Difference between revisions of "Team:NYU Shanghai/Protocols"

 
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<div id="overview">
 
<div id="overview">
 
   <h3>Protocols</h3>
 
   <h3>Protocols</h3>
−
  <p style="font-size: 17px">We built our constructs from pre-made biobrick parts. Our overall conclusion is that 3A assembly is generally inefficient, and an insufficient method for adding small parts (such as a terminator) to a larger construction within pSB1C3. We learned that ratios were extremely important in the process of 3A Assembly, and we made a summary sheet of the equations we used in pre-digest and pre-ligation that accounts for digest dilution and amount needed to ensure results are seen on a gel, not just ligation ratios. We wished we used gibson assembly. </p>
+
  <p style="font-size: 17px">We built our constructs from pre-made biobrick parts. Our overall conclusion is that 3A assembly is generally inefficient, and an insufficient method for adding small parts (such as a terminator) to a larger construction within pSB1C3. We learned that ratios were extremely important in the process of 3A Assembly, and we made a <a href="https://static.igem.org/mediawiki/2015/c/c0/NYU_Shanghai_3AAssemblyNumbers.pdf">summary sheet</a> of the equations we used in pre-digest and pre-ligation that accounts for digest dilution and amount needed to ensure results are seen on a gel, not just ligation ratios. We wished we used gibson assembly. </p>
 
<br>
 
<br>
 +
</div>
 +
<div id="makingColor">
 +
  <h4>Making Color</h4>
 +
 +
<div id="luc" class="collapsed">
 +
<h5 style="display:inline-block" onclick="expandluc()"><span class="noselect">Luciferase</span></h5>
 +
<div id="lucText" style="display:none">
 +
  <br>
 +
  <p><img width="800" src="https://static.igem.org/mediawiki/2015/1/12/NYU_Shanghai_Luciferase_Protein.png">
 +
  <br>
 +
    Overview
 +
    <ol>
 +
      <li>Luciferin substrate must be added.</li>
 +
      <li>D-Luciferin is too large of a chemical to cross the plasma membrane of E. Coli so cell lysis is required to extract luciferase.</li>
 +
      <li>After cell lysis, the reagent solution can be added to the lysis buffer. Light should be emitted within 5 to 10 seconds of adding the reagent solution.</li>
 +
      <li>The luciferase/luciferin reaction at 22.5 ºC theoretically offers the greatest light intensity.</li>
 +
      <li>Solutions of D-Luciferin should be aliquotted and stored in darkness at -80 ºC</li>
 +
      <li>We were only able to see the color in a very dark room.</li>
 +
    </ol>
 +
    </p>
 +
    <p>
 +
    <br>Materials
 +
      <li>D-Luciferin free acid</li>
 +
      <li>ATP</li>
 +
      <li>MgSO4 &#xB7; 7H2O</li>
 +
      <li>1M HEPES Buffer</li>
 +
      <li>Lysozyme</li>
 +
      <li>10 mM Tris-HCl</li>
 +
    </p>
 +
    <p>
 +
    <br>Lysis Buffer
 +
    <br>For E. coli cell lysis, use a freshly prepared lysozyme solution (10 mg/ml) in 10 mM Tris-HCl, pH 8.0.
 +
    </p>
 +
    <p>
 +
    <br>Reagent Solution
 +
    <br>Prepare using ATP free water. Combine 1 mM luciferin or luciferin salt, 3 mM ATP and 15 mM MgSO4 in 30mM HEPES buffer, pH 7.8.  Store substrate solution at -20ºC in polypropylene or glass.
 +
    </p>
 +
    <p>
 +
    <br>Preparing 1mM D-Luciferin
 +
    <br>Directions for a 5 mg sample: Dissolve 0.034 gr dithiothreitol in 22 mL of QH2O. Add 0.2 mL of this DTT solution to the 5 mg d-luciferin. Add 4μl of 10M NaOH to dissolve the luciferin. Dilute this into the remaining 21.8 mL of DTT solution and store as aliquots at - 80 ºC in darkness until use.
 +
    </p>
 +
    <p>
 +
    <br>Procedure<br>
 +
    Bacterial lysis:
 +
    <ol>
 +
      <li>After 12-18 hours of inoculation of bacteria expressing luciferase plasmid, pipette 2 mL of cell culture into a clean 2 mL tube. Centrifuge at 10,000 rpm for 1 minute. Pour out liquid into a collection beaker. Continue this process until all of the cell culture (in the inoculation tube) is gone.</li>
 +
      <li>Resuspend the pellets in 350 ml of STET buffer (10 mM Tris-HCl with 1 mM EDTA)</li>
 +
      <li>Add 25μl - 30μl of lysozyme buffer to the resuspended pellet.</li>
 +
      <li>Mix by vortexing for 3 seconds.</li>
 +
      <li>Incubate for 2 hours at room temperature.</li>
 +
      <li>If the reagent is not added immediately, store the lysed bacteria in the -20 ºC freezer until use.</li>
 +
    </ol>
 +
    </p>
 +
    <p>
 +
    Addition of Reagent Solution:
 +
    <ol>
 +
      <li>Following the above instructions, prepare a 1mM sample of D-Luciferin.</li>
 +
      <li>Following the above recipe, prepare the reagent solution.</li>
 +
      <li>In a dark room, add about 250-350μl of reagent solution to each sample of lysis product.</li>
 +
      <li>Light should be emitted within two-three seconds.</li>
 +
    </ol>
 +
    </p>
 +
    <p>
 +
    <br>Example Calculations<br>
 +
    Lysis Buffer (Desired Total Volume: 15mL)<br>
 +
    <table border="1">
 +
      <tr>
 +
        <td><font color="#d66">Chemical Name</font></td>
 +
        <td>Tris-HCl</td>
 +
        <td>EDTA</td>
 +
        <td>NaCl</td>
 +
      </tr>
 +
      <tr>
 +
        <td><font color="#d66">Molecular Weight</font></td>
 +
        <td>N/A</td>
 +
        <td>292.23 g/mol</td>
 +
        <td>58.44 g/mol</td>
 +
      </tr>
 +
      <tr>
 +
        <td><font color="#d66">Molarity Desired</font></td>
 +
        <td>10 mM</td>
 +
        <td>1mM</td>
 +
        <td>0.1M</td>
 +
      </tr>
 +
      <tr>
 +
        <td><font color="#d66">Calculation</font></td>
 +
        <td>Dilute 1M Tris-HCl:</td>
 +
        <td> </td>
 +
        <td> </td>
 +
      </tr>
 +
      <tr>
 +
        <td><font color="#d66">Final Amount</font></td>
 +
        <td>150μl (+14.85 mL ddH2O)</td>
 +
        <td>0.00438 g</td>
 +
        <td>0.08766 g</td>
 +
      </tr>
 +
    </table>
 +
    </p>
 +
    <p>
 +
    Lysozyme Solution (Desired Total Volume: 15mL)<br>
 +
    <table border="1">
 +
      <tr>
 +
        <td><font color="#d66">Lysozyme Solubility</font> </td>
 +
        <td>10 mg lysozyme in 1 mL of 10 mM Tris-HCl</td>
 +
      </tr>
 +
      <tr>
 +
        <td><font color="#d66">Desired Amount of Lysozyme Solution to Make</font></td>
 +
        <td>15 mL</td>
 +
      </tr>
 +
      <tr>
 +
        <td><font color="#d66">Amount of Lysozyme Needed</font></td>
 +
        <td>10 mg x 15 = <b>150 mg</b></td>
 +
      </tr>
 +
      <tr>
 +
        <td><font color="#d66">Amount of 10 mM Tris-HCl Needed</font> </td>
 +
        <td><b>15 mL</b></td>
 +
      </tr>
 +
    </table>
 +
    </p>
 +
    <p>
 +
    Reagent Solution (Desired total volume: 22 mL)<br>
 +
    <table border="1">
 +
      <tr>
 +
        <td><font color="#d66">Chemical Name</font></td>
 +
        <td>ATP disodium salt trihydrate</td>
 +
        <td>MgSO4•7H2O</td>
 +
        <td>HEPES Buffer</td>
 +
        <td>D-Luciferin free acid</td>
 +
      </tr>
 +
      <tr>
 +
        <td><font color="#d66">Molecular Weight</font></td>
 +
        <td>605.24 g/mol</td>
 +
        <td>246.5 g/mol</td>
 +
        <td>238.3 g/mol</td>
 +
        <td>280.33 g/mol</td>
 +
      </tr>
 +
      <tr>
 +
        <td><font color="#d66">Molarity Desired</font></td>
 +
        <td>3 mM</td>
 +
        <td>15 mM</td>
 +
        <td>30 mM</td>
 +
        <td>1 mM</td>
 +
      </tr>
 +
      <tr>
 +
        <td><font color="#d66">Calculation</font></td>
 +
        <td> </td>
 +
        <td> </td>
 +
        <td> </td>
 +
        <td>Use the 1 mM stock solution created earlier</td>
 +
      </tr>
 +
      <tr>
 +
        <td><font color="#d66">Final Amount</font></td>
 +
        <td>0.039945 g</td>
 +
        <td>0.081345 g</td>
 +
        <td>0.1573 g</td>
 +
        <td>22 mL</td>
 +
      </tr>
 +
    </table>
 +
    </p>
 +
    <p>
 +
    Controls
 +
    <li>No arabinose added during inoculation</li>
 +
    <li>Use bacteria without luciferase plasmid and go through steps to induce color</li>
 +
    </p>
 +
    </p>
 +
  </div>
 +
</div>
 +
 +
<div id="chromo" class="collapsed">
 +
<h5 style="display:inline-block" onclick="expandchromo()"><span class="noselect">Chromoproteins</span></h5> 
 +
<br>
 +
<div id="chromoText" style="display:none">
 +
    <p>
 +
    <h6><font color="#d66">Building our Construct: from biobrick parts in the kit</font></h6>
 +
    <p>Note: If using construct with pBAD promoter, DO NOT USE SOC MEDIA. Glucose inhibits the uptake of arabinose, and will inhibit promoter induction.
 +
    <br>Note: We should have used PCR to amplify linearized backbone.
 +
    <br>Note: Always use gel electrophoresis to check digest results.</p>
 +
    <br><img src="https://static.igem.org/mediawiki/2015/d/df/NYU_Shanghai_Chromo_Procedure.png" width="800">
 +
    <br><br><br>
 +
    <h6><font color="#d66">Building our Construct: from IDT gBlocks</font></h6>
 +
    <p>Note: We recommend adding a reporter gene to the construct.</p>
 +
    <br><img src="https://static.igem.org/mediawiki/2015/7/71/NYU_Shanghai_IDTprocedure.png" width="550">
 +
    <br><br><br>
 +
    <h6><font color="#d66">Expressing XJTLU's Construct</font></h6>
 +
    <br><img src="https://static.igem.org/mediawiki/2015/thumb/9/9a/NYU_Shanghai_Chromo_Procedure_2.png/573px-NYU_Shanghai_Chromo_Procedure_2.png">
 +
    </p>
 +
</div>
 +
</div>
 
</div>
 
</div>
  
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<div id="3a">
 
<div id="3a">
 
   <h4>3A Assembly</h4>
 
   <h4>3A Assembly</h4>
 +
<div id="calculations">
 +
  <h5><span><a href="https://static.igem.org/mediawiki/2015/c/c0/NYU_Shanghai_3AAssemblyNumbers.pdf">Calculations (pdf)</a></span></h5>
 
<div id="transform" class="collapsed">
 
<div id="transform" class="collapsed">
 
  <h5 style="display:inline-block" onclick="expandtransform()"><span class="noselect">Transformation</span></h5>   
 
  <h5 style="display:inline-block" onclick="expandtransform()"><span class="noselect">Transformation</span></h5>   
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     </table>
 
     </table>
 
     </p>
 
     </p>
 +
  <p>Controls
 +
  <li>DNA with known sites for the enzyme</li>
 +
  <li>If control DNA cleaved and experimental DNA resists cleavage, the two DNAs can be mixed to determine if an inhibitor is present in the experimental sample</li>
 +
  </p>
 
</div>
 
</div>
 
</div>
 
</div>
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       <li>Visualize the gel and record the results.</li>
 
       <li>Visualize the gel and record the results.</li>
 
     </ol>
 
     </ol>
 +
 +
    <br><p>Controls
 +
      <li>Uncut plasmid</li>
 +
      <li>Uncut insert DNA</li>
 +
      <li>Ladder DNA</li>
 +
    </p>
 
</div>
 
</div>
 
</div>
 
</div>
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     </ol>
 
     </ol>
 
     <br>
 
     <br>
 +
  </p>
 +
  <p>
 +
  Controls
 +
  <li>"Digest empty vector cut with a single enzyme, perform the gel extraction, and re-ligate it. A vector cut with one enzyme should re-ligate very easily and provide plenty of colonies on the plate. If it does, then the inability to clone the DNA may be related to some other factor, such as secondary structure of the DNA, repeat sequences causing instability in E.coli, or the DNA cloned codes for a protein that may be toxic in bacteria." <a href="http://bitesizebio.com/13506/10-tips-for-better-dna-gel-extraction-results/">Bitesize Bio</a>
 
   </p>
 
   </p>
 
</div>
 
</div>
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       </tr>
 
       </tr>
 
     </table>
 
     </table>
−
  </p>
+
     <br>
−
  <p>Controls
+
     <table>
−
  <li>No template control</li>
+
     <tr>
−
  <li>No enzyme control</li>
+
          <td><font color="#d66">Initial Denaturation</font></td>
−
  </p>
+
          <td>98C</td>
−
</div>
+
          <td>30s</td>
−
</div>
+
−
 
+
−
<div id="PCRclean" class="collapsed">
+
−
<h5 style="display:inline-block" onclick="expandPCRclean()"><span class="noselect">PCR Cleanup</span></h5> 
+
−
<br>
+
−
<div id="PCRcleanText" style="display:none">
+
−
     <p>We used <a href="https://static.igem.org/mediawiki/2015/d/d1/NYU_Shanghai_Tianquick.pdf">TIANquick Mini Purification Kit</a>.
+
−
</div>
+
−
</div>
+
−
</div>
+
−
 
+
−
<div id="makingColor">
+
−
  <h4>Making Color</h4>
+
−
 
+
−
<div id="luc" class="collapsed">
+
−
<h5 style="display:inline-block" onclick="expandluc()"><span class="noselect">Luciferase</span></h5>
+
−
<br>
+
−
<p><img id="lucText" style="display:none" width="800" src="https://static.igem.org/mediawiki/2015/1/12/NYU_Shanghai_Luciferase_Protein.png"></p>
+
−
<br>
+
−
<div id="lucText" style="display:none">
+
−
     <p>
+
−
    Overview
+
−
     <ol>
+
−
      <li>Luciferin substrate must be added.</li>
+
−
      <li>D-Luciferin is too large of a chemical to cross the plasma membrane of E. Coli so cell lysis is required to extract luciferase.</li>
+
−
      <li>After cell lysis, the reagent solution can be added to the lysis buffer. Light should be emitted within 5 to 10 seconds of adding the reagent solution.</li>
+
−
      <li>The luciferase/luciferin reaction at 22.5 ºC theoretically offers the greatest light intensity.</li>
+
−
      <li>Solutions of D-Luciferin should be aliquotted and stored in darkness at -80 ºC</li>
+
−
    </ol>
+
−
    </p>
+
−
    <p>
+
−
    <br>Materials
+
−
      <li>D-Luciferin free acid</li>
+
−
      <li>ATP</li>
+
−
      <li>MgSO4 &#xB7; 7H2O</li>
+
−
      <li>1M HEPES Buffer</li>
+
−
      <li>Lysozyme</li>
+
−
      <li>10 mM Tris-HCl</li>
+
−
    </p>
+
−
    <p>
+
−
    <br>Lysis Buffer
+
−
    <br>For E. coli cell lysis, use a freshly prepared lysozyme solution (10 mg/ml) in 10 mM Tris-HCl, pH 8.0.
+
−
    </p>
+
−
    <p>
+
−
    <br>Reagent Solution
+
−
    <br>Prepare using ATP free water. Combine 1 mM luciferin or luciferin salt, 3 mM ATP and 15 mM MgSO4 in 30mM HEPES buffer, pH 7.8.  Store substrate solution at -20ºC in polypropylene or glass.
+
−
    </p>
+
−
    <p>
+
−
    <br>Preparing 1mM D-Luciferin
+
−
    <br>Directions for a 5 mg sample: Dissolve 0.034 gr dithiothreitol in 22 mL of QH2O. Add 0.2 mL of this DTT solution to the 5 mg d-luciferin. Add 4μl of 10M NaOH to dissolve the luciferin. Dilute this into the remaining 21.8 mL of DTT solution and store as aliquots at - 80 ºC in darkness until use.
+
−
    </p>
+
−
    <p>
+
−
    <br>Procedure<br>
+
−
    Bacterial lysis:
+
−
    <ol>
+
−
      <li>After 12-18 hours of inoculation of bacteria expressing luciferase plasmid, pipette 2 mL of cell culture into a clean 2 mL tube. Centrifuge at 10,000 rpm for 1 minute. Pour out liquid into a collection beaker. Continue this process until all of the cell culture (in the inoculation tube) is gone.</li>
+
−
      <li>Resuspend the pellets in 350 ml of STET buffer (10 mM Tris-HCl with 1 mM EDTA)</li>
+
−
      <li>Add 25μl - 30μl of lysozyme buffer to the resuspended pellet.</li>
+
−
      <li>Mix by vortexing for 3 seconds.</li>
+
−
      <li>Incubate for 2 hours at room temperature.</li>
+
−
      <li>If the reagent is not added immediately, store the lysed bacteria in the -20 ºC freezer until use.</li>
+
−
    </ol>
+
−
    </p>
+
−
    <p>
+
−
    Addition of Reagent Solution:
+
−
    <ol>
+
−
      <li>Following the above instructions, prepare a 1mM sample of D-Luciferin.</li>
+
−
      <li>Following the above recipe, prepare the reagent solution.</li>
+
−
      <li>In a dark room, add about 250-350μl of reagent solution to each sample of lysis product.</li>
+
−
      <li>Light should be emitted within two-three seconds.</li>
+
−
    </ol>
+
−
    </p>
+
−
    <p>
+
−
    <br>Example Calculations<br>
+
−
    Lysis Buffer (Desired Total Volume: 15mL)<br>
+
−
    <table border="1">
+
−
      <tr>
+
−
        <td><font color="#d66">Chemical Name</font></td>
+
−
        <td>Tris-HCl</td>
+
−
        <td>EDTA</td>
+
−
        <td>NaCl</td>
+
 
       </tr>
 
       </tr>
 
       <tr>
 
       <tr>
−
        <td><font color="#d66">Molecular Weight</font></td>
+
          <td><font color="#d66">25 cycles</font></td>
−
        <td>N/A</td>
+
          <td>98C</td>
−
        <td>292.23 g/mol</td>
+
          <td>15s</td>
−
        <td>58.44 g/mol</td>
+
 
       </tr>
 
       </tr>
 
       <tr>
 
       <tr>
−
        <td><font color="#d66">Molarity Desired</font></td>
+
          <td>Annealing temp 1</td>
−
        <td>10 mM</td>
+
          <td>59.5C</td>
−
        <td>1mM</td>
+
          <td>30s</td>
−
        <td>0.1M</td>
+
 
       </tr>
 
       </tr>
 
       <tr>
 
       <tr>
−
        <td><font color="#d66">Calculation</font></td>
+
          <td>Annealing temp 2</td>
−
        <td>Dilute 1M Tris-HCl:</td>
+
          <td>56.3C</td>
−
        <td> </td>
+
          <td>30s</td>
−
        <td> </td>
+
 
       </tr>
 
       </tr>
 
       <tr>
 
       <tr>
−
        <td><font color="#d66">Final Amount</font></td>
+
          <td>Annealing temp 3</td>
−
        <td>150μl (+14.85 mL ddH2O)</td>
+
          <td>53.7C</td>
−
        <td>0.00438 g</td>
+
          <td>30s</td>
−
        <td>0.08766 g</td>
+
 
       </tr>
 
       </tr>
−
    </table>
 
−
    </p>
 
−
    <p>
 
−
    Lysozyme Solution (Desired Total Volume: 15mL)<br>
 
−
    <table border="1">
 
 
       <tr>
 
       <tr>
−
        <td><font color="#d66">Lysozyme Solubility</font> </td>
+
          <td>Extension</td>
−
        <td>10 mg lysozyme in 1 mL of 10 mM Tris-HCl</td>
+
          <td>72C</td>
 +
          <td>60s</td>
 
       </tr>
 
       </tr>
 
       <tr>
 
       <tr>
−
        <td><font color="#d66">Desired Amount of Lysozyme Solution to Make</font></td>
+
          <td><font color="#d66">Final Extension</font></td>
−
        <td>15 mL</td>
+
          <td>72C</td>
 +
          <td>2m</td>
 
       </tr>
 
       </tr>
 
       <tr>
 
       <tr>
−
        <td><font color="#d66">Amount of Lysozyme Needed</font></td>
+
          <td><font color="#d66">Hold</font></td>
−
        <td>10 mg x 15 = <b>150 mg</b></td>
+
          <td>4C</td>
−
      </tr>
+
          <td></td>
−
      <tr>
+
−
        <td><font color="#d66">Amount of 10 mM Tris-HCl Needed</font> </td>
+
−
        <td><b>15 mL</b></td>
+
 
       </tr>
 
       </tr>
 
     </table>
 
     </table>
−
    </p>
+
  </p>
−
    <p>
+
  <p>Controls
−
    Reagent Solution (Desired total volume: 22 mL)<br>
+
  <li>No template control</li>
−
    <table border="1">
+
  <li>No enzyme control</li>
−
      <tr>
+
  </p>
−
        <td><font color="#d66">Chemical Name</font></td>
+
</div>
−
        <td>ATP disodium salt trihydrate</td>
+
−
        <td>MgSO4•7H2O</td>
+
−
        <td>HEPES Buffer</td>
+
−
        <td>D-Luciferin free acid</td>
+
−
      </tr>
+
−
      <tr>
+
−
        <td><font color="#d66">Molecular Weight</font></td>
+
−
        <td>605.24 g/mol</td>
+
−
        <td>246.5 g/mol</td>
+
−
        <td>238.3 g/mol</td>
+
−
        <td>280.33 g/mol</td>
+
−
      </tr>
+
−
      <tr>
+
−
        <td><font color="#d66">Molarity Desired</font></td>
+
−
        <td>3 mM</td>
+
−
        <td>15 mM</td>
+
−
        <td>30 mM</td>
+
−
        <td>1 mM</td>
+
−
      </tr>
+
−
      <tr>
+
−
        <td><font color="#d66">Calculation</font></td>
+
−
        <td> </td>
+
−
        <td> </td>
+
−
        <td> </td>
+
−
        <td>Use the 1 mM stock solution created earlier</td>
+
−
      </tr>
+
−
      <tr>
+
−
        <td><font color="#d66">Final Amount</font></td>
+
−
        <td>0.039945 g</td>
+
−
        <td>0.081345 g</td>
+
−
        <td>0.1573 g</td>
+
−
        <td>22 mL</td>
+
−
      </tr>
+
−
    </table>
+
−
    </p>
+
−
    </p>
+
−
  </div>
+
 
</div>
 
</div>
  
−
<div id="chromo" class="collapsed">
+
<div id="PCRclean" class="collapsed">
−
  <h5 style="display:inline-block" onclick="expandchromo()"><span class="noselect">Chromoproteins</span></h5>   
+
  <h5 style="display:inline-block" onclick="expandPCRclean()"><span class="noselect">PCR Cleanup</span></h5>   
 
  <br>
 
  <br>
−
  <div id="chromoText" style="display:none">
+
  <div id="PCRcleanText" style="display:none">
−
     <p>
+
     <p>We used <a href="https://static.igem.org/mediawiki/2015/d/d1/NYU_Shanghai_Tianquick.pdf">TIANquick Mini Purification Kit</a>.
−
    <img src="https://static.igem.org/mediawiki/2015/d/df/NYU_Shanghai_Chromo_Procedure.png" width="700">
+
    <ol>
 +
    <li>Add ethanol (96-100%) to Buffer PW before use (see bottle label
 +
for volume).</li>
 +
    <li>Column equilibration: add 500μl Buffer BL to the Spin Column CB1 (put Spin Column CB1 into a collection tube). Centrifuge for 1min at 12,000 rpm. Discard the flow-through, and then place Spin Column CB1 back into the collection tube.</li>
 +
    <li>Add 5 volumes of Buffer PB to 1 volume of the PCR reaction or enzymatic reaction and mix.</li>
 +
    <li>Transfer the mixture to the Spin Column CB1, incubate at room temperature for 2min. Centrifuge for 30-60s at 12,000rpm. Discard the flow-through, and then place Spin Column CB1 back into the same collection tube.
 +
    <br>The maximum loading volume of the column is 800μl. For sample volumes greater than 800 μl simply load again.</li>
 +
    <li>Add 600 μl Buffer PW (ensure that ethanol has been added) to the Spin Column CB1 and centrifuge for 30-60s at 12,000 rpm. Discard the flow-through, and place Spin Column CB1 back in the same collection tube.
 +
    <br>Note: If the purified DNA is used for the subsequent salt sensitive experiments, such as ligation or sequencing experiment, it is suggested to stand for 2-5min after adding Buffer PW, and then centrifuge.</li>
 +
    <li>Repeat step 4.</li>
 +
    <li>Centrifuge at 12,000 rpm for 2min to remove residual Buffer PW. Discard the flow-through, and allow the column to air dry with the cap open for several minutes to dry the membrane.</li>
 +
    <li>Place the Spin Column CB1 in a clean 1.5ml microcentrifuge tube. Add 20μl Buffer EB to the center of membrane, incubate for 2min, and centrifuge for 2min at 12,000 rpm</li>
 +
    </ol>
 
     </p>
 
     </p>
 
</div>
 
</div>

Latest revision as of 16:56, 18 September 2015

Protocols

We built our constructs from pre-made biobrick parts. Our overall conclusion is that 3A assembly is generally inefficient, and an insufficient method for adding small parts (such as a terminator) to a larger construction within pSB1C3. We learned that ratios were extremely important in the process of 3A Assembly, and we made a summary sheet of the equations we used in pre-digest and pre-ligation that accounts for digest dilution and amount needed to ensure results are seen on a gel, not just ligation ratios. We wished we used gibson assembly.


Making Color

Recipes

3A Assembly

Calculations (pdf)