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| Line 263: |
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| | <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>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. | | <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>
| |
| − | </p>
| |
| − | </div>
| |
| − | </div>
| |
| − | </div>
| |
| − | </div>
| |
| − | <div class="panel panel-default">
| |
| − | <div class="panel-heading" role="tab" id="headingFive">
| |
| − | <h4 class="panel-title">
| |
| − | <a class="collapsed" role="button" data-toggle="collapse" data-parent="#accordion" href="#collapseFive" aria-expanded="false" aria-controls="collapseFive">
| |
| − | Glycerol Stock
| |
| − | </a>
| |
| − | </h4>
| |
| − | </div>
| |
| − | <div id="collapseFive" class="panel-collapse collapse" role="tabpanel" aria-labelledby="headingFive">
| |
| − | <div class="panel-body">
| |
| − | <div class="col-md-6">
| |
| − | <p>
| |
| − | <h2>Materials</h2>
| |
| − | <ul>
| |
| − | <li>500µl 50% glycerol solution
| |
| − | <li>500µl cultured cells
| |
| − | </ul>
| |
| − | </p>
| |
| − | </div>
| |
| − | <div class="col-md-6">
| |
| − | <p>
| |
| − | <h2>Procedure</h2>
| |
| − | <ul>
| |
| − | <li>1. Add the 50% glycerol to a sterile Eppendorf tube.
| |
| − | <li>2. Add 500µl of cells to the tube and vortex to mix.
| |
| − | <li>3. Freeze at -80°C.
| |
| − | </uL>
| |
| − | </p>
| |
| − | </div>
| |
| − | </div>
| |
| − | </div>
| |
| − | </div>
| |
| − | <div class="panel panel-default">
| |
| − | <div class="panel-heading" role="tab" id="headingSix">
| |
| − | <h4 class="panel-title">
| |
| − | <a class="collapsed" role="button" data-toggle="collapse" data-parent="#accordion" href="#collapseSix" aria-expanded="false" aria-controls="collapseSix">
| |
| − | Heat Shock Transformation
| |
| − | </a>
| |
| − | </h4>
| |
| − | </div>
| |
| − | <div id="collapseSix" class="panel-collapse collapse" role="tabpanel" aria-labelledby="headingSix">
| |
| − | <div class="panel-body">
| |
| − | <div class="col-md-6">
| |
| − | <p>
| |
| − | <h2>Materials</h2>
| |
| − | <ul>
| |
| − | <li>2.5µl ligated DNA or 0.5µl purified plasmid
| |
| − | <li>50µl chemically competent DH5α E.coli cells
| |
| − | <li>250µl Luria Broth
| |
| − | <li>Petri dish with LB agar and specific antibiotic (chloramphenicol or ampicillin)
| |
| − | </ul>
| |
| − | </p>
| |
| − | </div>
| |
| − | <div class="col-md-6">
| |
| − | <p>
| |
| − | <h2>Procedure</h2>
| |
| − | <ul>
| |
| − | <li>1. Thaw the competent cells on ice.
| |
| − | <li>2. Add 50µl of cells to a pre-chilled, labelled microcentrifuge tube.
| |
| − | <li>3. Add 2.5µl of DNA suspension to the tube. Pipette up and down to mix.
| |
| − | <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>
| |
| − | </p>
| |
| − | </div>
| |
| − | </div>
| |
| − | </div>
| |
| − | </div>
| |
| − | <div class="panel panel-default">
| |
| − | <div class="panel-heading" role="tab" id="headingSeven">
| |
| − | <h4 class="panel-title">
| |
| − | <a class="collapsed" role="button" data-toggle="collapse" data-parent="#accordion" href="#collapseSeven" aria-expanded="false" aria-controls="collapseSeven">
| |
| − | Miniprep using QIAprep Spin Miniprep Kit
| |
| − | </a>
| |
| − | </h4>
| |
| − | </div>
| |
| − | <div id="collapseSeven" class="panel-collapse collapse" role="tabpanel" aria-labelledby="headingSeven">
| |
| − | <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>
| |
| − | </div>
| |
| − | </div>
| |
| − | <div class="panel panel-default">
| |
| − | <div class="panel-heading" role="tab" id="headingEight">
| |
| − | <h4 class="panel-title">
| |
| − | <a class="collapsed" role="button" data-toggle="collapse" data-parent="#accordion" href="#collapseEight" aria-expanded="false" aria-controls="collapseEight">
| |
| − | Nanodrop
| |
| − | </a>
| |
| − | </h4>
| |
| − | </div>
| |
| − | <div id="collapseEight" class="panel-collapse collapse" role="tabpanel" aria-labelledby="headingEight">
| |
| − | <div class="panel-body">
| |
| − | <div class="col-md-6">
| |
| − | <p>
| |
| − | <h2>Materials</h2>
| |
| − | <ul>
| |
| − | <li>Buffer EB to blank
| |
| − | <li>DNA sample
| |
| − | </ul>
| |
| − | </p>
| |
| − | </div>
| |
| − | <div class="col-md-6">
| |
| − | <p>
| |
| − | <h2>Procedure</h2>
| |
| − | <ul>
| |
| − | <li>1. Wipe the NanoDrop 2000 clean before pipetting 1µl of buffer EB and lowering the arm. Blank the NanoDrop spectrophotometer.
| |
| − | <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. Clean the NanoDrop pedestal after use.
| |
| − | </uL>
| |
| − | </p>
| |
| − | </div>
| |
| − | </div>
| |
| − | </div>
| |
| − | </div>
| |
| − | <div class="panel panel-default">
| |
| − | <div class="panel-heading" role="tab" id="headingNine">
| |
| − | <h4 class="panel-title">
| |
| − | <a class="collapsed" role="button" data-toggle="collapse" data-parent="#accordion" href="#collapseNine" aria-expanded="false" aria-controls="collapseNine">
| |
| − | PCR Purification using QIAquick PCR Purification Kit
| |
| − | </a>
| |
| − | </h4>
| |
| − | </div>
| |
| − | <div id="collapseNine" class="panel-collapse collapse" role="tabpanel" aria-labelledby="headingNine">
| |
| − | <div class="panel-body">
| |
| − | <div class="col-md-6">
| |
| − | <p>
| |
| − | <h2>Materials</h2>
| |
| − | <ul>
| |
| − | <li>Buffer PB
| |
| − | <li>10µl of 3M sodium acetate
| |
| − | <li>750µl buffer PE
| |
| − | <li>25µl buffer EB
| |
| − | </ul>
| |
| − | </p>
| |
| − | </div>
| |
| − | <div class="col-md-6">
| |
| − | <p>
| |
| − | <h2>Procedure</h2>
| |
| − | All centrifugation steps are carried out at 17,900 x g (13,000 rpm)
| |
| − | <ul>
| |
| − | <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. 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. Place a QIAquick spin column into a 2ml collection tube.
| |
| − | <li>4. Apply the sample to the centre of the column and centrifuge for 60s.
| |
| − | <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>
| |
| − | </p>
| |
| − | </div>
| |
| − | </div>
| |
| − | </div>
| |
| − | </div>
| |
| − | <div class="panel panel-default">
| |
| − | <div class="panel-heading" role="tab" id="headingTen">
| |
| − | <h4 class="panel-title">
| |
| − | <a class="collapsed" role="button" data-toggle="collapse" data-parent="#accordion" href="#collapseTen" aria-expanded="false" aria-controls="collapseTen">
| |
| − | Resuspending gBlocks
| |
| − | </a>
| |
| − | </h4>
| |
| − | </div>
| |
| − | <div id="collapseTen" class="panel-collapse collapse" role="tabpanel" aria-labelledby="headingTen">
| |
| − | <div class="panel-body">
| |
| − | <div class="col-md-6">
| |
| − | <p>
| |
| − | <h2>Materials</h2>
| |
| − | <ul>
| |
| − | <li>gBlocks dry gene fragment
| |
| − | <li>100µl distilled water
| |
| − | </ul>
| |
| − | </p>
| |
| − | </div>
| |
| − | <div class="col-md-6">
| |
| − | <p>
| |
| − | <h2>Procedure</h2>
| |
| − | <ul>
| |
| − | <li>1. Centrifuge the tube containing the gBlocks gene fragment pellet at 3000 x g for 5s.
| |
| − | <li>2. Add 100µl water to the tube.
| |
| − | <li>3. Vortex to mix.
| |
| − | <li>5. Incubate at 50°C for 20 mins.
| |
| − | <li>6. Briefly vortex and centrifuge.
| |
| − | </uL>
| |
| − | </p>
| |
| − | </div>
| |
| − | </div>
| |
| − | </div>
| |
| − | </div>
| |
| − | <div class="panel panel-default">
| |
| − | <div class="panel-heading" role="tab" id="headingEleven">
| |
| − | <h4 class="panel-title">
| |
| − | <a class="collapsed" role="button" data-toggle="collapse" data-parent="#accordion" href="#collapseEleven" aria-expanded="false" aria-controls="collapseEleven">
| |
| − | Sequencing
| |
| − | </a>
| |
| − | </h4>
| |
| − | </div>
| |
| − | <div id="collapseEleven" class="panel-collapse collapse" role="tabpanel" aria-labelledby="headingEleven">
| |
| − | <div class="panel-body">
| |
| − | <div class="col-md-6">
| |
| − | <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> | | </uL> |
| | </p> | | </p> |