Difference between revisions of "Team:CHINA CD UESTC/Protocol"

Line 100: Line 100:
 
<h4>Biography</h4>
 
<h4>Biography</h4>
 
<p>Morbi rutrum, elit ac fermentum egestas, tortor ante vestibulum est, eget scelerisque nisl velit eget tellus. Fusce porta facilisis luctus. Integer neque dolor, rhoncus nec euismod eget, pharetra et tortor. Nulla id pulvinar nunc. Vestibulum auctor nisl vel lectus ullamcorper sed pellentesque dolor eleifend. Praesent lobortis magna vel diam mattis sagittis.</p>
 
<p>Morbi rutrum, elit ac fermentum egestas, tortor ante vestibulum est, eget scelerisque nisl velit eget tellus. Fusce porta facilisis luctus. Integer neque dolor, rhoncus nec euismod eget, pharetra et tortor. Nulla id pulvinar nunc. Vestibulum auctor nisl vel lectus ullamcorper sed pellentesque dolor eleifend. Praesent lobortis magna vel diam mattis sagittis.</p>
−
<p>Mauris porta odio eu risus scelerisque id facilisis ipsum dictum vitae volutpat. Lorem ipsum dolor sit amet, consectetur adipiscing elit. Sed pulvinar neque eu purus sollicitudin et sollicitudin dui ultricies. Maecenas cursus auctor tellus sit amet blandit. Maecenas a erat ac nibh molestie interdum. Class aptent taciti sociosqu ad litora torquent per conubia nostra, per inceptos himenaeos. Sed lorem enim, ultricies sed sodales id, convallis molestie ipsum. Morbi eget dolor ligula. Vivamus accumsan rutrum nisi nec elementum. Pellentesque at nunc risus. Phasellus ullamcorper bibendum varius.  Quisque quis ligula sit amet felis ornare porta. Aenean viverra lacus et mi elementum mollis. Praesent eu justo elit.</p>
 
 
</div>
 
</div>
  
Line 120: Line 119:
 
     </ul>
 
     </ul>
 
   </div>
 
   </div>
−
  <script type="text/javascript">
+
 
−
    function showprot(pid){
+
−
      $(".protocolactive").each(function() {
+
−
        $(this).addClass("protocol");
+
−
        $(this).removeClass("protocolactive");
+
−
      });
+
−
      $(pid).addClass("protocolactive");
+
−
    }
+
−
   
+
−
    document.onkeydown = function(evt) {
+
−
      evt = evt || window.event;
+
−
      switch (evt.keyCode) {
+
−
          case 37:
+
−
              leftArrowPressed();
+
−
              break;
+
−
          case 39:
+
−
            rightArrowPressed();
+
−
            break;
+
−
      }
+
−
  };
+
−
  </script>
+
 
   <div class="rightside">
 
   <div class="rightside">
 
     <div id="prot1" class="protocolactive">
 
     <div id="prot1" class="protocolactive">
Line 210: Line 189:
 
       </ol></p>
 
       </ol></p>
 
     </div>
 
     </div>
−
 
−
<!-- Row Highlight Javascript -->
 
−
<script type="text/javascript">
 
−
  window.onload=function(){
 
−
  var tfrow = document.getElementById('retable').rows.length;
 
−
  var tbRow=[];
 
−
  for (var i=1;i<tfrow;i++) {
 
−
    tbRow[i]=document.getElementById('retable').rows[i];
 
−
    tbRow[i].onmouseover = function(){
 
−
    this.style.backgroundColor = '#f3f8aa';
 
−
    };
 
−
    tbRow[i].onmouseout = function() {
 
−
    this.style.backgroundColor = '#ffffff';
 
−
    };
 
−
    }
 
−
    };
 
−
    </script>
 
−
 
−
<center>
 
−
  <table id="retable" class="tftable" border="1">
 
−
    <tr><th>Reagent</th><th>Volume (ul)</th></tr>
 
−
    <tr><td>Forward Primer</td><td>1.0</td></tr>
 
−
    <tr><td>Reverse Primer</td><td>1.0</td></tr>
 
−
    <tr><td>Template DNA</td><td>2.0</td></tr>
 
−
    <tr><td>Quick-Load Taq 2x Master Mix</td><td>10</td></tr>
 
−
    <tr><td>Nuclease-free water</td><td>6</td></tr>
 
−
    <tr><td>Total Volume</td><td>20</td></tr>
 
−
  </table>
 
−
</center>
 
−
<div style="float:right;font-size:10px;margin-right:5%;">Created with the <a href="http://www.textfixer.com/html/html-table-generator.php" target="_blank">HTML Table Generator</a></div>
 
−
<div style="clear: both;"></div>
 
−
      </p>
 
−
      <h3> Thermocycler Protocol: NEB Quick-Load</h3>
 
−
<script type="text/javascript">
 
−
  window.onload=function(){
 
−
  var tfrow = document.getElementById('nebtable').rows.length;
 
−
  var tbRow=[];
 
−
  for (var i=1;i<tfrow;i++) {
 
−
    tbRow[i]=document.getElementById('nebtable').rows[i];
 
−
    tbRow[i].onmouseover = function(){
 
−
    this.style.backgroundColor = '#f3f8aa';
 
−
    };
 
−
    tbRow[i].onmouseout = function() {
 
−
    this.style.backgroundColor = '#ffffff';
 
−
    };
 
−
    }
 
−
    };
 
−
    </script>
 
 
     </div>
 
     </div>
  
Line 277: Line 208:
 
</div>
 
</div>
 
</div>
 
</div>
−
 
+
  <script type="text/javascript">
 +
    function showprot(pid){
 +
      $(".protocolactive").each(function() {
 +
        $(this).addClass("protocol");
 +
        $(this).removeClass("protocolactive");
 +
      });
 +
      $(pid).addClass("protocolactive");
 +
    }
 +
   
 +
    document.onkeydown = function(evt) {
 +
      evt = evt || window.event;
 +
      switch (evt.keyCode) {
 +
          case 37:
 +
              leftArrowPressed();
 +
              break;
 +
          case 39:
 +
            rightArrowPressed();
 +
            break;
 +
      }
 +
  };
 +
  </script>
 
</body>
 
</body>
 
</html>
 
</html>

Revision as of 17:07, 20 August 2015

<!DOCTYPE html>

PROTOCOL

  We are a skillful and persistent group of nine Finns. We started as a group of students who didn't really know each other, assuming that we were going to spend our summer studying synthetic biology with strange colleagues. In the end we got a bunch of new friends and (in addition to studying synthetic biology) we just might have spent one of the best summers of our lives.

Protocols

Biography

Morbi rutrum, elit ac fermentum egestas, tortor ante vestibulum est, eget scelerisque nisl velit eget tellus. Fusce porta facilisis luctus. Integer neque dolor, rhoncus nec euismod eget, pharetra et tortor. Nulla id pulvinar nunc. Vestibulum auctor nisl vel lectus ullamcorper sed pellentesque dolor eleifend. Praesent lobortis magna vel diam mattis sagittis.

Heat Shock Transformation of E. coli

This protocol can be used to transform chemically competent (i.e. from CaCl2) with a miniprepped plasmid or a ligation product.

Note: Never vortex competent cells. Mix cells by gentle shaking.

  1. Thaw competent cells on ice. These can be prepared using the CaCl2 protocol.
  2. Place 20 ul of cells in a pre-chilled Eppendorf tube.
    • For an Intact Vector: Add 0.5 ul or less to the chilled cells
    • For a Ligation Product: Add 2-3 ul to the chilled cells.
  3. Mix gently by flicking the tube.
  4. Chill on ice for 10 minutes. This step is optional, but can improve yields when transforming a ligation product.
  5. Heat shock at 42 °C for 30 seconds.
  6. Return to ice for 2 minutes.
  7. Add 200 ul LB medium and recover the cells by shaking at 37 °C.
    Another rich medium can substitute for the recovery.
    The recovery time varies with the antibiotic selection.
    Ampicillin: 15-30 minutes
    Kanamycin or Spectinomycin: 30-60 minutes
    Chloramphenicol: 60-120 minutes
  8. Plate out the cells on selective LB.
    Use glass beads to spread the cells.
    The volume of cells plated depends on what is being transformed.
    • For an Intact Vector: High transformation efficiencies are expected. Plating out 10 ul of recovered cells should produce many colonies.
    • For a Ligation Product: Lower transformation efficiencies are expected. Therefore you can plate the entire 200 ul volume of recovered cells.
    Note: 200 ul is the maximum volume of liquid that an LB plate can absorb.
  9. Incubate at 37 °C. Transformants should appear within 12 hrs.

CaCl2 Competent Cells

This protocol makes 4 ml of competent cells, and can be easily scaled up to make more. The cells are typically stored in 110 ul aliquots, so this will make about 35 tubes. A typical transformation uses 20 ul of cells.

Note: Never vortex competent cells. Resuspend by pipetting with large Pasteur pipettes.

  1. The night before, inoculate a 5 ml culture and grow overnight with selection.
  2. The day of the experiment dilute cells ~ 1:200 into selective media.
    For this example add 250 ul to 50 ml of selective media.
    Note: The protocol is easily scaled to increase the number of cells.
  3. Grow the cells to an OD600 of 0.6 – 0.7.
    Use a large flask, 500ml, for good aeration.
    Use a baffled flask for fastest growth.
    This takes about 3 hours depending on the cells.
    Medium-heavy cloudiness by eye is fine.
  4. Spin down the cells at 4 ºC, 4000 rpm, 15 minutes. Note: Keep the cells at 4 ºC from now on.
  5. Resuspend cells in 15 ml, ice-cold 100 mM CaCl2. Leave on ice 4 hours to overnight.
  6. Spin down the cells at 4 ºC, 4000 rpm, 15 minutes.
  7. Resuspend cells in 4 ml, ice-cold 100 mM CaCl2 + 15% glycerol.
  8. Aliquot into pre-chilled Eppendorf tubes. Use immediately or store at -80ºC.
    Note: Frozen cells are only good once.Do not refreeze cells once thawed.

Glycerol Stocks

  1. Pick Single colonies from agar plates
  2. Innoculate 5ml LB broth overnight.
  3. Add 750ml of overnight culture to 250ml of 60% glycerol in a cryotube.
  4. Make two sets of Glycerol stocks freeze one at -20ºC and the other at -80ºC.





</div> </div> </div> 

 <script type="text/javascript">
   function showprot(pid){
     $(".protocolactive").each(function() {
        $(this).addClass("protocol");
        $(this).removeClass("protocolactive");
     });
     $(pid).addClass("protocolactive");
   }
   
   document.onkeydown = function(evt) {
      evt = evt || window.event;
      switch (evt.keyCode) {
         case 37:
             leftArrowPressed();
             break;
         case 39:
           rightArrowPressed();
           break;
     }
  };
 </script>

</body> </html>