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

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     <div id="prot1" class="protocolactive">
      <h2> Heat Shock Transformation of <i>E. coli</i></h2>
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<object data="https://static.igem.org/mediawiki/2014/d/da/10-16-14_Spec_Sheet_V2.pdf" type="application/pdf" width="100%" height="800px">
      <p>This protocol can be used to transform chemically competent (i.e. from CaCl2) with a miniprepped plasmid or a ligation product.</p>
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<p>It appears you don't have a PDF plugin for this browser.
      <h5>Note: Never vortex competent cells. Mix cells by gentle shaking.</h5>
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  No biggie... you can <a href="myfile.pdf">click here to
      <p><ol>
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  download the PDF file.</a></p>
  <li>Thaw competent cells on ice. These can be prepared using the CaCl2 protocol.</li>
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  <li>Place 20 ul of cells in a pre-chilled Eppendorf tube.
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    <ul>
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              <li><u>For an Intact Vector:</u> Add 0.5 ul or less to the chilled cells</li>
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              <li><u>For a Ligation Product:</u> Add 2-3 ul to the chilled cells.</li>
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    </ul>
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  </li>
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  <li>Mix gently by flicking the tube.</li>
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  <li>Chill on ice for 10 minutes. <em>This step is optional, but can improve yields when transforming a ligation product.</em></li>
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  <li>Heat shock at 42 &deg;C for 30 seconds.</li>
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  <li>Return to ice for 2 minutes.</li>
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  <li>Add 200 ul LB medium and recover the cells by shaking at 37 &deg;C.<br />
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    Another rich medium can substitute for the recovery.<br />
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    The recovery time varies with the antibiotic selection.<br />
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    Ampicillin: 15-30 minutes<br />
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    Kanamycin or Spectinomycin: 30-60 minutes<br />
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    Chloramphenicol: 60-120 minutes
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  </li>
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  <li>Plate out the cells on selective LB.<br />
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    Use glass beads to spread the cells.<br />
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    The volume of cells plated depends on what is being transformed.<br />
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    <ul>
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              <li><u>For an Intact Vector:</u> High transformation efficiencies are expected. Plating out 10 ul of recovered cells should produce many colonies.</li>
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              <li><u>For a Ligation Product:</u> Lower transformation efficiencies are expected. Therefore you can plate the entire 200 ul volume of recovered cells.</li>
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    </ul>
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    Note: 200 ul is the maximum volume of liquid that an LB plate can absorb.
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  </li>
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  <li>Incubate at 37 &deg;C. Transformants should appear within 12 hrs.</li>
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      </ol></p>
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     <div id="prot2" class="protocol">
 
     <div id="prot2" class="protocol">
      <h2> CaCl2 Competent Cells </h2>
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<object data="https://static.igem.org/mediawiki/2014/d/da/10-16-14_Spec_Sheet_V2.pdf" type="application/pdf" width="100%" height="800px">
      <p>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.</p>
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<p>It appears you don't have a PDF plugin for this browser.
      <h5> Note: Never vortex competent cells. Resuspend by pipetting with large Pasteur pipettes.</h5>
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  No biggie... you can <a href="myfile.pdf">click here to
      <p><ol>
+
download the PDF file.</a></p>
  <li><b><u>The night before</b></u>, inoculate a 5 ml culture and grow overnight with selection.</li>
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</object>
  <li> <b><u>The day of</b></u> the experiment dilute cells ~ 1:200 into selective media.<br>For this example add 250 ul to 50 ml of selective media.<br>Note: The protocol is easily scaled to increase the number of cells.</li>
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  <li> Grow the cells to an OD600 of 0.6 – 0.7.
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    <br>Use a large flask, 500ml, for good aeration.
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    <br>Use a baffled flask for fastest growth.
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    <br>This takes about 3 hours depending on the cells.
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    <br>Medium-heavy cloudiness by eye is fine.</li>
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  <li>Spin down the cells at 4 ºC, 4000 rpm, 15 minutes.
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    Note: Keep the cells at 4 ºC from now on.</li>
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  <li>Resuspend cells in 15 ml, ice-cold 100 mM CaCl2.
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    Leave on ice 4 hours to overnight.</li>
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  <li>Spin down the cells at 4 ºC, 4000 rpm, 15 minutes.</li>
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  <li>Resuspend cells in 4 ml, ice-cold 100 mM CaCl2 + 15% glycerol.</li>
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  <li>Aliquot into pre-chilled Eppendorf tubes. Use immediately or store at -80ºC.<br />
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    Note: Frozen cells are only good once.Do not refreeze cells once thawed.</li>
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      </ol></p>
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     <div id="prot3" class="protocol">
<object data="https://static.igem.org/mediawiki/2014/d/da/10-16-14_Spec_Sheet_V2.pdf" type="application/pdf" width="100%" height="800px">
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<object data="https://static.igem.org/mediawiki/2014/d/da/10-16-14_Spec_Sheet_V2.pdf" type="application/pdf" width="100%" height="800px">
<p>It appears you don't have a PDF plugin for this browser.
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<p>It appears you don't have a PDF plugin for this browser.
  No biggie... you can <a href="myfile.pdf">click here to
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  No biggie... you can <a href="myfile.pdf">click here to
  download the PDF file.</a></p>
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  download the PDF file.</a></p>
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Revision as of 17:30, 20 August 2015

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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

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