http://2012.igem.org/wiki/index.php?title=Team:Washington/Protocols/Plas_DNA.&feed=atom&action=historyTeam:Washington/Protocols/Plas DNA. - Revision history2024-03-28T15:02:39ZRevision history for this page on the wikiMediaWiki 1.16.0http://2012.igem.org/wiki/index.php?title=Team:Washington/Protocols/Plas_DNA.&diff=252614&oldid=prevAcker: /* Equipment Necessary */2012-10-03T00:50:50Z<p><span class="autocomment">Equipment Necessary</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#Centrifuge</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#Centrifuge</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>#<del class="diffchange diffchange-inline">QIAGEM </del>Miniprep Kit</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>#<ins class="diffchange diffchange-inline">QIAGEN </ins>Miniprep Kit</div></td></tr>
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</table>Ackerhttp://2012.igem.org/wiki/index.php?title=Team:Washington/Protocols/Plas_DNA.&diff=252609&oldid=prevAcker: /* Equipment Necessary */2012-10-03T00:50:29Z<p><span class="autocomment">Equipment Necessary</span></p>
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</table>Ackerhttp://2012.igem.org/wiki/index.php?title=Team:Washington/Protocols/Plas_DNA.&diff=252552&oldid=prevAcker: /* Isolation of Plasmid DNA (miniprep) */2012-10-03T00:41:24Z<p><span class="autocomment">Isolation of Plasmid DNA (miniprep)</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#* Pipet 1 ul of a miniprep sample onto the machine. Hit "collect" in order to run the sample to clean the machine.Wipe off the remaining sample with a kimwipe, and record the concentration of the miniprep (ng/ul).</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#* Pipet 1 ul of a miniprep sample onto the machine. Hit "collect" in order to run the sample to clean the machine.Wipe off the remaining sample with a kimwipe, and record the concentration of the miniprep (ng/ul).</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#* Repeat the process of pipeting a sample and "collected" until all samples have been measured.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#* Repeat the process of pipeting a sample and "collected" until all samples have been measured.</div></td></tr>
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<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">#Miniprep Kit</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">#Microcentrifuge tubes</ins></div></td></tr>
</table>Ackerhttp://2012.igem.org/wiki/index.php?title=Team:Washington/Protocols/Plas_DNA.&diff=241181&oldid=prevTjiang: Created page with "{{Template:Team:Washington/Templates/Top}} =Isolation of Plasmid DNA (miniprep)= # Gently vortex overnight culture(s) to mix cells. # Pipet ~1 mL of cells into a labeled micro..."2012-09-29T06:14:46Z<p>Created page with "{{Template:Team:Washington/Templates/Top}} =Isolation of Plasmid DNA (miniprep)= # Gently vortex overnight culture(s) to mix cells. # Pipet ~1 mL of cells into a labeled micro..."</p>
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=Isolation of Plasmid DNA (miniprep)=<br />
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# Gently vortex overnight culture(s) to mix cells.<br />
# Pipet ~1 mL of cells into a labeled microcentrifuge tube. Pellet the cells by centrifuging at 6000 X g. <br />
# Carefully pour off the supernatant without disturbing the pelleted cells.<br />
# Resuspend the pelleted cells in 250 μL of refrigerated buffer P1. <br />
# Add 250 μL of buffer P2 and thoroughly mix the tube by inverting 4-6 times. <br />
# Add 350 μL of buffer N3 and immediately (but gently) mix the tube by inverting 4-6 times. <br />
# Centrifuge the sample at ~17000 X g for 10 minutes. <br />
# Pour/Pipet the supernatant into a spin column (blue).<br />
# Centrifuge the sample for ~ 1 minute. Discard the flow-through. <br />
# Wash the sample by adding 500 μL buffer PB and centrifuge for ~ 1 minute. <br />
# Wash the sample by adding 750 μLbuffer PE and centrifuge for ~ 1 minute.<br />
# Discard the flow-through and centrifuge the sample for an additional 1 minute.<br />
# To Elute the DNA, place the spin column in a clean 1.5 μL (labeled) microcentrifuge. Add 30 μL of buffer EB and '''let Stand for 1 minute!''' <br />
# Centrifuge the sample for 1 minute.<br />
# Record the DNA concentration using the nanodrop program.<br />
#* Open the nanodrop program on the lab computer.<br />
#* Select "DNA"<br />
#* Once the program loads, pipet 1 ul of distilled H20 onto the machine. <br />
#* Hit "collect" in order to run the sample to clean the machine.Wipe off the remaining water with a kimwipe.<br />
#* Pipet 1 ul of EB buffer onto the machine. Hit "collect" in order to run the sample to clean the machine.Wipe off the remaining buffer with a kimwipe.<br />
#* Pipet 1 ul of a miniprep sample onto the machine. Hit "collect" in order to run the sample to clean the machine.Wipe off the remaining sample with a kimwipe, and record the concentration of the miniprep (ng/ul).<br />
#* Repeat the process of pipeting a sample and "collected" until all samples have been measured.</div>Tjiang