Difference between revisions of "Team:BostonU/Protocols"

Line 211: Line 211:
 
     </table>
 
     </table>
 
   </div>
 
   </div>
−
   <h3 class="inline-heading-type">KOD PCR</h3>
+
   <h3 class="inline-heading-type">INSERT</h3>
 
   <div>
 
   <div>
 
     <p class="body-type">
 
     <p class="body-type">
−
     Nam enim risus, molestie et, porta ac, aliquam ac, risus. Quisque lobortis.
+
     Materials
−
    Phasellus pellentesque purus in massa. Aenean in pede. Phasellus ac libero
+
−
    ac tellus pellentesque semper. Sed ac felis. Sed commodo, magna quis
+
−
    lacinia ornare, quam ante aliquam nisi, eu iaculis leo purus venenatis dui.
+
−
    </p>
+
−
  </div>
+
−
  <h3 class="inline-heading-type">Overlap Extension PCR</h3>
+
−
  <div>
+
−
    <p class="body-type">
+
−
    Nam enim risus, molestie et, porta ac, aliquam ac, risus. Quisque lobortis.
+
−
    Phasellus pellentesque purus in massa. Aenean in pede. Phasellus ac libero
+
−
    ac tellus pellentesque semper. Sed ac felis. Sed commodo, magna quis
+
−
    lacinia ornare, quam ante aliquam nisi, eu iaculis leo purus venenatis dui.
+
 
     </p>
 
     </p>
 
     <ul class="body-type">
 
     <ul class="body-type">
−
       <li>List item one</li>
+
       <li>5% DMSO</li>
−
       <li>List item two</li>
+
       <li>2x KOD Hot Start Master Mix</li>
−
       <li>List item three</li>
+
       <li>PCR Tubes</li>
 +
      <li>Forward Primers(s) [10 uM]</li>
 +
      <li>Reverse Primer(s) [10 uM]</li>
 +
      <li>DNA Template</li>
 +
      <li>Deionized water</li>
 
     </ul>
 
     </ul>
−
  </div>
 
−
  <h3 class="inline-heading-type">Agarose Gel Preparation</h3>
 
−
  <div>
 
 
     <p class="body-type">
 
     <p class="body-type">
−
     Nam enim risus, molestie et, porta ac, aliquam ac, risus. Quisque lobortis.
+
     Procedure
−
    Phasellus pellentesque purus in massa. Aenean in pede. Phasellus ac libero
+
−
    ac tellus pellentesque semper. Sed ac felis. Sed commodo, magna quis
+
−
    lacinia ornare, quam ante aliquam nisi, eu iaculis leo purus venenatis dui.
+
 
     </p>
 
     </p>
−
     <ul class="body-type">
+
     <ol class="body-type" type="1">
−
       <li>List item one</li>
+
       <li>Prepare the DNA template by picking a colony from a plate and mixing it into 10 uL of deionized water in a PCR tube</li>
−
       <li>List item two</li>
+
       <li>Add 17.5 µl of deionized water to another PCR tube</li>
−
       <li>List item three</li>
+
       <li>Add 1.5 µl of both the requisite Forward and Reverse Primers to the second PCR tube</li>
−
    </ul>
+
      <li>Add 2 ul of water containing cells with DNA template sample</li>
−
  </div>
+
      <li>Add 27.5 µl of KOD Hot Start Master Mix/li>
−
  <h3 class="inline-heading-type">Agarose Gel Electrophoresis</h3>
+
      <li>Centrifuge for 10 seconds to remove air bubbles</li>
−
  <div>
+
      <li>Place in Thermocycler</li>
−
     <p class="body-type">
+
     </ol>
−
     Nam enim risus, molestie et, porta ac, aliquam ac, risus. Quisque lobortis.
+
<p class="body-type">
−
    Phasellus pellentesque purus in massa. Aenean in pede. Phasellus ac libero
+
     Thermocycler Conditions
−
    ac tellus pellentesque semper. Sed ac felis. Sed commodo, magna quis
+
−
    lacinia ornare, quam ante aliquam nisi, eu iaculis leo purus venenatis dui.
+
 
     </p>
 
     </p>
−
     <ul class="body-type">
+
     <table class="body-type" frame="box">
−
       <li>List item one</li>
+
       <tr>
−
       <li>List item two</li>
+
        <th>Number of Cycles</th>
−
       <li>List item three</li>
+
        <td>From 20 - 40</td>
−
     </ul>
+
       </tr>
 +
      <tr>
 +
        <th>Annealing Temperature</th>
 +
        <td>Set temperature to the lowest primer melt temperature</td>
 +
       </tr>
 +
      <tr>
 +
        <th>Extension Time</th>
 +
        <td>If target size is: <BR>< 500 bp run 10 sec/kb<BR>500-1000 bp run 15 sec/kb<BR>1000-3000 bp run 20 sec/kb<BR>< 3000 bp run 20 sec/kp</td>
 +
      </tr>
 +
     </table>
 
   </div>
 
   </div>
−
  <h3 class="inline-heading-type">Agarose Gel Extraction</h3>
 
−
  <div>
 
−
    <p class="body-type">
 
−
    Nam enim risus, molestie et, porta ac, aliquam ac, risus. Quisque lobortis.
 
−
    Phasellus pellentesque purus in massa. Aenean in pede. Phasellus ac libero
 
−
    ac tellus pellentesque semper. Sed ac felis. Sed commodo, magna quis
 
−
    lacinia ornare, quam ante aliquam nisi, eu iaculis leo purus venenatis dui.
 
−
    </p>
 
−
    <ul class="body-type">
 
−
      <li>List item one</li>
 
−
      <li>List item two</li>
 
−
      <li>List item three</li>
 
−
    </ul>
 
−
  </div>
 
−
  <h3 class="inline-heading-type">Cell Stock Preparation</h3>
 
−
  <div>
 
−
    <p class="body-type">
 
−
    Nam enim risus, molestie et, porta ac, aliquam ac, risus. Quisque lobortis.
 
−
    Phasellus pellentesque purus in massa. Aenean in pede. Phasellus ac libero
 
−
    ac tellus pellentesque semper. Sed ac felis. Sed commodo, magna quis
 
−
    lacinia ornare, quam ante aliquam nisi, eu iaculis leo purus venenatis dui.
 
−
    </p>
 
−
    <ul class="body-type">
 
−
      <li>List item one</li>
 
−
      <li>List item two</li>
 
−
      <li>List item three</li>
 
−
    </ul>
 
−
  </div>
 
−
  <h3 class="inline-heading-type">PCR Cleanup</h3>
 
−
  <div>
 
−
    <p class="body-type">
 
−
    Nam enim risus, molestie et, porta ac, aliquam ac, risus. Quisque lobortis.
 
−
    Phasellus pellentesque purus in massa. Aenean in pede. Phasellus ac libero
 
−
    ac tellus pellentesque semper. Sed ac felis. Sed commodo, magna quis
 
−
    lacinia ornare, quam ante aliquam nisi, eu iaculis leo purus venenatis dui.
 
−
    </p>
 
−
    <ul class="body-type">
 
−
      <li>List item one</li>
 
−
      <li>List item two</li>
 
−
      <li>List item three</li>
 
−
    </ul>
 
−
  </div>
 
−
  <h3 class="inline-heading-type">Miniprep</h3>
 
−
  <div>
 
−
    <p class="body-type">
 
−
    Nam enim risus, molestie et, porta ac, aliquam ac, risus. Quisque lobortis.
 
−
    Phasellus pellentesque purus in massa. Aenean in pede. Phasellus ac libero
 
−
    ac tellus pellentesque semper. Sed ac felis. Sed commodo, magna quis
 
−
    lacinia ornare, quam ante aliquam nisi, eu iaculis leo purus venenatis dui.
 
−
    </p>
 
−
    <ul class="body-type">
 
−
      <li>List item one</li>
 
−
      <li>List item two</li>
 
−
      <li>List item three</li>
 
−
    </ul>
 
−
  </div>
 
−
    <h3 class="inline-heading-type">Ligation</h3>
 
−
  <div>
 
−
    <p class="body-type">
 
−
    Nam enim risus, molestie et, porta ac, aliquam ac, risus. Quisque lobortis.
 
−
    Phasellus pellentesque purus in massa. Aenean in pede. Phasellus ac libero
 
−
    ac tellus pellentesque semper. Sed ac felis. Sed commodo, magna quis
 
−
    lacinia ornare, quam ante aliquam nisi, eu iaculis leo purus venenatis dui.
 
−
    </p>
 
−
    <ul class="body-type">
 
−
      <li>List item one</li>
 
−
      <li>List item two</li>
 
−
      <li>List item three</li>
 
−
    </ul>
 
−
  </div>
 
−
    <h3 class="inline-heading-type">Transformation</h3>
 
−
  <div>
 
−
    <p class="body-type">
 
−
    Nam enim risus, molestie et, porta ac, aliquam ac, risus. Quisque lobortis.
 
−
    Phasellus pellentesque purus in massa. Aenean in pede. Phasellus ac libero
 
−
    ac tellus pellentesque semper. Sed ac felis. Sed commodo, magna quis
 
−
    lacinia ornare, quam ante aliquam nisi, eu iaculis leo purus venenatis dui.
 
−
    </p>
 
−
    <ul class="body-type">
 
−
      <li>List item one</li>
 
−
      <li>List item two</li>
 
−
      <li>List item three</li>
 
−
    </ul>
 
−
  </div>
 
−
    <h3 class="inline-heading-type">Liquid Culture</h3>
 
−
  <div>
 
−
    <p class="body-type">
 
−
    Nam enim risus, molestie et, porta ac, aliquam ac, risus. Quisque lobortis.
 
−
    Phasellus pellentesque purus in massa. Aenean in pede. Phasellus ac libero
 
−
    ac tellus pellentesque semper. Sed ac felis. Sed commodo, magna quis
 
−
    lacinia ornare, quam ante aliquam nisi, eu iaculis leo purus venenatis dui.
 
−
    </p>
 
−
    <ul class="body-type">
 
−
      <li>List item one</li>
 
−
      <li>List item two</li>
 
−
      <li>List item three</li>
 
−
    </ul>
 
−
  </div>
 
−
    <h3 class="inline-heading-type">Restriction Digestion</h3>
 
−
  <div>
 
−
    <p class="body-type">
 
−
    Nam enim risus, molestie et, porta ac, aliquam ac, risus. Quisque lobortis.
 
−
    Phasellus pellentesque purus in massa. Aenean in pede. Phasellus ac libero
 
−
    ac tellus pellentesque semper. Sed ac felis. Sed commodo, magna quis
 
−
    lacinia ornare, quam ante aliquam nisi, eu iaculis leo purus venenatis dui.
 
−
    </p>
 
−
    <ul class="body-type">
 
−
      <li>List item one</li>
 
−
      <li>List item two</li>
 
−
      <li>List item three</li>
 
−
    </ul>
 
−
  </div>
 
−
    <h3 class="inline-heading-type">Test Cut Digestion</h3>
 
−
  <div>
 
−
    <p class="body-type">
 
−
    Nam enim risus, molestie et, porta ac, aliquam ac, risus. Quisque lobortis.
 
−
    Phasellus pellentesque purus in massa. Aenean in pede. Phasellus ac libero
 
−
    ac tellus pellentesque semper. Sed ac felis. Sed commodo, magna quis
 
−
    lacinia ornare, quam ante aliquam nisi, eu iaculis leo purus venenatis dui.
 
−
    </p>
 
−
    <ul class="body-type">
 
−
      <li>List item one</li>
 
−
      <li>List item two</li>
 
−
      <li>List item three</li>
 
−
    </ul>
 
−
  </div>
 
−
    <h3 class="inline-heading-type">Gibson Assembly</h3>
 
−
  <div>
 
−
    <p class="body-type">
 
−
    Nam enim risus, molestie et, porta ac, aliquam ac, risus. Quisque lobortis.
 
−
    Phasellus pellentesque purus in massa. Aenean in pede. Phasellus ac libero
 
−
    ac tellus pellentesque semper. Sed ac felis. Sed commodo, magna quis
 
−
    lacinia ornare, quam ante aliquam nisi, eu iaculis leo purus venenatis dui.
 
−
    </p>
 
−
    <ul class="body-type">
 
−
      <li>List item one</li>
 
−
      <li>List item two</li>
 
−
      <li>List item three</li>
 
−
    </ul>
 
−
  </div>
 
−
  <h3 class="inline-heading-type">Recombination Reaction</h3>
 
−
  <div>
 
−
    <p class="body-type">
 
−
    Nam enim risus, molestie et, porta ac, aliquam ac, risus. Quisque lobortis.
 
−
    Phasellus pellentesque purus in massa. Aenean in pede. Phasellus ac libero
 
−
    ac tellus pellentesque semper. Sed ac felis. Sed commodo, magna quis
 
−
    lacinia ornare, quam ante aliquam nisi, eu iaculis leo purus venenatis dui.
 
−
    </p>
 
−
    <ul class="body-type">
 
−
      <li>List item one</li>
 
−
      <li>List item two</li>
 
−
      <li>List item three</li>
 
−
    </ul>
 
−
  </div><h3 class="inline-heading-type">Cell-Free TX-TL Reaction</h3>
 
−
  <div>
 
−
    <p class="body-type">
 
−
    Nam enim risus, molestie et, porta ac, aliquam ac, risus. Quisque lobortis.
 
−
    Phasellus pellentesque purus in massa. Aenean in pede. Phasellus ac libero
 
−
    ac tellus pellentesque semper. Sed ac felis. Sed commodo, magna quis
 
−
    lacinia ornare, quam ante aliquam nisi, eu iaculis leo purus venenatis dui.
 
−
    </p>
 
−
    <ul class="body-type">
 
−
      <li>List item one</li>
 
−
      <li>List item two</li>
 
−
      <li>List item three</li>
 
−
    </ul>
 
−
  </div><h3 class="inline-heading-type">Cell-Free TX-TL Master Mix Preparation</h3>
 
−
  <div>
 
−
    <p class="body-type">
 
−
    Nam enim risus, molestie et, porta ac, aliquam ac, risus. Quisque lobortis.
 
−
    Phasellus pellentesque purus in massa. Aenean in pede. Phasellus ac libero
 
−
    ac tellus pellentesque semper. Sed ac felis. Sed commodo, magna quis
 
−
    lacinia ornare, quam ante aliquam nisi, eu iaculis leo purus venenatis dui.
 
−
    </p>
 
−
    <ul class="body-type">
 
−
      <li>List item one</li>
 
−
      <li>List item two</li>
 
−
      <li>List item three</li>
 
−
    </ul>
 
−
    </div>
 
 
</div>
 
</div>
 
</div>
 
</div>

Revision as of 16:04, 28 October 2017

OUR TEAM

Below is an interactive list of the laboratory techniques employed by the team. Click to expand for details.

Colony PCR

Materials

  • 5% DMSO
  • 2x KOD Hot Start Master Mix
  • PCR Tubes
  • Forward Primers(s) [10 uM]
  • Reverse Primer(s) [10 uM]
  • DNA Template
  • Deionized water

Procedure

  1. Add 18.5 uL of deionized water to PCR tube
  2. Add 1.5 µl of both the requisite Forward and Reverse Primers to PCR tube
  3. Add 1 ul of DNA template
  4. Add 27.5 µl of KOD Hot start Master Mix to PCR tube
  5. Centrifuge for 10 seconds to remove air bubbles
  6. Place in Thermocycler

Thermocycler Conditions

Number of Cycles From 20 - 40
Annealing Temperature Set temperature to the lowest primer melt temperature
Extension Time If target size is:
< 500 bp run 10 sec/kb
500-1000 bp run 15 sec/kb
1000-3000 bp run 20 sec/kb
< 3000 bp run 20 sec/kp

INSERT

Materials

  • 5% DMSO
  • 2x KOD Hot Start Master Mix
  • PCR Tubes
  • Forward Primers(s) [10 uM]
  • Reverse Primer(s) [10 uM]
  • DNA Template
  • Deionized water

Procedure

  1. Prepare the DNA template by picking a colony from a plate and mixing it into 10 uL of deionized water in a PCR tube
  2. Add 17.5 µl of deionized water to another PCR tube
  3. Add 1.5 µl of both the requisite Forward and Reverse Primers to the second PCR tube
  4. Add 2 ul of water containing cells with DNA template sample
  5. Add 27.5 µl of KOD Hot Start Master Mix/li>
  6. Centrifuge for 10 seconds to remove air bubbles
  7. Place in Thermocycler

Thermocycler Conditions

Number of Cycles From 20 - 40
Annealing Temperature Set temperature to the lowest primer melt temperature
Extension Time If target size is:
< 500 bp run 10 sec/kb
500-1000 bp run 15 sec/kb
1000-3000 bp run 20 sec/kb
< 3000 bp run 20 sec/kp