http://2013.igem.org/wiki/index.php?title=Team:Paris_Saclay/extraction&feed=atom&action=historyTeam:Paris Saclay/extraction - Revision history2024-03-28T21:02:26ZRevision history for this page on the wikiMediaWiki 1.16.5http://2013.igem.org/wiki/index.php?title=Team:Paris_Saclay/extraction&diff=288772&oldid=prevThaonguyenlelam: /* Extraction of the Genomic DNA from Bacteria by using NucleoSpin® Tissue */2013-10-04T09:22:04Z<p><span class="autocomment">Extraction of the Genomic DNA from Bacteria by using NucleoSpin® Tissue</span></p>
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<td colspan='2' style="background-color: white; color:black;">← Older revision</td>
<td colspan='2' style="background-color: white; color:black;">Revision as of 09:22, 4 October 2013</td>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Add 500 µl Wash Buffer BW to the column and centrifuge for 1 min at 13,200*g.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Add 500 µl Wash Buffer BW to the column and centrifuge for 1 min at 13,200*g.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Discard the flow-though and place the column back into the collection tube.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Discard the flow-though and place the column back into the collection tube.</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"></del></div></td><td colspan="2"> </td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>2nd wash:</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>2nd wash:</div></td></tr>
</table>Thaonguyenlelamhttp://2013.igem.org/wiki/index.php?title=Team:Paris_Saclay/extraction&diff=248220&oldid=prevSolenneParisSaclay: /* Extraction of the Genomic DNA from Bacteria by using NucleoSpin® Tissue */2013-09-29T12:50:39Z<p><span class="autocomment">Extraction of the Genomic DNA from Bacteria by using NucleoSpin® Tissue</span></p>
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<td colspan='2' style="background-color: white; color:black;">← Older revision</td>
<td colspan='2' style="background-color: white; color:black;">Revision as of 12:50, 29 September 2013</td>
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<td colspan="2" class="diff-lineno">Line 6:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><u>1. Prepare sample:</u></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><u><ins class="diffchange diffchange-inline">'''</ins>1. Prepare sample:<ins class="diffchange diffchange-inline">'''</ins></u></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Take 1 ml of bacteria culture in a 1.5 ml microcentrifuge tube (eppendorf), prepare one <del class="diffchange diffchange-inline">samples</del>.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Take 1 ml of bacteria culture in a 1.5 ml microcentrifuge tube (eppendorf), prepare one <ins class="diffchange diffchange-inline">sample</ins>.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Centrifuge the culture for 2 min at 13,200*g.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Centrifuge the culture for 2 min at 13,200*g.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Remove and discard supernatant.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Remove and discard supernatant.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><u>2. Pre-lyse sample:</u></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><u><ins class="diffchange diffchange-inline">'''</ins>2. Pre-lyse sample:<ins class="diffchange diffchange-inline">'''</ins></u></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Resuspend and dissolve the pellet in 180 µl Lysis Buffer T1 by pipetting up and down.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Resuspend and dissolve the pellet in 180 µl Lysis Buffer T1 by pipetting up and down.</div></td></tr>
<tr><td colspan="2" class="diff-lineno">Line 20:</td>
<td colspan="2" class="diff-lineno">Line 20:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Heat up the Elution Buffer BE at 70°C.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Heat up the Elution Buffer BE at 70°C.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><u>3. Lyse sample:</u></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><u><ins class="diffchange diffchange-inline">'''</ins>3. Lyse sample:<ins class="diffchange diffchange-inline">'''</ins></u></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Vortex the sample.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Vortex the sample.</div></td></tr>
<tr><td colspan="2" class="diff-lineno">Line 27:</td>
<td colspan="2" class="diff-lineno">Line 27:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Discard the insoluble particles.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Discard the insoluble particles.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><u>4. Adjust DNA binding conditions:</u></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><u><ins class="diffchange diffchange-inline">'''</ins>4. Adjust DNA binding conditions:<ins class="diffchange diffchange-inline">'''</ins></u></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Add 210 µl ethanol 100% to the sample, stringy precipitates are visible.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Add 210 µl ethanol 100% to the sample, stringy precipitates are visible.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Agitate vigorously with hand.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Agitate vigorously with hand.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><u>5. Bind DNA:</u></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><u><ins class="diffchange diffchange-inline">'''</ins>5. Bind DNA:<ins class="diffchange diffchange-inline">'''</ins></u></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Place a NucleoSpin® Tissue Column into a collection tube.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Place a NucleoSpin® Tissue Column into a collection tube.</div></td></tr>
<tr><td colspan="2" class="diff-lineno">Line 39:</td>
<td colspan="2" class="diff-lineno">Line 39:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Discard the flow-though and place the column back into the collection tube.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Discard the flow-though and place the column back into the collection tube.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><u>6. Wash silica membrane:</u></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><u><ins class="diffchange diffchange-inline">'''</ins>6. Wash silica membrane:<ins class="diffchange diffchange-inline">'''</ins></u></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>1st wash:</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>1st wash:</div></td></tr>
<tr><td colspan="2" class="diff-lineno">Line 52:</td>
<td colspan="2" class="diff-lineno">Line 52:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Discard the flow-though and place the column back into the collection tube.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Discard the flow-though and place the column back into the collection tube.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><u>7. Dry silica membrane:</u></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><u><ins class="diffchange diffchange-inline">'''</ins>7. Dry silica membrane:<ins class="diffchange diffchange-inline">'''</ins></u></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Centrifuge the column for 1 min at 13,200*g to remove the residual ethanol.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Centrifuge the column for 1 min at 13,200*g to remove the residual ethanol.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Discard the flow-though.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Discard the flow-though.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><u>8. Elute highly pure DNA:</u></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><u><ins class="diffchange diffchange-inline">'''</ins>8. Elute highly pure DNA:<ins class="diffchange diffchange-inline">'''</ins></u></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Place the column into a 1.5 ml microcentrifuge tube, add 100 µl Elution Buffer BE (pre-warmed at 70°C).</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Place the column into a 1.5 ml microcentrifuge tube, add 100 µl Elution Buffer BE (pre-warmed at 70°C).</div></td></tr>
<tr><td colspan="2" class="diff-lineno">Line 65:</td>
<td colspan="2" class="diff-lineno">Line 65:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Discard the column.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Discard the column.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><u>9. Analysis:</u></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><u><ins class="diffchange diffchange-inline">'''</ins>9. Analysis:<ins class="diffchange diffchange-inline">'''</ins></u></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Check the presence of DNA and determine its concentration by using a nanodrop spectrophotometer.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Check the presence of DNA and determine its concentration by using a nanodrop spectrophotometer.</div></td></tr>
</table>SolenneParisSaclayhttp://2013.igem.org/wiki/index.php?title=Team:Paris_Saclay/extraction&diff=146856&oldid=prevCarolineMir: Created page with "{{Team:Paris_Saclay/incl_debut_generique}} = '''Extraction of the Genomic DNA from Bacteria by using NucleoSpin® Tissue''' = The experiment is performed with buffer solutions ..."2013-09-22T10:47:29Z<p>Created page with "{{Team:Paris_Saclay/incl_debut_generique}} = '''Extraction of the Genomic DNA from Bacteria by using NucleoSpin® Tissue''' = The experiment is performed with buffer solutions ..."</p>
<p><b>New page</b></p><div>{{Team:Paris_Saclay/incl_debut_generique}}<br />
= '''Extraction of the Genomic DNA from Bacteria by using NucleoSpin® Tissue''' =<br />
<br />
<br />
The experiment is performed with buffer solutions of NucleoSpin® Tissue, protocol 6.2 for bacteria.<br />
<br />
<br />
<u>1. Prepare sample:</u><br />
<br />
Take 1 ml of bacteria culture in a 1.5 ml microcentrifuge tube (eppendorf), prepare one samples.<br />
Centrifuge the culture for 2 min at 13,200*g.<br />
Remove and discard supernatant.<br />
<br />
<u>2. Pre-lyse sample:</u><br />
<br />
Resuspend and dissolve the pellet in 180 µl Lysis Buffer T1 by pipetting up and down.<br />
Add 25 µl Proteinase Buffer PB (Proteinase K).<br />
Agitate vigorously with vortex and incubate at 56°C for 1 h (until complete lysis).<br />
Vortex every 10 min during incubation.<br />
Heat up the Elution Buffer BE at 70°C.<br />
<br />
<u>3. Lyse sample:</u><br />
<br />
Vortex the sample.<br />
Add 200 µl Lysis Buffer B3, Agitate vigorously with hand and incubate at 70°C for 10 min.<br />
If the insoluble particles are visible, centrifuge the samples for 5 min at 13,200*g and transfer the supernatant to a new 1.5 ml microcentrifuge tube.<br />
Discard the insoluble particles.<br />
<br />
<u>4. Adjust DNA binding conditions:</u><br />
<br />
Add 210 µl ethanol 100% to the sample, stringy precipitates are visible.<br />
Agitate vigorously with hand.<br />
<br />
<u>5. Bind DNA:</u><br />
<br />
Place a NucleoSpin® Tissue Column into a collection tube.<br />
Apply the sample to the column, make sure to load all of the precipitate.<br />
Centrifuge for 1 min at 13,200*g.<br />
Discard the flow-though and place the column back into the collection tube.<br />
<br />
<u>6. Wash silica membrane:</u><br />
<br />
1st wash:<br />
<br />
Add 500 µl Wash Buffer BW to the column and centrifuge for 1 min at 13,200*g.<br />
Discard the flow-though and place the column back into the collection tube.<br />
<br />
<br />
2nd wash:<br />
<br />
Add 600 µl Wash Buffer B5 to the column and centrifuge for 1 min at 13,200*g.<br />
Discard the flow-though and place the column back into the collection tube.<br />
<br />
<u>7. Dry silica membrane:</u><br />
<br />
Centrifuge the column for 1 min at 13,200*g to remove the residual ethanol.<br />
Discard the flow-though.<br />
<br />
<u>8. Elute highly pure DNA:</u><br />
<br />
Place the column into a 1.5 ml microcentrifuge tube, add 100 µl Elution Buffer BE (pre-warmed at 70°C).<br />
Incubate at room temperature for 1 min.<br />
Centrifuge for 1 min at 13,200*g.<br />
<br />
Discard the column.<br />
<br />
<u>9. Analysis:</u><br />
<br />
Check the presence of DNA and determine its concentration by using a nanodrop spectrophotometer.<br />
<br />
<br />
[[File:Psextraction.jpg|center|300px|caption]]<br />
<br />
<br />
<br />
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{{Team:Paris_Saclay/incl_fin}}</div>CarolineMir