat imperdiet sed. Donec aliquet, velit ac semper fermentum, quam leo eleifend nisl, non rhoncus sapien nibh ac massa. Integer mattis arcu nec suscipit laoreet. Cras nec libero leo. Nulla bibendum ligula ut nisi dictum, et interdum ipsum scelerisque. Nullam a volutpat tellus. Maecenas nisi est, ultricies in neque in, aliquet dictum odio. Etiam suscipit sapien purus, suscipit pharetra tellus lobortis ut. Maecenas commodo feugiat justo ac ullamcorper. In volutpat nec velit posuere placerat. Suspendisse rutrum dolor sed semper pharetra.
−
    </tr>
+
The Bike
−
    <tr>
+
 
−
      <td>Total Volume</td>
+
Lorem ipsum dolor sit amet, consectetur adipiscing elit. Ut egestas consectetur ante a rhoncus. Praesent a sapien vulputate, vehicula risus et, congue mi. Praesent quis auctor enim. Aenean gravida, libero ut dignissim porta, elit nisi faucibus nisi, vitae faucibus lorem eros in augue. Pellentesque habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas. In ac varius diam. Mauris consectetur sed erat at dapibus. Fusce pulvinar est eu orci lacinia egestas.
−
      <td>20 μl***</td>
+
 
−
      <td>20 μl***</td>
+
Phasellus facilisis nulla quis tortor adipiscing, eu blandit tortor pulvinar. In lacus purus, aliquet eu ante ut, tempor ultricies diam. Donec cursus neque neque, eget vehicula nisl commodo at. Fusce eu urna tempus, egestas ante id, facilisis dolor. Pellentesque venenatis ante convallis, ullamcorper erat at, malesuada elit. Interdum et malesuada fames ac ante ipsum primis in faucibus. Aliquam commodo lectus ut luctus malesuada. Donec leo nisl, dignissim et ullamcorper ut, porta id ante. Pellentesque habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas. Vestibulum eros felis, consequat sed augue non, semper eleifend nisi.
−
      <td>20 μl</td>
+
 
−
    </tr>
+
Description lists
−
    </table>
+
    A description list is perfect for defining terms.
−
  </li>
+
Euismod
−
  <li>Source
+
     Vestibulum id ligula porta felis euismod semper eget lacinia odio sem nec elit.
−
    <ol type=i>
+
     Donec id elit non mi porta gravida at eg</div>
−
    <li>https://www.neb.com/-/media/catalog/datacards-or-manuals/manuale2621.pdf</li>
+
 
−
    </ol>
+
<div id="snavtest">
−
  </li>
+
<h3>THIS IS MY ID TARGET</h3>
−
  </ol>
+
<a href="#snavtest">test this page</a><br>
−
</li>
+
</div>
−
<br>
+
 
−
<li id="nav4"><b>Preparing competent <i>E. coli</i> DH5α cells</b>
+
<div>faucibus nisi, vitae faucibus lorem eros in augue. Pellentesque habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas. In ac varius diam. Mauris consectetur sed erat at dapibus. Fusce pulvinar est eu orci lacinia egestas.
−
  <ol type=a>
+
 
−
  <li>Materials
+
Phasellus facilisis nulla quis tortor adipiscing, eu blandit tortor pulvinar. In lacus purus, aliquet eu ante ut, tempor ultricies diam. Donec cursus neque neque, eget vehicula nisl commodo at. Fusce eu urna tempus, egestas ante id, facilisis dolor. Pellentesque venenatis ante convallis, ullamcorper erat at, malesuada elit. Interdum et malesuada fames ac ante ipsum primis in faucibus. Aliquam commodo lectus ut luctus malesuada. Donec leo nisl, dignissim et ullamcorper ut, porta id ante. Pellentesque habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas. Vestibulum eros felis, consequat sed augue non, semper eleifend nisi.
−
    <ol type=i>
+
 
−
    <li>Plate or stock of <i>E.coli</i> DH5α cell</li>
+
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−
    <li>LB</li>
+
Sub heading style
−
    <li><a class="mouseover" title="5g PEG 8000
+
 
−
1.5 mL 1M MgCl2 (or 0.30g MgCl2*6H20)
+
Nunc ut ornare odio. Proin massa turpis, porta at dolor non, tempus sagittis risus. Maecenas ornare, eros eget commodo sagittis, neque orci consectetur risus, sed convallis purus
−
2.5 mL DMSO
+
# First Name Last Name Username
−
Add LB to 50 mL
+
1 Mark Otto @mdo
−
Filter sterilize (0.22 μm filter)">TSS buffer</a></li>
+
2 Jacob Thornton @fat
−
    <li>Ice</li>
+
3 Larry the Bird @twitter
−
    </ol>
+
The Route
−
  </li>
+
 
−
  <li>Procedure
+
Lorem ipsum dolor sit amet, consectetur adipiscing elit. Ut egestas consectetur ante a rhoncus. Praesent a sapien vulputate, vehicula risus et, congue mi. Praesent quis auctor enim. Aenean gravida, libero ut dignissim porta, elit nisi faucibus nisi, vitae faucibus lorem eros in augue. Pellentesque habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas. In ac varius diam. Mauris consectetur sed erat at dapibus. Fusce pulvinar est eu orci lacinia egestas.
−
    <ol type=i>
+
Hotel.
−
    <li>Grow 5ml overnight culture of cells in LB media, dilute this culture back into 25-50ml of fresh LB media in a 200ml conical flask.</li>
+
795 Folsom Ave, Suite 600
−
    <li>In the morning You should aim to dilute the overnight culture by at least 1/100.</li>
+
San Francisco, CA 94107
−
    <li>Grow the diluted culture to an OD600 of 0.2 - 0.5. (You will get a very small pellet if you grow 25ml to OD600 0.2)</li>
+
P: (123) 456-7890
−
    <li>Put Eppendorf tubes on ice now so that they are cold when cells are aliquoted into them later. If your culture is X ml, you will need X tubes. At this point you should also make sure that your TSS is being cooled (it should be stored at 4°C but if you have just made it fresh then put it in an ice bath).</li>
+
Full Name
−
    <li>Split the culture into two 50ml falcon tubes and incubate on ice for 10 min.</li>
+
first.last@example.com
−
    <li>All subsequent steps should be carried out at 4°C and the cells should be kept on ice whenever possible.</li>
+
 
−
    <li>Centrifuge for 10 minutes at 3000 rpm and 4°C.</li>
+
Phasellus facilisis nulla quis tortor adipiscing, eu blandit tortor pulvinar. In lacus purus, aliquet eu ante ut, tempor ultricies diam. Donec cursus neque neque, eget vehicula nisl commodo at. Fusce eu urna tempus, egestas ante id, facilisis dolor. Pellentesque venenatis ante convallis, ullamcorper erat at, malesuada elit. Interdum et malesuada fames ac ante ipsum primis in faucibus. Aliquam commodo lectus ut luctus malesuada. Donec leo nisl, dignissim et ullamcorper ut, porta id ante. Pellentesque habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas. Vestibulum eros felis, consequat sed augue non, semper eleifend nisi.
−
    <li>Decant supernatant, remove leftover media by carefully pipetting</li>
+
 
−
    <li>Resuspend in TSS buffer (10% of original volume), vortex gently</li>
+
Maecenas tincidunt, ante in ultricies porta, magna ipsum convallis augue, non ornare turpis orci ac risus. Duis non enim sit amet libero ultricies egestas. Duis bibendum felis est, nec feugiat erat imperdiet sed. Donec aliquet, velit ac semper fermentum, quam leo eleifend nisl, non rhoncus sapien nibh ac massa. Integer mattis arcu nec suscipit laoreet. Cras nec libero leo. Nulla bibendum ligula ut nisi dictum, et interdum ipsum scelerisque. Nullam a volutpat tellus. Maecenas nisi est, ultricies in neque in, aliquet dictum odio. Etiam suscipit sapien purus, suscipit pharetra tellus lobortis ut. Maecenas commodo feugiat justo ac ullamcorper. In volutpat nec velit posuere placerat. Suspendisse rutrum dolor sed semper pharetra.
−
    <li>Add 100 μl aliquots to chilled Eppendorfs, flash freeze and store at – 80°C in 200 µl aliquots.</li>
+
The Bike
−
    </ol>
+
 
−
  </li>
+
Lorem ipsum dolor sit amet, consectetur adipiscing elit. Ut egestas consectetur ante a rhoncus. Praesent a sapien vulputate, vehicula risus et, congue mi. Praesent quis auctor enim. Aenean gravida, libero ut dignissim porta, elit nisi faucibus nisi, vitae faucibus lorem eros in augue. Pellentesque habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas. In ac varius diam. Mauris consectetur sed erat at dapibus. Fusce pulvinar est eu orci lacinia egestas.
−
  <li>Source
+
 
−
    <ol type=i>
+
Phasellus facilisis nulla quis tortor adipiscing, eu blandit tortor pulvinar. In lacus purus, aliquet eu ante ut, tempor ultricies diam. Donec cursus neque neque, eget vehicula nisl commodo at. Fusce eu urna tempus, egestas ante id, facilisis dolor. Pellentesque venenatis ante convallis, ullamcorper erat at, malesuada elit. Interdum et malesuada fames ac ante ipsum primis in faucibus. Aliquam commodo lectus ut luctus malesuada. Donec leo nisl, dignissim et ullamcorper ut, porta id ante. Pellentesque habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas. Vestibulum eros felis, consequat sed augue non, semper eleifend nisi.
−
    <li>https://openwetware.org/wiki/Preparing_chemically_competent_cells</li>
+
 
−
    </ol>
+
Description lists
−
  </li>
+
     A description list is perfect for defining terms.
−
  </ol> 
+
Euismod
−
</li>
+
     Vestibulum id ligula porta felis euismod semper eget lacinia odio sem nec elit.
−
<br>
+
     Donec id elit non mi porta gravida at eg</div>
−
<li id="nav5"><b>Making competent Lactococcus <i>L. lactis</i> NZ9000 cells</b>
+
 
−
  <ol type=a>
+
−
  <li>Materials
+
−
    <ol type=i>
+
−
    <li><a class="mouseover" title="M17
+
−
        0.5% glucose">GM17</a></li>
+
−
    <li><a class="mouseover" title="M17
+
−
        0.5 M sucrose
+
−
        0.5 % glucose
+
−
        2% glycine">SGM17 + glycine</a></li>
+
−
    <li><a class="mouseover" title="0.5 M sucrose
+
−
10% glycerol">Electroporation buffer</a></li>
+
−
    </ol>
+
−
  </li>
+
−
    <li>Procedure
+
−
    <ol type=a>
+
−
      <li>Grow cells overnight in 25ml of GM17</li>
+
−
      <li>Add 1ml of overnight culture into 25ml SGM17 + glycine</li>
+
−
      <li>Grow for ~4 hours until OD600 ~ 0.7</li>
+
−
      <li>Chill culture on ice for 10 mins</li>
+
−
      <li>Centrifuge cells for 15 mins at 3000g</li>
+
−
      <li>Gently shake to resuspend pellet in 3ml Electroporation Buffer</li>
+
−
      <li>Centrifuge cells for 15 mins at 3000g</li>
+
−
      <li>Resuspend pellet in 3ml Electroporation Buffer</li>
+
−
      <li>Centrifuge cells for 15 mins at 3000g</li>
+
−
      <li>Resuspend pellet in 500 µl Electroporation Buffer</li>
+
−
      <li>Separate into 100 µl aliquots and store at -80°C until use.</li>
+
−
    </ol>
+
−
    </li>
+
−
    <li>Source
+
−
    <ol type=i>
+
−
      <li>https://openwetware.org/wiki/Lactococcus_transformation</li>
+
−
    </ol>
+
−
    </li>
+
−
  </ol>
+
−
<br>
+
−
</li>
+
−
<li id="nav6"><b>Transformation <i>E.coli</i> DH5α</b>
+
−
  <ol type=a>
+
−
  <li>Materials
+
−
    <ol type=i>
+
−
    <li>Plasmid DNA</li>
+
−
    <li>Competent <i>E. coli</i> DH5α cells: 50 µl per transformation</li>
+
−
    <li><a class="mouseover" title="1% Yeast Extract
+
−
4% Tryptone
+
−
40 mM Glucose
+
−
Store at 4°C">2x SOC stock</a></li>
+
−
    <li><a class="mouseover" title="1 M NaCl
+
−
250 mM KCl
+
−
1 M MgCl2
+
−
1 M MgSO4
+
−
Store at 4°C">SOC salt stocks</a></li>
+
−
    <li>Sterile MQ</li>
+
−
    <li>LB agar selection plates: Two per transformation</li>
+
−
    <li>Eppendorf tubes</li>
+
−
    <li>Floater</li>
+
−
    <li>Ice</li>
+
−
    <li>42°C water bath</li>
+
−
    <li>37°C incubator: both shaker and stove</li>
+
−
    <li>Sterile spreader/glass beads</li>
+
−
    </ol>
+
−
  </li>
+
−
  <li>Procedure (on ice)
+
−
    <ol type=i>
+
−
    <li>Thaw competent cells on ice. When using multiple aliquots, first pool all cells into a single volume to homonogize the solution. Dispose of unused competent cells. Do not refreeze since reusing thawed cells, will drastically reduce transformation efficiency.</li>
+
−
    <li>Pipet 50 µl of competent cells into Eppendorf tube for each transformation (labeled, prechilled, in floating rack), don’t forget control tubes</li>
+
−
    <li>Pipet 1-100 ng of DNA as well as control into tubes and gently mix with tip</li>
+
−
    <li>incubate on ice for 30 min, tubes may be gently flicked, return to ice ASAP</li>
+
−
    <li>Meanwhile, for every transformation, add 2 µl of each SOC salt solution into 100 µl 2x SOC stock and ad to 200 µl with sterile MQ. Place the SOC medium on ice till use</li> 
+
−
    <li>Heat shock tubes at 42°C for 30 seconds (precisely)</li>
+
−
    <li>Incubate on ice for 5 min</li>
+
−
    <li>Add 200 µl of SOC medium to each transformation</li>
+
−
    <li>Incubate at 37°C for 1 hours, shaker or rotor recommended</li>
+
−
    <li>Pipet 20 µl & 200 µl transformation mixture onto petri plates and spread with sterilized spreader or glass beads. Let the plates dry near the flame before placing them in the incubator</li>
+
−
    <li>Incubate plates upside down overnight (14-18hr) at 37°C</li>
+
−
    <li>Pick single colonies</li>
+
−
    <li>Perform Colony PCR to verify</li>
+
−
    <li>Grow cells & miniprep</li>
+
−
    <li>Calculate efficiency by counting colonies (expected value: 1.5x10^8 to 6x10^8 cfu/µg DNA)</li>
+
−
    </ol>
+
−
  </li>
+
−
  <li>Source
+
−
    <ol type=i>
+
−
    <li>http://parts.igem.org/Help:Protocols/Transformation</li>
+
−
    </ol>
+
−
  </li>
+
−
  </ol> 
+
−
</li>
+
−
<br>
+
−
<li id="nav7"><b>Electrotransformation <i>L. Lactis</i></b>
+
−
  <ol type=a>
+
−
  <li>Materials
+
−
    <ol type=i>
+
−
    <li>Plasmid DNA (preferably without salts from buffers. These can be removed by incubating the DNA on a filter, which is floating on MQ for 10 minutes at room temperature</li>
+
−
    <li>Electrocompetent <i>L. lactis</i> cells: 50 µl per transformation</li>
+
−
    <li><a class="mouseover" title="M17
+
−
        0.5% glucose
+
−
        0.5 M sucrose
+
−
        2 mM MgCl2
+
−
        2 mM CaCl2">Recovery medium</a></li>
+
−
    <li>Electroporation Cuvettes</li>
+
−
    <li>Electroporator</li>
+
−
    <li>SGM17 agar selection plates</li>
+
−
    <li>Eppendorf tubes</li>
+
−
    <li>Ice</li>
+
−
    <li>Eppendorf centrifuge</li>
+
−
    <li>30°C incubator</li>
+
−
    <li>Sterile spreader/glass beads</li> 
+
−
    </ol>
+
−
  </li>
+
−
  <li>Procedure
+
−
    <ol type=i>
+
−
    <li>Mix all elements of the recovery medium (1 ml per transformation) and place it on ice together with the electroporation cuvettes</li>
+
−
    <li>Thaw competent cells on ice. When using multiple aliquots, first pool all cells into a single volume to homonogize the solution. Dispose of unused competent cells. Do not refreeze since reusing thawed cells, will drastically reduce transformation efficiency.</li>
+
−
    <li>Pipet 50 µl of competent cells into Eppendorf tube for each transformation (labeled, prechilled, in floating rack), don’t forget control tubes</li>
+
−
    <li>Add at most 5 µl of plasmid DNA to the competent cells and incubate on ice for 10 minutes</li>
+
−
    <li>Dry the cuvette, electroporate at 2500 V and carefully add 950 µl recovery medium to the cells. Place the cuvette back on ice and incubate for 10 minutes</li>
+
−
    <li>Transfer the cell suspension with careful mixing into Eppendorf tubes and incubate for 2 hours at 30°C</li>
+
−
    <li>Plate 100 µl of the recovered cells onto a selection plate and spin down the remaining cells at 6000 rpm for 5 minutes</li>
+
−
    <li>Resuspend cell pellet into 50-100µl and plate on a selection plate</li>
+
−
    <li>Incubate the plates at 30°C for 24 - 48 hours</li>
+
−
    </ol>
+
−
  </li>
+
−
  <li>Source
+
−
     <ol type=i>
+
−
    <li>https://openwetware.org/wiki/Lactococcus_transformation</li>
+
−
     </ol>
+
−
  </li>
+
−
  </ol>
+
−
</li>
+
−
<br>
+
−
<li id="nav8"><b>Colony PCR</b>
+
−
  <ol type=a>
+
−
  <li>Materials
+
−
    <ol type=i>
+
−
    <li>10X Standard Taq Reaction Buffer</li>
+
−
    <li>10 mM dNTPs</li>
+
−
    <li>10 µM Forward Primer</li>
+
−
    <li>10 µM Reverse Primer</li>
+
−
    <li>Template DNA (colony resuspended in MQ / plasmid DNA)</li>
+
−
    <li>Taq DNA Polymerase</li>
+
−
    <li>Nuclease-free water</li>
+
−
    <li>PCR tubes</li>
+
−
    <li>Ice</li>
+
−
    <li>PCR tube rack</li>
+
−
    </ol>
+
−
  </li>
+
−
  <li>Procedure
+
−
    <ol type=i>
+
−
    <li>Suspend a colony in 10 µl sterile MQ</li>
+
−
    <li>Prepare Mastermix for 10 reactions according to:
+
−
      <table style="width:100%">
+
−
      <tr>
+
−
        <th>Component</th>
+
−
        <th>220 µl = 10 colonies</th>  
+
−
        <th>Final Concentration</th>
+
−
      </tr>
+
−
      <tr>
+
−
        <td>10X Standard Taq (Mg-free) Reaction Buffer</td>
+
−
        <td>22 µl</td>
+
−
        <td>1X</td>
+
−
      </tr>
+
−
      <tr>
+
−
        <td>25 mM MgCl2</td>
+
−
        <td>13,2 µl</td>
+
−
        <td>1.5 mM</td>
+
−
      </tr>
+
−
      <tr>
+
−
        <td>10 mM dNTPs</td>
+
−
        <td>4,4 µl</td>
+
−
        <td>200 µM</td>
+
−
      </tr>
+
−
      <tr>
+
−
        <td>10 µM pJET fw</td>
+
−
        <td>4,4 µl</td>
+
−
        <td>0.2 µM (0.05–1 µM, typically 0.1-0.5µM)</td>
+
−
      </tr>
+
−
      <tr>
+
−
        <td>10 µM pJET rv</td>
+
−
        <td>4,4 µl</td>
+
−
        <td>0.2 µM (0.05–1 µM, typically 0.1-0.5µM)</td>
+
−
      </tr>
+
−
      <tr>
+
−
        <td>Taq DNA Polymerase</td>
+
−
        <td>1,1 µl</td>
+
−
        <td>1.25 units/50 µl PCR</td>
+
−
      </tr>
+
−
      <tr>
+
−
        <td>Nuclease-free water</td>
+
−
        <td>148,5 µl</td>
+
−
        <td>-</td>
+
−
      </tr>
+
−
      </table>
+
−
    </li>
+
−
    <li>Put 19 µl of mastermix in each reaction tube & add 2 µl suspended colony mixture</li>
+
−
    <li>Add 2 µl plasmid DNA for positive control and 2 µl MQ for the negative control</li>
+
−
    <li>Place the tubes in the PCR machine (Taq program)</li>
+
−
    <li>Once the PCR is done, mix 10 µl of PCR product with 2 µl 6X purple gel loading dye and run it on a gel for 50 minutes at 130 Volts</li>
+
−
    <li>If the correct products are present in the gel samples, inoculate overnight cultures from the original plates.</li>
+
−
    <li>Mix 5 ml LB with appropriate antibiotic. Scoop a colony from the plate and drop the tip into the medium. Incubate the tube at 37°C overnight to let the culture grow</li>
+
−
    </ol>
+
−
  </li>
+
−
  <li>Source
+
−
    <ol type=i>
+
−
    <li>https://www.qiagen.com/us/resources/download.aspx?id=c73208eb-a83e-40c4-a9b6-ea5c4c94b9f4&lang=en</li>
+
−
    </ol>
+
−
  </li>
+
−
  </ol>
+
−
</li>
+
−
<br>
+
−
<li id="nav9"><b>Quickchange PCR</li></b>
+
−
<br>
+
−
<li id="nav10"><b>Taq PCR</b>
+
−
  <ol type=a >
+
−
  <li>Materials according to table</li>
+
−
  <li>Procedure
+
−
    <ol type=i>
+
−
    <li>Mix according to table
+
−
      <table style="width:100%">
+
−
      <tr>
+
−
        <th>Component</th>
+
−
        <th>25 μl reaction</th>
+
−
        <th>50 μl reaction</th>
+
−
        <th>Final Concentration</th>
+
−
      </tr>
+
−
      <tr>
+
−
        <td>10X Standard Taq Reaction Buffer</td>
+
−
        <td>2.5 μl</td>
+
−
        <td>5 μ</td>
+
−
        <td>1X</td>
+
−
      </tr>
+
−
      <tr>
+
−
        <td>10 mM dNTPs</td>
+
−
        <td>0.5 µl</td>
+
−
        <td>1 μl</td>
+
−
        <td>200 µM</td>
+
−
      </tr>
+
−
      <tr>
+
−
        <td>10 µM Forward Primer</td>
+
−
        <td>0.5 µl</td>
+
−
        <td>1 μl</td>
+
−
        <td>0.2 µM (0.05–1 µM)</td>
+
−
      </tr>
+
−
      <tr>
+
−
        <td>10 µM Reverse Primer</td>
+
−
        <td>0.5 µl</td>
+
−
        <td>1 μl</td>
+
−
        <td>0.2 µM (0.05–1 µM)</td>
+
−
      </tr>
+
−
      <tr>
+
−
        <td>Template DNA</td>
+
−
        <td>variable</td>
+
−
        <td>variable</td>
+
−
        <td>1,000 ng</td>
+
−
      </tr>
+
−
      <tr>
+
−
        <td>Taq DNA Polymerase</td>
+
−
        <td>0.125 µl</td>
+
−
        <td>0.25 µl</td>
+
−
        <td>1.25 units/50 µl PCR</td>
+
−
      </tr>
+
−
      <tr>
+
−
        <td>Nuclease-free water</td>
+
−
        <td>to 25 µl</td>
+
−
        <td>to 50 µl</td>
+
−
        <td>-</td>
+
−
      </tr>
+
−
      </table>
+
−
    </li>
+
−
    <li>PCR cycler conditions
+
−
      <table style="width:100%">
+
−
      <tr>
+
−
        <th>Step</th>
+
−
        <th>Temperature</th>
+
−
        <th>Time</th>
+
−
      </tr>
+
−
      <tr>
+
−
        <td>Initial Denaturation</td>
+
−
        <td>95°C</td>
+
−
        <td>30 sec</td>
+
−
      </tr>
+
−
      <tr>
+
−
        <td>30 cycles</td>
+
−
        <td>95°C</td>
+
−
        <td>15-30 sec</td>
+
−
      </tr>
+
−
      <tr>
+
−
        <td>30 cycles</td>
+
−
        <td>45°C-68°C</td>
+
−
        <td>15-60 sec</td>
+
−
      </tr>
+
−
      <tr>
+
−
        <td>30 cycles</td>
+
−
        <td>68°C</td>
+
−
        <td>1 min/ kb</td>
+
−
      </tr>
+
−
      <tr>
+
−
        <td>Final extension</td>
+
−
        <td>68°C</td>
+
−
        <td>5 min</td>
+
−
      </tr>
+
−
      <tr>
+
−
        <td>Hold</td>
+
−
        <td>4-10°C</td>
+
−
        <td>-</td>
+
−
      </tr>
+
−
      </table>
+
−
    </li>
+
−
    </ol>
+
−
    <li>Source
+
−
    <ol type=i>
+
−
      <li>NEB https://www.neb.com/protocols/1/01/01/taq-dna-polymerase-with-standard-taq-buffer-m0273</li>
+
−
    </ol>
+
−
    </li>
+
−
  </li>
+
−
  </ol>
+
−
</li>
+
−
<br>
+
−
<li id="nav11"><b>Phusion PCR</b>
+
−
  <ol type=a >
+
−
  <li>Materials according to table</li>
+
−
  <li>Procedure
+
−
    <table style="width:100%">
+
−
    <tr>
+
−
      <th>Component</th>
+
−
      <th>20 μl reaction</th>
+
−
      <th>50 μl reaction</th>
+
−
      <th>Final Concentration</th>
+
−
    </tr>
+
−
    <tr>
+
−
      <td>5X Phusion HF/ GC Buffer</td>
+
−
      <td>4 μl</td>
+
−
      <td>10 μl</td>
+
−
      <td>1X</td>
+
−
    </tr>
+
−
    <tr>
+
−
      <td>10 mM dNTPs</td>
+
−
      <td>0.4 µl</td>
+
−
      <td>1 μl</td>
+
−
      <td>200 µM</td>
+
−
    </tr>
+
−
    <tr>
+
−
      <td>10 µM Forward Primer</td>
+
−
      <td>1 µl</td>
+
−
      <td>2,5 μl</td>
+
−
      <td>0.5 µM (0.05–1 µM)</td>
+
−
    </tr>
+
−
    <tr>
+
−
      <td>10 µM Reverse Primer</td>
+
−
      <td>1 µl</td>
+
−
      <td>2,5 μl</td>
+
−
      <td>0.5 µM (0.05–1 µM)</td>
+
−
    </tr>
+
−
    <tr>
+
−
      <td>Template DNA</td>
+
−
      <td>variable</td>
+
−
      <td>variable</td>
+
−
      <td><250 ng</td>
+
−
    </tr>
+
−
    <tr>
+
−
      <td>Phusion DNA Polymerase</td>
+
−
      <td>0.2 µl</td>
+
−
      <td>0.5 µl</td>
+
−
      <td>1. units/50 µl PCR</td>
+
−
    </tr>
+
−
    <tr>
+
−
      <td>Nuclease-free water</td>
+
−
      <td>to 20 µl</td>
+
−
      <td>to 50 µl</td>
+
−
      <td>-</td>
+
−
    </tr>
+
−
    </table>
+
−
  </li>
+
−
  <li>PCR cycler conditions</li>
+
−
  <li>
+
−
    <table style="width:100%">
+
−
    <tr>
+
−
      <th>Step</th>
+
−
      <th>Temperature</th>
+
−
      <th>Time</th>
+
−
    </tr>
+
−
    <tr>
+
−
      <td>Initial Denaturation</td>
+
−
      <td>98°C</td>
+
−
      <td>30 sec</td>
+
−
    </tr>
+
−
    <tr>
+
−
      <td>30 cycles</td>
+
−
      <td>98°C</td>
+
−
      <td>5-10 sec</td>
+
−
    </tr>
+
−
    <tr>
+
−
      <td>30 cycles</td>
+
−
      <td>45°C-72°C</td>
+
−
      <td>10-30 sec</td>
+
−
    </tr>
+
−
    <tr>
+
−
      <td>30 cycles</td>
+
−
      <td>72°C</td>
+
−
      <td>15-30 sec/ kb</td>
+
−
    </tr>
+
−
    <tr>
+
−
      <td>Final extension</td>
+
−
      <td>72°C</td>
+
−
      <td>5-10 min</td>
+
−
    </tr>
+
−
    <tr>
+
−
      <td>Hold</td>
+
−
      <td>4-10°C</td>
+
−
      <td>-</td>
+
−
    </tr>
+
−
    </table>
+
−
  </li>
+
−
  <li>Source
+
−
    <ol type=i>
+
−
    <li>https://www.neb.com/protocols/1/01/01/pcr-protocol-m0530</li>
+
−
    </ol>
+
−
  </li>
+
−
  </ol>
+
−
</li>
+
−
<br>
+
−
<li id="nav12"><b>PCR cleanup, Gel extraction, minipreps was performed by using kits provided by Qiagen. Standard procedure was followed.</b></li>
+
−
<br>
+
−
<li id="nav13"><b>Media</b>
+
−
  <ol type=a>
+
−
  <li>Lysogeny Broth (LB)
+
−
    <ol type=i>
+
−
    <li>Dissolve the following in 1L ddH2O</li>
+
−
    <li>10g bacto-tryptone</li>
+
−
    <li>5g bacto-yeast extract</li>
+
−
    <li>10g NaCl (5g in some recipes)</li>
+
−
    <li>Adjust to pH 7.0 with NaOH</li>
+
−
    </ol>
+
−
  </li>
+
−
  <li>M17
+
−
    <ol type=i>
+
−
    <li>Dissolve the following in 1L ddH2O</li>
+
−
    <li>5.0 g Pancreatic Digest of Casein</li>
+
−
    <li>5.0 g Soy Peptone</li>
+
−
    <li>5.0 g Beef Extract</li>
+
−
    <li>2.5 g Yeast Extract</li>
+
−
    <li>0.5 g Ascorbic Acid</li>
+
−
    <li>0.25 g Magnesium Sulfate</li>
+
−
    <li>10.0 g Disodium-β-glycerophosphate</li>
+
−
    <li>11.0 g Agar</li>
+
−
    </ol>
+
−
  </li>
+
−
  <li>SOC
+
−
     <ol type=i>
+
−
    <li>Dissolve the following in 1L ddH2O</li>
+
−
    <li>20g Bacto Tryptone</li>
+
−
    <li>5g Bacto Yeast Extract</li>
+
−
    <li>2ml of 5M NaCl</li>
+
−
    <li>2.5ml of 1M KC</li>
+
−
    <li>10ml of 1M MgCl2</li>
+
−
    <li>10ml of 1M MgSO4</li>
+
−
    <li>20ml of 1M glucose</li>
+
−
     </ol>
+
−
  </li>
+
−
  </ol>
+
−
</li>
+
−
<br>
+
−
<li id="nav14"><b>Antibiotic</b>
+
−
  <ol type=a>
+
−
     <li>Chloramphenicol</li>
+
−
    <li>Erythromycin</li>
+
−
    <li>Ampicillin</li>
+
−
  </ol>
+
−
</li>
+
−
</ol>
+
  
−
</div> <!-- margin -->
 
 
</div> <!-- main-col -->
 
</div> <!-- main-col -->
  

Latest revision as of 19:33, 31 October 2017


back to test 1

hicula risus et, congue mi. Praesent quis auctor enim. Aenean gravida, libero ut dignissim porta, elit nisi faucibus nisi, vitae faucibus lorem eros in augue. Pellentesque habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas. In ac varius diam. Mauris consectetur sed erat at dapibus. Fusce pulvinar est eu orci lacinia egestas. Phasellus facilisis nulla quis tortor adipiscing, eu blandit tortor pulvinar. In lacus purus, aliquet eu ante ut, tempor ultricies diam. Donec cursus neque neque, eget vehicula nisl commodo at. Fusce eu urna tempus, egestas ante id, facilisis dolor. Pellentesque venenatis ante convallis, ullamcorper erat at, malesuada elit. Interdum et malesuada fames ac ante ipsum primis in faucibus. Aliquam commodo lectus ut luctus malesuada. Donec leo nisl, dignissim et ullamcorper ut, porta id ante. Pellentesque habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas. Vestibulum eros felis, consequat sed augue non, semper eleifend nisi. Maecenas tincidunt, ante in ultricies porta, magna ipsum convallis augue, non ornare turpis orci ac risus. Duis non enim sit amet libero ultricies egestas. Duis bibendum felis est, nec feugiat erat imperdiet sed. Donec aliquet, velit ac semper fermentum, quam leo eleifend nisl, non rhoncus sapien nibh ac massa. Integer mattis arcu nec suscipit laoreet. Sub heading style Nunc ut ornare odio. Proin massa turpis, porta at dolor non, tempus sagittis risus. Maecenas ornare, eros eget commodo sagittis, neque orci consectetur risus, sed convallis purus # First Name Last Name Username 1 Mark Otto @mdo 2 Jacob Thornton @fat 3 Larry the Bird @twitter The Route Lorem ipsum dolor sit amet, consectetur adipiscing elit. Ut egestas consectetur ante a rhoncus. Praesent a sapien vulputate, vehicula risus et, congue mi. Praesent quis auctor enim. Aenean gravida, libero ut dignissim porta, elit nisi faucibus nisi, vitae faucibus lorem eros in augue. Pellentesque habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas. In ac varius diam. Mauris consectetur sed erat at dapibus. Fusce pulvinar est eu orci lacinia egestas. Hotel. 795 Folsom Ave, Suite 600 San Francisco, CA 94107 P: (123) 456-7890 Full Name first.last@example.com Phasellus facilisis nulla quis tortor adipiscing, eu blandit tortor pulvinar. In lacus purus, aliquet eu ante ut, tempor ultricies diam. Donec cursus neque neque, eget vehicula nisl commodo at. Fusce eu urna tempus, egestas ante id, facilisis dolor. Pellentesque venenatis ante convallis, ullamcorper erat at, malesuada elit. Interdum et malesuada fames ac ante ipsum primis in faucibus. Aliquam commodo lectus ut luctus malesuada. Donec leo nisl, dignissim et ullamcorper ut, porta id ante. Pellentesque habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas. Vestibulum eros felis, consequat sed augue non, semper eleifend nisi. Maecenas tincidunt, ante in ultricies porta, magna ipsum convallis augue, non ornare turpis orci ac risus. Duis non enim sit amet libero ultricies egestas. Duis bibendum felis est, nec feugiat erat imperdiet sed. Donec aliquet, velit ac semper fermentum, quam leo eleifend nisl, non rhoncus sapien nibh ac massa. Integer mattis arcu nec suscipit laoreet. Cras nec libero leo. Nulla bibendum ligula ut nisi dictum, et interdum ipsum scelerisque. Nullam a volutpat tellus. Maecenas nisi est, ultricies in neque in, aliquet dictum odio. Etiam suscipit sapien purus, suscipit pharetra tellus lobortis ut. Maecenas commodo feugiat justo ac ullamcorper. In volutpat nec velit posuere placerat. Suspendisse rutrum dolor sed semper pharetra. The Bike Lorem ipsum dolor sit amet, consectetur adipiscing elit. Ut egestas consectetur ante a rhoncus. Praesent a sapien vulputate, vehicula risus et, congue mi. Praesent quis auctor enim. Aenean gravida, libero ut dignissim porta, elit nisi faucibus nisi, vitae faucibus lorem eros in augue. Pellentesque habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas. In ac varius diam. Mauris consectetur sed erat at dapibus. Fusce pulvinar est eu orci lacinia egestas. Phasellus facilisis nulla quis tortor adipiscing, eu blandit tortor pulvinar. In lacus purus, aliquet eu ante ut, tempor ultricies diam. Donec cursus neque neque, eget vehicula nisl commodo at. Fusce eu urna tempus, egestas ante id, facilisis dolor. Pellentesque venenatis ante convallis, ullamcorper erat at, malesuada elit. Interdum et malesuada fames ac ante ipsum primis in faucibus. Aliquam commodo lectus ut luctus malesuada. Donec leo nisl, dignissim et ullamcorper ut, porta id ante. Pellentesque habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas. Vestibulum eros felis, consequat sed augue non, semper eleifend nisi. Description lists A description list is perfect for defining terms. Euismod Vestibulum id ligula porta felis euismod semper eget lacinia odio sem nec elit. Donec id elit non mi porta gravida at eg

THIS IS MY ID TARGET

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faucibus nisi, vitae faucibus lorem eros in augue. Pellentesque habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas. In ac varius diam. Mauris consectetur sed erat at dapibus. Fusce pulvinar est eu orci lacinia egestas. Phasellus facilisis nulla quis tortor adipiscing, eu blandit tortor pulvinar. In lacus purus, aliquet eu ante ut, tempor ultricies diam. Donec cursus neque neque, eget vehicula nisl commodo at. Fusce eu urna tempus, egestas ante id, facilisis dolor. Pellentesque venenatis ante convallis, ullamcorper erat at, malesuada elit. Interdum et malesuada fames ac ante ipsum primis in faucibus. Aliquam commodo lectus ut luctus malesuada. Donec leo nisl, dignissim et ullamcorper ut, porta id ante. Pellentesque habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas. Vestibulum eros felis, consequat sed augue non, semper eleifend nisi. Maecenas tincidunt, ante in ultricies porta, magna ipsum convallis augue, non ornare turpis orci ac risus. Duis non enim sit amet libero ultricies egestas. Duis bibendum felis est, nec feugiat erat imperdiet sed. Donec aliquet, velit ac semper fermentum, quam leo eleifend nisl, non rhoncus sapien nibh ac massa. Integer mattis arcu nec suscipit laoreet. Sub heading style Nunc ut ornare odio. Proin massa turpis, porta at dolor non, tempus sagittis risus. Maecenas ornare, eros eget commodo sagittis, neque orci consectetur risus, sed convallis purus # First Name Last Name Username 1 Mark Otto @mdo 2 Jacob Thornton @fat 3 Larry the Bird @twitter The Route Lorem ipsum dolor sit amet, consectetur adipiscing elit. Ut egestas consectetur ante a rhoncus. Praesent a sapien vulputate, vehicula risus et, congue mi. Praesent quis auctor enim. Aenean gravida, libero ut dignissim porta, elit nisi faucibus nisi, vitae faucibus lorem eros in augue. Pellentesque habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas. In ac varius diam. Mauris consectetur sed erat at dapibus. Fusce pulvinar est eu orci lacinia egestas. Hotel. 795 Folsom Ave, Suite 600 San Francisco, CA 94107 P: (123) 456-7890 Full Name first.last@example.com Phasellus facilisis nulla quis tortor adipiscing, eu blandit tortor pulvinar. In lacus purus, aliquet eu ante ut, tempor ultricies diam. Donec cursus neque neque, eget vehicula nisl commodo at. Fusce eu urna tempus, egestas ante id, facilisis dolor. Pellentesque venenatis ante convallis, ullamcorper erat at, malesuada elit. Interdum et malesuada fames ac ante ipsum primis in faucibus. Aliquam commodo lectus ut luctus malesuada. Donec leo nisl, dignissim et ullamcorper ut, porta id ante. Pellentesque habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas. Vestibulum eros felis, consequat sed augue non, semper eleifend nisi. Maecenas tincidunt, ante in ultricies porta, magna ipsum convallis augue, non ornare turpis orci ac risus. Duis non enim sit amet libero ultricies egestas. Duis bibendum felis est, nec feugiat erat imperdiet sed. Donec aliquet, velit ac semper fermentum, quam leo eleifend nisl, non rhoncus sapien nibh ac massa. Integer mattis arcu nec suscipit laoreet. Cras nec libero leo. Nulla bibendum ligula ut nisi dictum, et interdum ipsum scelerisque. Nullam a volutpat tellus. Maecenas nisi est, ultricies in neque in, aliquet dictum odio. Etiam suscipit sapien purus, suscipit pharetra tellus lobortis ut. Maecenas commodo feugiat justo ac ullamcorper. In volutpat nec velit posuere placerat. Suspendisse rutrum dolor sed semper pharetra. The Bike Lorem ipsum dolor sit amet, consectetur adipiscing elit. Ut egestas consectetur ante a rhoncus. Praesent a sapien vulputate, vehicula risus et, congue mi. Praesent quis auctor enim. Aenean gravida, libero ut dignissim porta, elit nisi faucibus nisi, vitae faucibus lorem eros in augue. Pellentesque habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas. In ac varius diam. Mauris consectetur sed erat at dapibus. Fusce pulvinar est eu orci lacinia egestas. Phasellus facilisis nulla quis tortor adipiscing, eu blandit tortor pulvinar. In lacus purus, aliquet eu ante ut, tempor ultricies diam. Donec cursus neque neque, eget vehicula nisl commodo at. Fusce eu urna tempus, egestas ante id, facilisis dolor. Pellentesque venenatis ante convallis, ullamcorper erat at, malesuada elit. Interdum et malesuada fames ac ante ipsum primis in faucibus. Aliquam commodo lectus ut luctus malesuada. Donec leo nisl, dignissim et ullamcorper ut, porta id ante. Pellentesque habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas. Vestibulum eros felis, consequat sed augue non, semper eleifend nisi. Description lists A description list is perfect for defining terms. Euismod Vestibulum id ligula porta felis euismod semper eget lacinia odio sem nec elit. Donec id elit non mi porta gravida at eg
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