User:Neil J. Parikh/Ethanol Precipitation Explained: Difference between revisions

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Revision as of 12:04, 11 March 2008

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<div class=Section1>

<h2>Ethanol Precipitation (for &gt; 200bp)</h2>

<p class=MsoListParagraphCxSpFirst style='margin-left:.75in;mso-add-space:auto; text-indent:-.25in;mso-list:l3 level1 lfo2'><![if !supportLists]><span style='font-family:Symbol;mso-fareast-font-family:Symbol;mso-bidi-font-family: Symbol;mso-bidi-font-weight:bold'><span style='mso-list:Ignore'>®<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]>Ethanol Precipitation is used to concentrate DNA or RNA from dilute, aqueous solution. Such is needed for nearly all protocols involving purification of nucleic acids from cells.<b><o:p></o:p></b></p>

<p class=MsoListParagraphCxSpMiddle style='margin-left:.75in;mso-add-space: auto;text-indent:-.25in;mso-list:l3 level1 lfo2'><![if !supportLists]><span style='font-family:Symbol;mso-fareast-font-family:Symbol;mso-bidi-font-family: Symbol;mso-bidi-font-weight:bold'><span style='mso-list:Ignore'>®<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]>Salt and ethanol are added to the aqueous solution, which forces the nucleic acid to precipitate out of solution. This precipitated nucleic acid (DNA) can now be separated from the solution by centrifuging it.<b><o:p></o:p></b></p>

<p class=MsoListParagraphCxSpLast style='margin-left:.75in;mso-add-space:auto; text-indent:-.25in;mso-list:l3 level1 lfo2'><![if !supportLists]><span style='font-family:Symbol;mso-fareast-font-family:Symbol;mso-bidi-font-family: Symbol'><span style='mso-list:Ignore'>®<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><b style='mso-bidi-font-weight:normal'><i style='mso-bidi-font-style:normal'>Use: 3 volumes of COLD 100% Ethanol (EtOH), 1/10 volume 3M Sodium Acetate, 1µl</i></b><i style='mso-bidi-font-style:normal'> <b style='mso-bidi-font-weight:normal'>GlycoBlue</b></i></p>

<h3 style='margin-left:.5in'>Procedure:</h3>

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0in 5.4pt 0in 5.4pt'>
<tr style='mso-yfti-irow:0;mso-yfti-firstrow:yes'>
 <td width=26 valign=top style='width:19.2pt;border:solid black 1.0pt;
 mso-border-themecolor:text1;mso-border-alt:solid black .5pt;mso-border-themecolor:
 text1;padding:0in 5.4pt 0in 5.4pt'>
 <p class=MsoListParagraphCxSpFirst style='margin:0in;margin-bottom:.0001pt;
 mso-add-space:auto;line-height:normal'><o:p>&nbsp;</o:p></p>
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 <td width=262 valign=top style='width:196.8pt;border:solid black 1.0pt;
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 Procedure<o:p></o:p></i></p>
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 mso-border-left-themecolor:text1;mso-border-alt:solid black .5pt;mso-border-themecolor:
 text1;padding:0in 5.4pt 0in 5.4pt'>
 <p class=MsoListParagraphCxSpMiddle style='margin:0in;margin-bottom:.0001pt;
 mso-add-space:auto;line-height:normal'><i style='mso-bidi-font-style:normal'>For
 <span style='mso-spacerun:yes'> </span>50µl<o:p></o:p></i></p>
 </td>
 <td width=127 valign=top style='width:94.95pt;border:solid black 1.0pt;
 mso-border-themecolor:text1;border-left:none;mso-border-left-alt:solid black .5pt;
 mso-border-left-themecolor:text1;mso-border-alt:solid black .5pt;mso-border-themecolor:
 text1;padding:0in 5.4pt 0in 5.4pt'>
 <p class=MsoListParagraphCxSpLast style='margin:0in;margin-bottom:.0001pt;
 mso-add-space:auto;line-height:normal'><i style='mso-bidi-font-style:normal'>For
 <span style='mso-spacerun:yes'> </span>100µl<o:p></o:p></i></p>
 </td>
</tr>
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 <td width=26 valign=top style='width:19.2pt;border:solid black 1.0pt;
 mso-border-themecolor:text1;border-top:none;mso-border-top-alt:solid black .5pt;
 mso-border-top-themecolor:text1;mso-border-alt:solid black .5pt;mso-border-themecolor:
 text1;padding:0in 5.4pt 0in 5.4pt'>
 <p class=MsoListParagraphCxSpFirst style='margin:0in;margin-bottom:.0001pt;
 mso-add-space:auto;line-height:normal'>1.</p>
 </td>
 <td width=262 valign=top style='width:196.8pt;border-top:none;border-left:
 none;border-bottom:solid black 1.0pt;mso-border-bottom-themecolor:text1;
 border-right:solid black 1.0pt;mso-border-right-themecolor:text1;mso-border-top-alt:
 solid black .5pt;mso-border-top-themecolor:text1;mso-border-left-alt:solid black .5pt;
 mso-border-left-themecolor:text1;mso-border-alt:solid black .5pt;mso-border-themecolor:
 text1;padding:0in 5.4pt 0in 5.4pt'>
 <p class=MsoListParagraphCxSpMiddle style='margin:0in;margin-bottom:.0001pt;
 mso-add-space:auto;line-height:normal'>Add 3 volumes COLD 100% EtOH</p>
 </td>
 <td width=121 valign=top style='width:90.45pt;border-top:none;border-left:
 none;border-bottom:solid black 1.0pt;mso-border-bottom-themecolor:text1;
 border-right:solid black 1.0pt;mso-border-right-themecolor:text1;mso-border-top-alt:
 solid black .5pt;mso-border-top-themecolor:text1;mso-border-left-alt:solid black .5pt;
 mso-border-left-themecolor:text1;mso-border-alt:solid black .5pt;mso-border-themecolor:
 text1;padding:0in 5.4pt 0in 5.4pt'>
 <p class=MsoListParagraphCxSpMiddle style='margin:0in;margin-bottom:.0001pt;
 mso-add-space:auto;line-height:normal'>Add 150µl EtOH</p>
 </td>
 <td width=127 valign=top style='width:94.95pt;border-top:none;border-left:
 none;border-bottom:solid black 1.0pt;mso-border-bottom-themecolor:text1;
 border-right:solid black 1.0pt;mso-border-right-themecolor:text1;mso-border-top-alt:
 solid black .5pt;mso-border-top-themecolor:text1;mso-border-left-alt:solid black .5pt;
 mso-border-left-themecolor:text1;mso-border-alt:solid black .5pt;mso-border-themecolor:
 text1;padding:0in 5.4pt 0in 5.4pt'>
 <p class=MsoListParagraphCxSpLast style='margin:0in;margin-bottom:.0001pt;
 mso-add-space:auto;line-height:normal'>Add 300µl EtOH</p>
 </td>
</tr>
<tr style='mso-yfti-irow:2'>
 <td width=26 valign=top style='width:19.2pt;border:solid black 1.0pt;
 mso-border-themecolor:text1;border-top:none;mso-border-top-alt:solid black .5pt;
 mso-border-top-themecolor:text1;mso-border-alt:solid black .5pt;mso-border-themecolor:
 text1;padding:0in 5.4pt 0in 5.4pt'>
 <p class=MsoListParagraphCxSpFirst style='margin:0in;margin-bottom:.0001pt;
 mso-add-space:auto;line-height:normal'>2.</p>
 </td>
 <td width=262 valign=top style='width:196.8pt;border-top:none;border-left:
 none;border-bottom:solid black 1.0pt;mso-border-bottom-themecolor:text1;
 border-right:solid black 1.0pt;mso-border-right-themecolor:text1;mso-border-top-alt:
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 text1;padding:0in 5.4pt 0in 5.4pt'>
 <p class=MsoListParagraphCxSpMiddle style='margin:0in;margin-bottom:.0001pt;
 mso-add-space:auto;line-height:normal'>Add 1/10 volume sodium acetate (NaAc)</p>
 </td>
 <td width=121 valign=top style='width:90.45pt;border-top:none;border-left:
 none;border-bottom:solid black 1.0pt;mso-border-bottom-themecolor:text1;
 border-right:solid black 1.0pt;mso-border-right-themecolor:text1;mso-border-top-alt:
 solid black .5pt;mso-border-top-themecolor:text1;mso-border-left-alt:solid black .5pt;
 mso-border-left-themecolor:text1;mso-border-alt:solid black .5pt;mso-border-themecolor:
 text1;padding:0in 5.4pt 0in 5.4pt'>
 <p class=MsoListParagraphCxSpMiddle style='margin:0in;margin-bottom:.0001pt;
 mso-add-space:auto;line-height:normal'>Add 20 µl NaAc</p>
 </td>
 <td width=127 valign=top style='width:94.95pt;border-top:none;border-left:
 none;border-bottom:solid black 1.0pt;mso-border-bottom-themecolor:text1;
 border-right:solid black 1.0pt;mso-border-right-themecolor:text1;mso-border-top-alt:
 solid black .5pt;mso-border-top-themecolor:text1;mso-border-left-alt:solid black .5pt;
 mso-border-left-themecolor:text1;mso-border-alt:solid black .5pt;mso-border-themecolor:
 text1;padding:0in 5.4pt 0in 5.4pt'>
 <p class=MsoListParagraphCxSpLast style='margin:0in;margin-bottom:.0001pt;
 mso-add-space:auto;line-height:normal'>Add 40µl NaAc</p>
 </td>
</tr>
<tr style='mso-yfti-irow:3'>
 <td width=26 valign=top style='width:19.2pt;border:solid black 1.0pt;
 mso-border-themecolor:text1;border-top:none;mso-border-top-alt:solid black .5pt;
 mso-border-top-themecolor:text1;mso-border-alt:solid black .5pt;mso-border-themecolor:
 text1;padding:0in 5.4pt 0in 5.4pt'>
 <p class=MsoListParagraphCxSpFirst style='margin:0in;margin-bottom:.0001pt;
 mso-add-space:auto;line-height:normal'>3.</p>
 </td>
 <td width=510 colspan=3 valign=top style='width:382.2pt;border-top:none;
 border-left:none;border-bottom:solid black 1.0pt;mso-border-bottom-themecolor:
 text1;border-right:solid black 1.0pt;mso-border-right-themecolor:text1;
 mso-border-top-alt:solid black .5pt;mso-border-top-themecolor:text1;
 mso-border-left-alt:solid black .5pt;mso-border-left-themecolor:text1;
 mso-border-alt:solid black .5pt;mso-border-themecolor:text1;padding:0in 5.4pt 0in 5.4pt'>
 <p class=MsoListParagraphCxSpLast style='margin:0in;margin-bottom:.0001pt;
 mso-add-space:auto;line-height:normal'>Add 1µl GlycoBlue</p>
 </td>
</tr>
<tr style='mso-yfti-irow:4'>
 <td width=26 valign=top style='width:19.2pt;border:solid black 1.0pt;
 mso-border-themecolor:text1;border-top:none;mso-border-top-alt:solid black .5pt;
 mso-border-top-themecolor:text1;mso-border-alt:solid black .5pt;mso-border-themecolor:
 text1;padding:0in 5.4pt 0in 5.4pt'>
 <p class=MsoListParagraphCxSpFirst style='margin:0in;margin-bottom:.0001pt;
 mso-add-space:auto;line-height:normal'>4.</p>
 </td>
 <td width=510 colspan=3 valign=top style='width:382.2pt;border-top:none;
 border-left:none;border-bottom:solid black 1.0pt;mso-border-bottom-themecolor:
 text1;border-right:solid black 1.0pt;mso-border-right-themecolor:text1;
 mso-border-top-alt:solid black .5pt;mso-border-top-themecolor:text1;
 mso-border-left-alt:solid black .5pt;mso-border-left-themecolor:text1;
 mso-border-alt:solid black .5pt;mso-border-themecolor:text1;padding:0in 5.4pt 0in 5.4pt'>
 <p class=MsoListParagraphCxSpLast style='margin:0in;margin-bottom:.0001pt;
 mso-add-space:auto;line-height:normal'>Incubate for 1hour at <b
 style='mso-bidi-font-weight:normal'><i style='mso-bidi-font-style:normal'>-</i></b>80°C
 </p>
 </td>
</tr>
<tr style='mso-yfti-irow:5'>
 <td width=26 valign=top style='width:19.2pt;border:solid black 1.0pt;
 mso-border-themecolor:text1;border-top:none;mso-border-top-alt:solid black .5pt;
 mso-border-top-themecolor:text1;mso-border-alt:solid black .5pt;mso-border-themecolor:
 text1;padding:0in 5.4pt 0in 5.4pt'>
 <p class=MsoListParagraphCxSpFirst style='margin:0in;margin-bottom:.0001pt;
 mso-add-space:auto;line-height:normal'>5.</p>
 </td>
 <td width=510 colspan=3 valign=top style='width:382.2pt;border-top:none;
 border-left:none;border-bottom:solid black 1.0pt;mso-border-bottom-themecolor:
 text1;border-right:solid black 1.0pt;mso-border-right-themecolor:text1;
 mso-border-top-alt:solid black .5pt;mso-border-top-themecolor:text1;
 mso-border-left-alt:solid black .5pt;mso-border-left-themecolor:text1;
 mso-border-alt:solid black .5pt;mso-border-themecolor:text1;padding:0in 5.4pt 0in 5.4pt'>
 <p class=MsoListParagraphCxSpLast style='margin:0in;margin-bottom:.0001pt;
 mso-add-space:auto;line-height:normal'>Centrifuge for 30 minutes at 0°C</p>
 </td>
</tr>
<tr style='mso-yfti-irow:6'>
 <td width=26 valign=top style='width:19.2pt;border:solid black 1.0pt;
 mso-border-themecolor:text1;border-top:none;mso-border-top-alt:solid black .5pt;
 mso-border-top-themecolor:text1;mso-border-alt:solid black .5pt;mso-border-themecolor:
 text1;padding:0in 5.4pt 0in 5.4pt'>
 <p class=MsoListParagraphCxSpFirst style='margin:0in;margin-bottom:.0001pt;
 mso-add-space:auto;line-height:normal'>6.</p>
 </td>
 <td width=510 colspan=3 valign=top style='width:382.2pt;border-top:none;
 border-left:none;border-bottom:solid black 1.0pt;mso-border-bottom-themecolor:
 text1;border-right:solid black 1.0pt;mso-border-right-themecolor:text1;
 mso-border-top-alt:solid black .5pt;mso-border-top-themecolor:text1;
 mso-border-left-alt:solid black .5pt;mso-border-left-themecolor:text1;
 mso-border-alt:solid black .5pt;mso-border-themecolor:text1;padding:0in 5.4pt 0in 5.4pt'>
 <p class=MsoListParagraphCxSpLast style='margin:0in;margin-bottom:.0001pt;
 mso-add-space:auto;line-height:normal'>Remove the supernatant carefully,
 ensuring not to remove any solid</p>
 </td>
</tr>
<tr style='mso-yfti-irow:7'>
 <td width=26 valign=top style='width:19.2pt;border:solid black 1.0pt;
 mso-border-themecolor:text1;border-top:none;mso-border-top-alt:solid black .5pt;
 mso-border-top-themecolor:text1;mso-border-alt:solid black .5pt;mso-border-themecolor:
 text1;padding:0in 5.4pt 0in 5.4pt'>
 <p class=MsoListParagraphCxSpFirst style='margin:0in;margin-bottom:.0001pt;
 mso-add-space:auto;line-height:normal'>7.</p>
 </td>
 <td width=510 colspan=3 valign=top style='width:382.2pt;border-top:none;
 border-left:none;border-bottom:solid black 1.0pt;mso-border-bottom-themecolor:
 text1;border-right:solid black 1.0pt;mso-border-right-themecolor:text1;
 mso-border-top-alt:solid black .5pt;mso-border-top-themecolor:text1;
 mso-border-left-alt:solid black .5pt;mso-border-left-themecolor:text1;
 mso-border-alt:solid black .5pt;mso-border-themecolor:text1;padding:0in 5.4pt 0in 5.4pt'>
 <p class=MsoListParagraphCxSpLast style='margin:0in;margin-bottom:.0001pt;
 mso-add-space:auto;line-height:normal'>Air Dry to allow EtOH to evaporate</p>
 </td>
</tr>
<tr style='mso-yfti-irow:8;mso-yfti-lastrow:yes'>
 <td width=26 valign=top style='width:19.2pt;border:solid black 1.0pt;
 mso-border-themecolor:text1;border-top:none;mso-border-top-alt:solid black .5pt;
 mso-border-top-themecolor:text1;mso-border-alt:solid black .5pt;mso-border-themecolor:
 text1;padding:0in 5.4pt 0in 5.4pt'>
 <p class=MsoListParagraphCxSpFirst style='margin:0in;margin-bottom:.0001pt;
 mso-add-space:auto;line-height:normal'>8.</p>
 </td>
 <td width=510 colspan=3 valign=top style='width:382.2pt;border-top:none;
 border-left:none;border-bottom:solid black 1.0pt;mso-border-bottom-themecolor:
 text1;border-right:solid black 1.0pt;mso-border-right-themecolor:text1;
 mso-border-top-alt:solid black .5pt;mso-border-top-themecolor:text1;
 mso-border-left-alt:solid black .5pt;mso-border-left-themecolor:text1;
 mso-border-alt:solid black .5pt;mso-border-themecolor:text1;padding:0in 5.4pt 0in 5.4pt'>
 <p class=MsoListParagraphCxSpLast style='margin:0in;margin-bottom:.0001pt;
 mso-add-space:auto;line-height:normal'>Resuspend the DNA in the desired
 buffer (usually H<sub>2</sub>O)</p>
 </td>
</tr>

</table>

<h3><span style='mso-tab-count:1'> </span>How Does it Work:</h3>

<p class=MsoListParagraphCxSpFirst style='margin-left:1.25in;mso-add-space: auto;text-indent:-.25in;mso-list:l1 level1 lfo5'><![if !supportLists]><span style='font-family:Symbol;mso-fareast-font-family:Symbol;mso-bidi-font-family: Symbol'><span style='mso-list:Ignore'>®<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]>DNA is a polar molecule and is hydrophilic because of the negatively charged phosphate (PO3-) groups along the sugar phosphate backbone</p>

<p class=MsoListParagraphCxSpMiddle style='margin-left:1.25in;mso-add-space: auto;text-indent:-.25in;mso-list:l1 level1 lfo5'><![if !supportLists]><span style='font-family:Symbol;mso-fareast-font-family:Symbol;mso-bidi-font-family: Symbol'><span style='mso-list:Ignore'>®<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]>Water is polar and hydrophilic as well and as such DNA dissolves easily in H<sub>2</sub>O</p>

<p class=MsoListParagraphCxSpMiddle style='margin-left:1.25in;mso-add-space: auto;text-indent:-.25in;mso-list:l1 level1 lfo5'><![if !supportLists]><span style='font-family:Symbol;mso-fareast-font-family:Symbol;mso-bidi-font-family: Symbol'><span style='mso-list:Ignore'>®<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]>The salt, Sodium Acetate in this case, is used to neutralize the charges on the sugar phosphate backbone. In the solution, Sodium Acetate breaks up into Na<sup>+</sup> and [CH3COO]<sup>-</sup> and so the positive sodium atoms neutralize the<span style='mso-spacerun:yes'> </span>negative charges on the phosphate groups. The DNA becomes more hydro<i style='mso-bidi-font-style:normal'>phobic</i> now, and therefore less soluble in H<sub>2</sub>O.</p>

<p class=MsoListParagraphCxSpMiddle style='margin-left:1.25in;mso-add-space: auto;text-indent:-.25in;mso-list:l1 level1 lfo5'><![if !supportLists]><span style='font-family:Symbol;mso-fareast-font-family:Symbol;mso-bidi-font-family: Symbol'><span style='mso-list:Ignore'>®<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]>Ethanol, which is added first, is used to change the dielectric constant of the solution. In H<sub>2</sub>O, which has a high dielectric constant, it is much more difficult for the Na<sup>+</sup><sub> </sub>and the PO<sub>3</sub><sup>-</sup><span style='font-size:10.0pt;mso-bidi-font-size: 11.0pt;line-height:115%'><span style='mso-spacerun:yes'> </span></span>to come together. EtOH on the other hand, has a lower dielectric constant, and therefore the sodium can neutralize the phosphate’s charges much easier, thereby making the molecule less hydrophilic and more insoluble.</p>

<p class=MsoListParagraphCxSpMiddle style='margin-left:1.25in;mso-add-space: auto;text-indent:-.25in;mso-list:l1 level1 lfo5'><![if !supportLists]><span style='font-family:Symbol;mso-fareast-font-family:Symbol;mso-bidi-font-family: Symbol'><span style='mso-list:Ignore'>®<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]>Incubation at <b style='mso-bidi-font-weight: normal'><i style='mso-bidi-font-style:normal'>-</i></b>80°C is used to allow the nucleic acids to precipitate</p>

<p class=MsoListParagraphCxSpMiddle style='margin-left:1.25in;mso-add-space: auto;text-indent:-.25in;mso-list:l1 level1 lfo5'><![if !supportLists]><span style='font-family:Symbol;mso-fareast-font-family:Symbol;mso-bidi-font-family: Symbol'><span style='mso-list:Ignore'>®<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]>Centrifuging the solution will allow the DNA to form a clump at the bottom, and allows for the supernatant to be removed, leaving the DNA behind</p>

<p class=MsoListParagraphCxSpLast style='margin-left:1.25in;mso-add-space:auto; text-indent:-.25in;mso-list:l1 level1 lfo5'><![if !supportLists]><span style='font-family:Symbol;mso-fareast-font-family:Symbol;mso-bidi-font-family: Symbol'><span style='mso-list:Ignore'>®<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]>Air drying allows the EtOH to evaporate from the precipitate, thus leaving behind only DNA</p>

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