http://openwetware.org/index.php?title=Corum:DNA_Quantification&feed=atom&action=history
Corum:DNA Quantification - Revision history
2013-06-19T16:20:14Z
Revision history for this page on the wiki
MediaWiki 1.13.2
http://openwetware.org/index.php?title=Corum:DNA_Quantification&diff=630035&oldid=prev
Sean P Corum: /* Procedure */
2012-09-26T17:35:14Z
<p><span class="autocomment">Procedure</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 17:35, 26 September 2012</td>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Prepare 2.5X quantifluore from 200X stock for N samples.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Prepare 2.5X quantifluore from 200X stock for N samples.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#* V<sub>TE</sub> = 1.2 × [80 + 40 × N] μL = 1.2 × 40 × [N + 2] μL </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#* V<sub>TE</sub> = 1.2 × [80 + 40 × N] μL = 1.2 × 40 × [N + 2] μL </div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>#* V<sub>200X</sub> = [V<sub>TE</sub> / (200/5 - 1 ) μL = V<sub>TE</sub> / 39] μL</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>#* V<sub>200X</sub> = [V<sub>TE</sub> / (200/5 - 1 )<ins class="diffchange diffchange-inline">] </ins>μL = V<sub>TE</sub> / 39] μL</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#* V<sub>2.5X</sub> = [V<sub>TE</sub> + V<sub>200X</sub>] μL</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#* V<sub>2.5X</sub> = [V<sub>TE</sub> + V<sub>200X</sub>] μL</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Aliquot 80 v blank TE, standard DNA curve, and diluted DNA samples onto a 96 well plate. Add 20 μL 2.5X quantifluore per well. (Note: final quantifluore concentration is 0.5X, not 1X.)</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Aliquot 80 v blank TE, standard DNA curve, and diluted DNA samples onto a 96 well plate. Add 20 μL 2.5X quantifluore per well. (Note: final quantifluore concentration is 0.5X, not 1X.)</div></td></tr>
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Sean P Corum
http://openwetware.org/index.php?title=Corum:DNA_Quantification&diff=630034&oldid=prev
Sean P Corum: /* Procedure */
2012-09-26T17:34:54Z
<p><span class="autocomment">Procedure</span></p>
<table style="background-color: white; color:black;">
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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 17:34, 26 September 2012</td>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#* 1/4000 = 2.5 μL 1/100 DNA + 97.5 μL TE</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#* 1/4000 = 2.5 μL 1/100 DNA + 97.5 μL TE</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Prepare 2.5X quantifluore from 200X stock for N samples.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Prepare 2.5X quantifluore from 200X stock for N samples.</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>#* V<sub>TE</sub> = [<del class="diffchange diffchange-inline">(</del>80 + 40 × N<del class="diffchange diffchange-inline">) × </del>1.2] μL</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>#* V<sub>TE</sub> = <ins class="diffchange diffchange-inline">1.2 × </ins>[80 + 40 × N<ins class="diffchange diffchange-inline">] μL = </ins>1.<ins class="diffchange diffchange-inline">2 × 40 × [N + </ins>2] μL </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#* V<sub>200X</sub> = [V<sub>TE</sub> / (200/5 - 1 ) μL = V<sub>TE</sub> / 39] μL</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#* V<sub>200X</sub> = [V<sub>TE</sub> / (200/5 - 1 ) μL = V<sub>TE</sub> / 39] μL</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#* V<sub>2.5X</sub> = [V<sub>TE</sub> + V<sub>200X</sub>] μL</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#* V<sub>2.5X</sub> = [V<sub>TE</sub> + V<sub>200X</sub>] μL</div></td></tr>
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Sean P Corum
http://openwetware.org/index.php?title=Corum:DNA_Quantification&diff=610061&oldid=prev
Sean P Corum: New page: ==Overview== DNA quantification using fluorescent quantifluore dye. ==Materials== * 200X stock quantifluore dye * 100 ng/μL stock DNA standard (salmon sperm) * DNA samples ==Procedure=...
2012-06-25T21:44:30Z
<p>New page: ==Overview== DNA quantification using fluorescent quantifluore dye. ==Materials== * 200X stock quantifluore dye * 100 ng/μL stock DNA standard (salmon sperm) * DNA samples ==Procedure=...</p>
<p><b>New page</b></p><div>==Overview==<br />
DNA quantification using fluorescent quantifluore dye.<br />
<br />
==Materials==<br />
<br />
* 200X stock quantifluore dye<br />
* 100 ng/μL stock DNA standard (salmon sperm)<br />
* DNA samples<br />
<br />
==Procedure==<br />
<br />
# Prepare DNA standard curve:<br />
#* 1 ng/μL = 1 μL 100 ng/μL standard DNA + 99 μL TE<br />
#* 0.1 ng/μL = 10 μL 1 ng/μL standard DNA + 90 μL TE<br />
#* 0.01 ng/μL = 10 μL 0.1 ng/μL standard DNA + 90 μL TE<br />
#Prepare DNA dilution samples.<br />
#* 1/100 = 1 μL sample DNA + 99 μL TE<br />
#* 1/2000 = 5 μL 1/100 DNA + 95 μL TE<br />
#* 1/4000 = 2.5 μL 1/100 DNA + 97.5 μL TE<br />
# Prepare 2.5X quantifluore from 200X stock for N samples.<br />
#* V<sub>TE</sub> = [(80 + 40 × N) × 1.2] μL<br />
#* V<sub>200X</sub> = [V<sub>TE</sub> / (200/5 - 1 ) μL = V<sub>TE</sub> / 39] μL<br />
#* V<sub>2.5X</sub> = [V<sub>TE</sub> + V<sub>200X</sub>] μL<br />
# Aliquot 80 v blank TE, standard DNA curve, and diluted DNA samples onto a 96 well plate. Add 20 μL 2.5X quantifluore per well. (Note: final quantifluore concentration is 0.5X, not 1X.)<br />
# With the plate reader, shake double-orbital fast 30 s, pause 10 min, shake double-orbital fast 30 s again, and then read end-point fluorescence at 485/528 nm. The plate reader returns the data in ng/μL concentration values.<br />
# Convert values to nM:<br />
#* 1 nM = 10<sup>6</sup>/(640 L) ng/μL (L = length of dsDNA; use 320 for ssDNA)<br />
<br />
==Notes==<br />
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!<br />
#List troubleshooting tips here. <br />
#You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.<br />
#Anecdotal observations that might be of use to others can also be posted here. <br />
<br />
Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.<br />
<br />
==Contact==<br />
* [http://openwetware.org/wiki/User:Sean_P_Corum Sean P Corum]<br />
<br />
or instead, [[Talk:{{PAGENAME}}|discuss this protocol]]. <br />
<br />
<!-- You can tag this protocol with various categories. See the [[Categories]] page for more information. --><br />
<br />
==Digital Signature==<br />
*'''SC 17:44, 25 June 2012 (EDT)''':<br />
<br />
[[Category:Protocol]]<br />
<br />
[[Category:DNA]]<br />
<br />
[[Category:Quantification]]</div>
Sean P Corum