Bitan:Measuring DNA concentration by absorbance
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<o:DocumentProperties> <o:Author>Farid Rahimi</o:Author> <o:Template>Normal</o:Template> <o:LastAuthor>Farid Rahimi</o:LastAuthor> <o:Revision>6</o:Revision> <o:Created>2009-12-02T23:57:00Z</o:Created> <o:LastSaved>2009-12-03T00:00:00Z</o:LastSaved> <o:Pages>1</o:Pages> <o:Company>UCLA</o:Company> <o:Lines>1</o:Lines> <o:Paragraphs>1</o:Paragraphs> <o:Version>11.1282</o:Version> </o:DocumentProperties> <o:OfficeDocumentSettings> <o:AllowPNG/> </o:OfficeDocumentSettings>
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border:none;mso-border-alt:solid windowtext .5pt;mso-padding-alt:0cm 5.4pt 0cm 5.4pt'> <tr> <td width=443 valign=top style='width:442.8pt;border:solid windowtext .5pt; background:#666666;mso-shading:windowtext;mso-pattern:solid #666666; padding:0cm 5.4pt 0cm 5.4pt'> <p class=MsoNormal align=center style='text-align:center'><span style='font-size:14.0pt;font-family:Calibri;color:#CCFFFF'><b>Measuring DNA product by UV absorbance<o:p></o:p></b></span></p> <p class=MsoNormal><span style='font-family:Calibri;color:#CCFFFF'><![if !supportEmptyParas]> <![endif]><o:p></o:p></span></p> <p class=MsoNormal><span style='font-family:Calibri;color:#CCFFFF'>The Eppendorf BioPhotometer (in Dr. Spencer’s Lab.) was used.<span style="mso-spacerun: yes"> </span><o:p></o:p></span></p>
<p class=MsoNormal style='margin-left:18.0pt;text-indent:-18.0pt;mso-list: l11 level1 lfo1;tab-stops:list 18.0pt'><![if !supportLists]><span style='font-family:Calibri;color:#CCFFFF'>1.<span style='font:7.0pt "Times New Roman"'> </span></span><![endif]><span style='font-family:Calibri;color:#CCFFFF'>Switch ON.<o:p></o:p></span></p> <p class=MsoNormal style='margin-left:18.0pt;text-indent:-18.0pt;mso-list: l11 level1 lfo1'><![if !supportLists]><span style='font-family:Calibri; color:#CCFFFF'>2.<span style='font:7.0pt "Times New Roman"'> </span></span><![endif]><span style='font-family:Calibri;color:#CCFFFF'>Select dsDNA from the options, showing 1 OD<sub>260</sub> = 50 μg/ml.<o:p></o:p></span></p> <p class=MsoNormal style='margin-left:18.0pt;text-indent:-18.0pt;mso-list: l11 level1 lfo1'><![if !supportLists]><span style='font-family:Calibri; color:#CCFFFF'>3.<span style='font:7.0pt "Times New Roman"'>
</span></span><![endif]><span style='font-family:Calibri;color:#CCFFFF'>Add 100 ml water to the plastic cuvette appropriate for use on this machine, and press ‘Blank’.<span style="mso-spacerun: yes"> </span>The monitor should show ‘0.000’.<o:p></o:p></span></p> <p class=MsoNormal style='margin-left:18.0pt;text-indent:-18.0pt;mso-list: l11 level1 lfo1'><![if !supportLists]><span style='font-family:Calibri; color:#CCFFFF'>4.<span style='font:7.0pt "Times New Roman"'> </span></span><![endif]><span style='font-family:Calibri;color:#CCFFFF'>Add 50 or 100-fold diluted DNA sample in the cuvette, insert the cuvette into the reader chamber, and press Sample.<span style="mso-spacerun: yes"> </span>The minimum required for measurement is 50 μl.<span style="mso-spacerun: yes"> </span><o:p></o:p></span></p> <p class=MsoNormal style='margin-left:18.0pt;text-indent:-18.0pt;mso-list: l11 level1 lfo1'><![if !supportLists]><span style='font-family:Calibri; color:#CCFFFF'>5.<span style='font:7.0pt "Times New Roman"'>
</span></span><![endif]><span style='font-family:Calibri;color:#CCFFFF'>Aspirate the sample off the cuvette and add the next sample. There is no need to wash between sample measurements.<o:p></o:p></span></p> <p class=MsoNormal style='margin-left:18.0pt;text-indent:-18.0pt;mso-list: l11 level1 lfo1'><![if !supportLists]><span style='font-family:Calibri; color:#CCFFFF'>6.<span style='font:7.0pt "Times New Roman"'> </span></span><![endif]><span style='font-family:Calibri;color:#CCFFFF'>Record the results. Calculate the actual concentration of purified DNA for the next </span><span style='font-family:Calibri;color:#CCFFFF;mso-fareast-language:KO'><i>in vitro</i></span><span style='font-size:16.0pt;font-family:Calibri;color:#CCFFFF'> </span><span style='font-family:Calibri;color:#CCFFFF'>transcription step.<span style="mso-spacerun: yes"> </span>Take into account the dilution factor used. For reference, when A260/A280 is 1.8 for DNA or 2.0 for RNA, the sample is ultra-pure.<o:p></o:p></span></p> <p class=MsoNormal><span style='font-family:Calibri;color:#CCFFFF'>The same measurements can be performed in our lab using the old spectrophotometer and the Quartz cuvette. Make sure the cuvette is washed properly because the residual protein may lead to erroneous A260/A280 ratios.<o:p></o:p></span></p>
<p class=MsoNormal align=center style='text-align:center'><span style='font-family:Calibri;color:#CCFFFF'><![if !supportEmptyParas]> <![endif]><o:p></o:p></span></p> <p class=MsoNormal align=center style='text-align:center'><span style='font-family:Calibri;color:#CCFFFF'><a href="http://openwetware.org/wiki/Bitan:todo"><span style='color:#CCFFFF'>Back to To-Do List</span></a></span><span style='font-family:Calibri;color:#33CCCC'><o:p></o:p></span></p> </td> </tr>
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