Difference between revisions of "Team:Edinburgh/Basic Part"

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           <h4 class="panel-title">
 
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             <a role="button" data-toggle="collapse" data-parent="#accordion" href="#collapseOne" aria-expanded="false" aria-controls="collapseOne">
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             <a class="collapsed" role="button" data-toggle="collapse" data-parent="#accordion" href="#collapseFour" aria-expanded="false" aria-controls="collapseFour">
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              1% Agarose Gel
+
            Gel Purification using QIAquick Gel Extraction Kit
 
             </a>
 
             </a>
 
           </h4>
 
           </h4>
 
         </div>
 
         </div>
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           <div class="panel-body">
 
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             <div class="col-md-6">
 
             <div class="col-md-6">
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               <p style="color: black;">
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               <p>
 
               <h2>Materials</h2>
 
               <h2>Materials</h2>
 
               <ul>
 
               <ul>
−
                 <li>1g Agarose
+
                 <li>Buffer QG
−
                 <li>100ml 1X TAE buffer
+
                 <li>10µl 3M sodium acetate
−
                 <li>5µl GelRed stain
+
                 <li>Isopropanol
 +
                <li>750µl Buffer PE
 +
                <li>25µl Buffer EB
 
               </ul>
 
               </ul>
 
             </p>
 
             </p>
 
             </div>
 
             </div>
 
             <div class="col-md-6">
 
             <div class="col-md-6">
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               <p class="text-muted">
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               <p>
 
               <h2>Procedure</h2>
 
               <h2>Procedure</h2>
 +
              All centrifuge steps are carried out at 13,000 rpm.
 
               <ul>
 
               <ul>
−
                 <li>1. Mix the agarose with the 1X TAE buffer in a flask.
+
                 <li>1. Excise the region of gel containing the DNA fragment using a scalpel.  Cut close to the DNA to minimise the gel volume.
−
                 <li>2. Heat the mixture until all the agarose is dissolved.
+
                <li>2. Place the gel slice in a 1.5ml tube and weigh it. Record the volume of the gel.
−
                 <li>3. Swirl the flask under cold running water to cool the mixture.
+
                 <li>3.  Add 300µl of Buffer QG for each 100mg of gel.
−
                 <li> 4. Add the gel stain.
+
                <li>4. Incubate at 50°C for 10 minutes or until the gel has completely dissolved. Mix by vortexing the tube every 2 minutes during the incubation.
−
                 <li>5. Pour into an assembled gel tray and let it cool.
+
                <li>5. Once the gel is completely dissolved, the mixture should be yellow. If the mixture is orange or violet add 10 µl of 3M sodium acetate and mix until it turns yellow. Yellow colour indicates the solution is the optimum pH for DNA binding to the QIAquick membrane.
 +
                 <li>6. Add 1 gel volume of isopropanol to the solution and mix (1:1 volumes of isopropanol to gel slice).
 +
                <li>7. Place a QIAquick spin column in a 2ml collection tube.
 +
                <li>8. Pipette the sample onto the QIAquick column and centrifuge. Discard flow-through.
 +
                 <li>9. Place column back in same collection tube. Add 500µl of Buffer QG to the column and centrifuge for 1 minute to remove all traces of agarose.
 +
                 <li>10. Wash column by adding 750µl buffer PE. Let it stand for 2-5 min and then centrifuge for 1 minute.
 +
                <li>11. Discard the flow-through. Centrifuge for 1 minute to remove the residual buffer PE.
 +
                <li>12. Then place the column in a clean, labelled 1.5ml Eppendorf tube.
 +
                <li>13. To elute the DNA, add 25µl of Buffer EB to the centre of the column membrane, let it stand for 1 minute and then centrifuge for 1 minute.
 +
                <li>14. Using a pipette, transfer the flow-through back into the centre of the column. Let it stand for 1 minute and then centrifuge for 1 minute. The DNA will now be in the flow-through.
 
               </uL>
 
               </uL>
 
             </p>
 
             </p>
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             <a class="collapsed" role="button" data-toggle="collapse" data-parent="#accordion" href="#collapseFive" aria-expanded="false" aria-controls="collapseFive">
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             Agarose Gel Electrophoresis
+
             Glycerol Stock
 
             </a>
 
             </a>
 
           </h4>
 
           </h4>
 
         </div>
 
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               <h2>Materials</h2>
 
               <h2>Materials</h2>
 
               <ul>
 
               <ul>
−
                 <li>1% Agarose
+
                 <li>500µl 50% glycerol solution
−
                 <li>1X TAE buffer
+
                 <li>500µl cultured cells
−
                <li>5X loading dye
+
−
                <li>DNA ladder
+
−
                <li>DNA samples
+
 
               </ul>
 
               </ul>
 
             </p>
 
             </p>
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               <h2>Procedure</h2>
 
               <h2>Procedure</h2>
 
               <ul>
 
               <ul>
−
                 <li>1. Place gel tray into the electrophoresis apparatus.
+
                 <li>1. Add the 50% glycerol to a sterile Eppendorf tube.
−
                 <li>2. Pour 1X TAE so that the gel is covered by buffer.
+
                 <li>2. Add 500µl of cells to the tube and vortex to mix.
−
                 <li>3. Prepare the samples by adding the appropriate amount of loading dye.
+
                 <li>3. Freeze at -80°C.
−
                <li>4. Load samples and DNA ladder into wells on the gel.
+
−
                <li>5. Run the gel at roughly 100V for around an hour
+
−
 
+
 
               </uL>
 
               </uL>
 
             </p>
 
             </p>
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             <a class="collapsed" role="button" data-toggle="collapse" data-parent="#accordion" href="#collapseSix" aria-expanded="false" aria-controls="collapseSix">
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              Culture
+
            Heat Shock Transformation
 
             </a>
 
             </a>
 
           </h4>
 
           </h4>
 
         </div>
 
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            <div class="col-md-6">
 
               <p>
 
               <p>
 
               <h2>Materials</h2>
 
               <h2>Materials</h2>
 
               <ul>
 
               <ul>
−
                 <li>10ml Luria Broth (LB)
+
                 <li>2.5µl ligated DNA or 0.5µl purified plasmid
−
                 <li>10µl Specific Antibiotic at 1000x (Chloramphenicol, Ampicillin or Kanamycin)
+
                 <li>50µl chemically competent DH5α E.coli cells
−
                 <li>Loop (for picking colony)
+
                 <li>250µl Luria Broth
−
                 <li>Ethanol
+
                 <li>Petri dish with LB agar and specific antibiotic (chloramphenicol or ampicillin)
 
               </ul>
 
               </ul>
 
             </p>
 
             </p>
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               <h2>Procedure</h2>
 
               <h2>Procedure</h2>
 
               <ul>
 
               <ul>
−
                 <li>1. Pour 10ml of LB into a 50ml Falcon tube.  
+
                 <li>1. Thaw the competent cells on ice.
−
                 <li>2. Pipette 10µl of antibiotic into the broth.
+
                <li>2. Add 50µl of cells to a pre-chilled, labelled microcentrifuge tube.
−
                 <li>3. Dip loop in ethanol and flame to sterilise. Once it is cool, pick colony and transfer to a 50ml Falcon tube.
+
                 <li>3. Add 2.5µl of DNA suspension to the tube. Pipette up and down to mix.
−
                 <li>4. Incubate at 37°C overnight in a shaking incubator.
+
                 <li>4. Incubate tube on ice for 30 min.
 +
                <li>5. Heat shock in a water bath at 42°C for 60s.
 +
                <li>6. Place tube back on ice for 3 min.
 +
                <li>7. Add 250µl of LB to the tube.
 +
                <li>8. Incubate the tube at 37°C for 1 hour.
 +
                <li>9. Prepare two plates for each transformation, one plated with 200µl of cells and another plated with 100µl of cells.  
 +
                 <li>10. Incubate at 37°C overnight (12-14 hours).
 
               </uL>
 
               </uL>
 
             </p>
 
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       </div>
 
       </div>
 
       <div class="panel panel-default">
 
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         <div class="panel-heading" role="tab" id="headingFour">
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         <div class="panel-heading" role="tab" id="headingSeven">
 
           <h4 class="panel-title">
 
           <h4 class="panel-title">
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             <a class="collapsed" role="button" data-toggle="collapse" data-parent="#accordion" href="#collapseFour" aria-expanded="false" aria-controls="collapseFour">
+
             <a class="collapsed" role="button" data-toggle="collapse" data-parent="#accordion" href="#collapseSeven" aria-expanded="false" aria-controls="collapseSeven">
−
             Gel Purification using QIAquick Gel Extraction Kit
+
             Miniprep using QIAprep Spin Miniprep Kit
 
             </a>
 
             </a>
 
           </h4>
 
           </h4>
 +
        </div>
 +
        <div id="collapseSeven" class="panel-collapse collapse" role="tabpanel" aria-labelledby="headingSeven">
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          <div class="panel-body">
 +
            <div class="col-md-6">
 +
              <p>
 +
              <h2>Materials</h2>
 +
              <ul>
 +
                <li>10ml cell culture
 +
                <li>250µl Buffer P1
 +
                <li>250µl Buffer P2
 +
                <li>350µl Buffer N3
 +
                <li>750µl Buffer PE
 +
                <li>50µl Buffer EB
 +
              </ul>
 +
            </p>
 +
            </div>
 +
            <div class="col-md-6">
 +
              <p>
 +
              <h2>Procedure</h2>
 +
              <ul>
 +
                <li>1. Centrifuge 10ml cell culture at 4,500 x g for 5 minutes and pour off the supernatant. Centrifuge at 4,500 x g for 2 minutes and remove the supernatant by pipetting.
 +
                <li>2. Resuspend pelleted cells in 250 µl Buffer P1 and transfer the solution to a labelled Eppendorf tube.
 +
                <li>3. Add 250 µl Buffer P2 and gently invert the tube 4–6 times to mix. If necessary, continue inverting the tube until the solution becomes viscous and slightly clear. Do not allow the lysis reaction to proceed for more than 5 minutes.
 +
                <li>4. Add 350 µl Buffer N3 and invert the tube immediately but gently 4–6 times. The solution should become cloudy.
 +
                <li>5. Centrifuge for 10 min at 13,000 rpm in an Eppendorf tube. A compact white pellet will form.
 +
                <li>6. Apply the supernatant to the QIAprep Spin Column by pipetting.
 +
                <li>7. Centrifuge for 60s at 13,000 rpm. Discard the flow-through.
 +
                <li>8. Wash QIAprep Spin Column by adding 750µl Buffer PE and centrifuging for 60s at 13,000 rpm.
 +
                <li>9. Discard the flow-through, and centrifuge for an additional 60s at 13,000 rpm to remove residual wash buffer.
 +
                <li>10. Place the QIAprep column in a clean, labelled Eppendorf tube. To double elute DNA, add 50 µl Buffer EB to the centre of each QIAprep Spin Column, let stand for 1 min, and centrifuge for 1 min at 13,000 rpm. Re-apply flow-through to the centre of the QIAprep Spin Column, let stand for 1 min and centrifuge for 1 min at 13,000 rpm.
 +
                <li>11. Discard column. Flow-through contains the DNA.
 +
              </uL>
 +
            </p>
 +
            </div>
 +
          </div>
 
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    <h3 class="panel-title">Panel title</h3>
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  </div>
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</div>
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    <div class="panel-group" id="accordion" role="tablist" aria-multiselectable="true">
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         <div class="panel-heading" role="tab" id="headingOne">
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           <h4 class="panel-title">
 
           <h4 class="panel-title">
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             <a role="button" data-toggle="collapse" data-parent="#accordion" href="#collapseOne" aria-expanded="false" aria-controls="collapseOne">
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             <a class="collapsed" role="button" data-toggle="collapse" data-parent="#accordion" href="#collapseEight" aria-expanded="false" aria-controls="collapseEight">
−
              1% Agarose Gel
+
            Nanodrop
 
             </a>
 
             </a>
 
           </h4>
 
           </h4>
 
         </div>
 
         </div>
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         <div id="collapseOne" class="panel-collapse collapse in" role="tabpanel" aria-labelledby="headingOne">
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           <div class="panel-body">
 
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             <div class="col-md-6">
 
             <div class="col-md-6">
−
               <p style="color: black;">
+
               <p>
 
               <h2>Materials</h2>
 
               <h2>Materials</h2>
 
               <ul>
 
               <ul>
−
                 <li>1g Agarose
+
                 <li>Buffer EB to blank
−
                 <li>100ml 1X TAE buffer
+
                 <li>DNA sample
−
                <li>5µl GelRed stain
+
 
               </ul>
 
               </ul>
 
             </p>
 
             </p>
 
             </div>
 
             </div>
 
             <div class="col-md-6">
 
             <div class="col-md-6">
−
               <p class="text-muted">
+
               <p>
 
               <h2>Procedure</h2>
 
               <h2>Procedure</h2>
 
               <ul>
 
               <ul>
−
                 <li>1. Mix the agarose with the 1X TAE buffer in a flask.
+
                 <li>1. Wipe the NanoDrop 2000 clean before pipetting 1µl of buffer EB and lowering the arm. Blank the NanoDrop spectrophotometer.
−
                 <li>2. Heat the mixture until all the agarose is dissolved.
+
                 <li>2. Clean the buffer EB from the pedestal and apply 1µl of sample. Measure and record the absorbance, and repeat for remainder of samples.
−
                <li>3. Swirl the flask under cold running water to cool the mixture.
+
                 <li>3. Clean the NanoDrop pedestal after use.
−
                 <li> 4. Add the gel stain.
+
−
                <li>5. Pour into an assembled gel tray and let it cool.
+
 
               </uL>
 
               </uL>
 
             </p>
 
             </p>
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         <div class="panel-heading" role="tab" id="headingTwo">
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         <div class="panel-heading" role="tab" id="headingNine">
 
           <h4 class="panel-title">
 
           <h4 class="panel-title">
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             <a class="collapsed" role="button" data-toggle="collapse" data-parent="#accordion" href="#collapseTwo" aria-expanded="false" aria-controls="collapseTwo">
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−
             Agarose Gel Electrophoresis
+
             PCR Purification using QIAquick PCR Purification Kit
 
             </a>
 
             </a>
 
           </h4>
 
           </h4>
 
         </div>
 
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               <h2>Materials</h2>
 
               <h2>Materials</h2>
 
               <ul>
 
               <ul>
−
                 <li>1% Agarose
+
                 <li>Buffer PB
−
                 <li>1X TAE buffer
+
                 <li>10µl of 3M sodium acetate
−
                 <li>5X loading dye
+
                 <li>750µl buffer PE
−
                 <li>DNA ladder
+
                 <li>25µl buffer EB
−
                <li>DNA samples
+
 
               </ul>
 
               </ul>
 
             </p>
 
             </p>
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               <p>
 
               <p>
 
               <h2>Procedure</h2>
 
               <h2>Procedure</h2>
 +
              All centrifugation steps are carried out at 17,900 x g (13,000 rpm)
 
               <ul>
 
               <ul>
−
                 <li>1. Place gel tray into the electrophoresis apparatus.
+
                 <li>1. Add 5 volumes of buffer PB to 1 volume of PCR sample (e.g. 500µl PB to 100µl PCR sample) and mix.
−
                 <li>2. Pour 1X TAE so that the gel is covered by buffer.
+
                 <li>2. The pH indicator in buffer PB will cause the mixture to turn yellow. If the mixture is orange or violet add 10µl of 3M sodium acetate and mix until it turns yellow.
−
                 <li>3. Prepare the samples by adding the appropriate amount of loading dye.
+
                 <li>3. Place a QIAquick spin column into a 2ml collection tube.
−
                 <li>4. Load samples and DNA ladder into wells on the gel.
+
                <li>4. Apply the sample to the centre of the column and centrifuge for 60s.
−
                 <li>5. Run the gel at roughly 100V for around an hour
+
                 <li>5. Discard the flow-through and replace the column in the same collection tube.
−
 
+
                <li>6. Add 750µl of buffer PE to the column and centrifuge for 60s.
 +
                <li>7. Discard the flow-through, replace the column in the collection tube and centrifuge for a further 60s to remove residual buffer.
 +
                <li>8. Place the column in a clean 1.5ml Eppendorf tube.
 +
                <li>9. Elute the DNA by adding 25µl buffer EB to the centre of the membrane in the column. Let it stand for 1 minute then centrifuge for 1 minute.  
 +
                 <li>10. Using a pipette, transfer the flow-through back into the centre of the column. Let it stand for 1 minute and then centrifuge for 1 minute.
 +
                <li>11. The purified DNA is now present in the flow-through.
 
               </uL>
 
               </uL>
 
             </p>
 
             </p>
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−
              Culture
+
            Resuspending gBlocks
 
             </a>
 
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           </h4>
 
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               <p>
 
               <p>
 
               <h2>Materials</h2>
 
               <h2>Materials</h2>
 
               <ul>
 
               <ul>
−
                 <li>10ml Luria Broth (LB)
+
                 <li>gBlocks dry gene fragment
−
                 <li>10µl Specific Antibiotic at 1000x (Chloramphenicol, Ampicillin or Kanamycin)
+
                 <li>100µl distilled water
−
                <li>Loop (for picking colony)
+
−
                <li>Ethanol
+
 
               </ul>
 
               </ul>
 
             </p>
 
             </p>
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               <h2>Procedure</h2>
 
               <h2>Procedure</h2>
 
               <ul>
 
               <ul>
−
                 <li>1. Pour 10ml of LB into a 50ml Falcon tube.  
+
                 <li>1. Centrifuge the tube containing the gBlocks gene fragment pellet at 3000 x g for 5s.
−
                 <li>2. Pipette 10µl of antibiotic into the broth.
+
                 <li>2. Add 100µl water to the tube.
−
                 <li>3. Dip loop in ethanol and flame to sterilise. Once it is cool, pick colony and transfer to a 50ml Falcon tube.
+
                 <li>3. Vortex to mix.
−
                 <li>4. Incubate at 37°C overnight in a shaking incubator.
+
                 <li>5. Incubate at 50°C for 20 mins.
 +
                <li>6. Briefly vortex and centrifuge.
 
               </uL>
 
               </uL>
 
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−
             Gel Purification using QIAquick Gel Extraction Kit
+
             Sequencing
 
             </a>
 
             </a>
 
           </h4>
 
           </h4>
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              <p>
 +
              <h2>Materials</h2>
 +
              <ul>
 +
                <li>200-500ng DNA sample
 +
                <li>0.32µl 10µM Vf2 and Vr primers
 +
                <li>Water
 +
              </ul>
 +
            </p>
 +
            </div>
 +
            <div class="col-md-6">
 +
              <p>
 +
              <h2>Procedure</h2>
 +
              <ul>
 +
                <li>1. Prepare the DNA for sequencing by adding DNA, primers and water to a final volume of 6µl.
 +
                <li>2. Prepare two samples for each DNA sequence, one containing the forward primer, and another separate sample containing the reverse primer.
 +
                <li>3. Send the samples to be Sanger sequenced by Edinburgh Genomics using an ABI 3730 DNA analyser.
 +
                <li>4. Edinburgh Genomics will send an email with the sequence data.
 +
              </uL>
 +
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 +
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Revision as of 11:13, 1 September 2015

Basic Parts




Materials

  • 1g Agarose
  • 100ml 1X TAE buffer
  • 5µl GelRed stain

Procedure

  • 1. Mix the agarose with the 1X TAE buffer in a flask.
  • 2. Heat the mixture until all the agarose is dissolved.
  • 3. Swirl the flask under cold running water to cool the mixture.
  • 4. Add the gel stain.
  • 5. Pour into an assembled gel tray and let it cool.

Materials

  • 1% Agarose
  • 1X TAE buffer
  • 5X loading dye
  • DNA ladder
  • DNA samples

Procedure

  • 1. Place gel tray into the electrophoresis apparatus.
  • 2. Pour 1X TAE so that the gel is covered by buffer.
  • 3. Prepare the samples by adding the appropriate amount of loading dye.
  • 4. Load samples and DNA ladder into wells on the gel.
  • 5. Run the gel at roughly 100V for around an hour

Materials

  • 10ml Luria Broth (LB)
  • 10µl Specific Antibiotic at 1000x (Chloramphenicol, Ampicillin or Kanamycin)
  • Loop (for picking colony)
  • Ethanol

Procedure

  • 1. Pour 10ml of LB into a 50ml Falcon tube.
  • 2. Pipette 10µl of antibiotic into the broth.
  • 3. Dip loop in ethanol and flame to sterilise. Once it is cool, pick colony and transfer to a 50ml Falcon tube.
  • 4. Incubate at 37°C overnight in a shaking incubator.

Materials

  • Buffer QG
  • 10µl 3M sodium acetate
  • Isopropanol
  • 750µl Buffer PE
  • 25µl Buffer EB

Procedure

All centrifuge steps are carried out at 13,000 rpm.
  • 1. Excise the region of gel containing the DNA fragment using a scalpel. Cut close to the DNA to minimise the gel volume.
  • 2. Place the gel slice in a 1.5ml tube and weigh it. Record the volume of the gel.
  • 3. Add 300µl of Buffer QG for each 100mg of gel.
  • 4. Incubate at 50°C for 10 minutes or until the gel has completely dissolved. Mix by vortexing the tube every 2 minutes during the incubation.
  • 5. Once the gel is completely dissolved, the mixture should be yellow. If the mixture is orange or violet add 10 µl of 3M sodium acetate and mix until it turns yellow. Yellow colour indicates the solution is the optimum pH for DNA binding to the QIAquick membrane.
  • 6. Add 1 gel volume of isopropanol to the solution and mix (1:1 volumes of isopropanol to gel slice).
  • 7. Place a QIAquick spin column in a 2ml collection tube.
  • 8. Pipette the sample onto the QIAquick column and centrifuge. Discard flow-through.
  • 9. Place column back in same collection tube. Add 500µl of Buffer QG to the column and centrifuge for 1 minute to remove all traces of agarose.
  • 10. Wash column by adding 750µl buffer PE. Let it stand for 2-5 min and then centrifuge for 1 minute.
  • 11. Discard the flow-through. Centrifuge for 1 minute to remove the residual buffer PE.
  • 12. Then place the column in a clean, labelled 1.5ml Eppendorf tube.
  • 13. To elute the DNA, add 25µl of Buffer EB to the centre of the column membrane, let it stand for 1 minute and then centrifuge for 1 minute.
  • 14. Using a pipette, transfer the flow-through back into the centre of the column. Let it stand for 1 minute and then centrifuge for 1 minute. The DNA will now be in the flow-through.

Materials

  • 500µl 50% glycerol solution
  • 500µl cultured cells

Procedure

  • 1. Add the 50% glycerol to a sterile Eppendorf tube.
  • 2. Add 500µl of cells to the tube and vortex to mix.
  • 3. Freeze at -80°C.

Materials

  • 2.5µl ligated DNA or 0.5µl purified plasmid
  • 50µl chemically competent DH5α E.coli cells
  • 250µl Luria Broth
  • Petri dish with LB agar and specific antibiotic (chloramphenicol or ampicillin)

Procedure

  • 1. Thaw the competent cells on ice.
  • 2. Add 50µl of cells to a pre-chilled, labelled microcentrifuge tube.
  • 3. Add 2.5µl of DNA suspension to the tube. Pipette up and down to mix.
  • 4. Incubate tube on ice for 30 min.
  • 5. Heat shock in a water bath at 42°C for 60s.
  • 6. Place tube back on ice for 3 min.
  • 7. Add 250µl of LB to the tube.
  • 8. Incubate the tube at 37°C for 1 hour.
  • 9. Prepare two plates for each transformation, one plated with 200µl of cells and another plated with 100µl of cells.
  • 10. Incubate at 37°C overnight (12-14 hours).

Materials

  • 10ml cell culture
  • 250µl Buffer P1
  • 250µl Buffer P2
  • 350µl Buffer N3
  • 750µl Buffer PE
  • 50µl Buffer EB

Procedure

  • 1. Centrifuge 10ml cell culture at 4,500 x g for 5 minutes and pour off the supernatant. Centrifuge at 4,500 x g for 2 minutes and remove the supernatant by pipetting.
  • 2. Resuspend pelleted cells in 250 µl Buffer P1 and transfer the solution to a labelled Eppendorf tube.
  • 3. Add 250 µl Buffer P2 and gently invert the tube 4–6 times to mix. If necessary, continue inverting the tube until the solution becomes viscous and slightly clear. Do not allow the lysis reaction to proceed for more than 5 minutes.
  • 4. Add 350 µl Buffer N3 and invert the tube immediately but gently 4–6 times. The solution should become cloudy.
  • 5. Centrifuge for 10 min at 13,000 rpm in an Eppendorf tube. A compact white pellet will form.
  • 6. Apply the supernatant to the QIAprep Spin Column by pipetting.
  • 7. Centrifuge for 60s at 13,000 rpm. Discard the flow-through.
  • 8. Wash QIAprep Spin Column by adding 750µl Buffer PE and centrifuging for 60s at 13,000 rpm.
  • 9. Discard the flow-through, and centrifuge for an additional 60s at 13,000 rpm to remove residual wash buffer.
  • 10. Place the QIAprep column in a clean, labelled Eppendorf tube. To double elute DNA, add 50 µl Buffer EB to the centre of each QIAprep Spin Column, let stand for 1 min, and centrifuge for 1 min at 13,000 rpm. Re-apply flow-through to the centre of the QIAprep Spin Column, let stand for 1 min and centrifuge for 1 min at 13,000 rpm.
  • 11. Discard column. Flow-through contains the DNA.

Materials

  • Buffer EB to blank
  • DNA sample

Procedure

  • 1. Wipe the NanoDrop 2000 clean before pipetting 1µl of buffer EB and lowering the arm. Blank the NanoDrop spectrophotometer.
  • 2. Clean the buffer EB from the pedestal and apply 1µl of sample. Measure and record the absorbance, and repeat for remainder of samples.
  • 3. Clean the NanoDrop pedestal after use.

Materials

  • Buffer PB
  • 10µl of 3M sodium acetate
  • 750µl buffer PE
  • 25µl buffer EB

Procedure

All centrifugation steps are carried out at 17,900 x g (13,000 rpm)
  • 1. Add 5 volumes of buffer PB to 1 volume of PCR sample (e.g. 500µl PB to 100µl PCR sample) and mix.
  • 2. The pH indicator in buffer PB will cause the mixture to turn yellow. If the mixture is orange or violet add 10µl of 3M sodium acetate and mix until it turns yellow.
  • 3. Place a QIAquick spin column into a 2ml collection tube.
  • 4. Apply the sample to the centre of the column and centrifuge for 60s.
  • 5. Discard the flow-through and replace the column in the same collection tube.
  • 6. Add 750µl of buffer PE to the column and centrifuge for 60s.
  • 7. Discard the flow-through, replace the column in the collection tube and centrifuge for a further 60s to remove residual buffer.
  • 8. Place the column in a clean 1.5ml Eppendorf tube.
  • 9. Elute the DNA by adding 25µl buffer EB to the centre of the membrane in the column. Let it stand for 1 minute then centrifuge for 1 minute.
  • 10. Using a pipette, transfer the flow-through back into the centre of the column. Let it stand for 1 minute and then centrifuge for 1 minute.
  • 11. The purified DNA is now present in the flow-through.

Materials

  • gBlocks dry gene fragment
  • 100µl distilled water

Procedure

  • 1. Centrifuge the tube containing the gBlocks gene fragment pellet at 3000 x g for 5s.
  • 2. Add 100µl water to the tube.
  • 3. Vortex to mix.
  • 5. Incubate at 50°C for 20 mins.
  • 6. Briefly vortex and centrifuge.

Materials

  • 200-500ng DNA sample
  • 0.32µl 10µM Vf2 and Vr primers
  • Water

Procedure

  • 1. Prepare the DNA for sequencing by adding DNA, primers and water to a final volume of 6µl.
  • 2. Prepare two samples for each DNA sequence, one containing the forward primer, and another separate sample containing the reverse primer.
  • 3. Send the samples to be Sanger sequenced by Edinburgh Genomics using an ABI 3730 DNA analyser.
  • 4. Edinburgh Genomics will send an email with the sequence data.