Ethanol precipitation of small DNA fragments protocol

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(New page: <html><h2>Solutions/reagents:</h2><ul type="circle"><li>absolute ethanol stored at -20°C</li><li>DNA sample</li><li>95% ethanol stored at room temperature</li><li>water</li></ul><h2>Equip...)
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<html><h2>Solutions/reagents:</h2><ul type="circle"><li>absolute ethanol stored at -20°C</li><li>DNA sample</li><li>95% ethanol stored at room temperature</li><li>water</li></ul><h2>Equipment:</h2><ul type="circle"><li>Incubator</li><li>Centrifuge</li><li>Eppendorf tubes</li></ul><h2>Steps:</h2><ol><p><li>Measure out DNA sample into an Eppendorf tube.<br>Add  <b><font color=#357EC7>2</font></b> volumes <font color=#357EC7>absolute ethanol</font>.<br><font color = "#800517"><i>Generally the sample is in a 1.5 mL eppendorf tube. I recommend storing the absolute ethanol at -20°C.</i></font><br></li></p><p><li>Incubate at <b><font color=#357EC7>-80°C</font></b> for <b><font color=#357EC7>1 hr</font></b>.<br><font color = "#800517"><i>The long incubation time is critical for small fragments.</i></font><br></li></p><p><li>Centrifuge at a speed of at least <font color=#357EC7>10000 Xg</font> for <b><font color=#357EC7>30 mins</font></b> at <b><font color=#357EC7>0°C</font></b>, gently aspirate out the supernatant and discard it.<br></li></p><p><li>Add <b><font color=#357EC7>750 - 1000 µl</font></b> of <font color=#357EC7>95% ethanol</font>.<br><font color = "#800517"><i>Another critical step for small fragments under 200 base pairs. Generally washing involves adding the ethanol and inverting several times.</i></font><br></li></p><p><li>Centrifuge at a speed of at least <font color=#357EC7>10000 Xg</font> for <b><font color=#357EC7>10 mins</font></b> at <b><font color=#357EC7>4°C</font></b>, gently aspirate out the supernatant and discard it.<br></li></p><p><li>Dry the pellet in air. <br><font color = "#800517"><i>I generally let the pellet air dry completely such that it becomes white so that all residual ethanol is eliminated.</i></font><br></li></p><p><li>Add <font color=#357EC7>water</font> to pellet.<br><font color = "#800517"><i>Use appropriate volume of water.</i></font><br>Resuspend the pellet by vortexing/by shaking vigorously.<br><font color = "#800517"><i>Many protocols recommend resuspending in 10 mM Tris-HCl or TE. The advantage of TE is that EDTA chelates magnesium ions which makes it more difficult for residual DNases to degrade the DNA. I generally prefer H2O and don't seem to experience problems of this sort. If you plan to ultimately use electroporation to transform your DNA then resuspending in H2O has the advantage of keeping the salt content of your ligation reaction down.</i></font><br></li></p></ol></html>
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<html><h2>Solutions/reagents:</h2><ul type="circle"><li>absolute ethanol stored at -20°C</li><li>DNA sample</li><li>95% ethanol stored at room temperature</li><li>water</li></ul><h2>Equipment:</h2><ul type="circle"><li>Incubator</li><li>Centrifuge</li><li>Eppendorf tubes</li></ul><h2>Steps:</h2><ol><p><li>Measure out DNA sample into an Eppendorf tube.<br>Add  <b><font color=#357EC7>2</font></b> volumes <font color=#357EC7>absolute ethanol</font>.<br><font color = "#800517"><i>Generally the sample is in a 1.5 mL eppendorf tube. I recommend storing the absolute ethanol at -20°C.</i></font><br></li></p><p><li>Incubate at <b><font color=#357EC7>-80°C</font></b> for <b><font color=#357EC7>1 hr</font></b>.<br><font color = "#800517"><i>The long incubation time is critical for small fragments.</i></font><br></li></p><p><li>Centrifuge at a speed of at least <font color=#357EC7>10000 Xg</font> for <b><font color=#357EC7>30 mins</font></b> at <b><font color=#357EC7>0°C</font></b>, gently aspirate out the supernatant and discard it.<br></li></p><p><li>Add <b><font color=#357EC7>750 - 1000 µl</font></b> of <font color=#357EC7>95% ethanol</font>.<br><font color = "#800517"><i>Another critical step for small fragments under 200 base pairs. Generally washing involves adding the ethanol and inverting several times.</i></font><br></li></p><p><li>Centrifuge at a speed of at least <font color=#357EC7>10000 Xg</font> for <b><font color=#357EC7>10 mins</font></b> at <b><font color=#357EC7>4°C</font></b>, gently aspirate out the supernatant and discard it.<br></li></p><p><li>Dry the pellet in air. <br><font color = "#800517"><i>I generally let the pellet air dry completely such that it becomes white so that all residual ethanol is eliminated.</i></font><br></li></p><p><li>Add <font color=#357EC7>water</font> to pellet.<br><font color = "#800517"><i>Use appropriate volume of water.</i></font><br>Resuspend the pellet by vortexing/by shaking vigorously.<br><font color = "#800517"><i>Many protocols recommend resuspending in 10 mM Tris-HCl or TE. The advantage of TE is that EDTA chelates magnesium ions which makes it more difficult for residual DNases to degrade the DNA. I generally prefer H<sub>2</sub>O and don't seem to experience problems of this sort. If you plan to ultimately use electroporation to transform your DNA then resuspending in H<sub>2</sub>O has the advantage of keeping the salt content of your ligation reaction down.</i></font><br></li></p></ol></html>

Revision as of 01:41, 1 October 2009

Solutions/reagents:

  • absolute ethanol stored at -20°C
  • DNA sample
  • 95% ethanol stored at room temperature
  • water

Equipment:

  • Incubator
  • Centrifuge
  • Eppendorf tubes

Steps:

  1. Measure out DNA sample into an Eppendorf tube.
    Add 2 volumes absolute ethanol.
    Generally the sample is in a 1.5 mL eppendorf tube. I recommend storing the absolute ethanol at -20°C.
  2. Incubate at -80°C for 1 hr.
    The long incubation time is critical for small fragments.
  3. Centrifuge at a speed of at least 10000 Xg for 30 mins at 0°C, gently aspirate out the supernatant and discard it.
  4. Add 750 - 1000 µl of 95% ethanol.
    Another critical step for small fragments under 200 base pairs. Generally washing involves adding the ethanol and inverting several times.
  5. Centrifuge at a speed of at least 10000 Xg for 10 mins at 4°C, gently aspirate out the supernatant and discard it.
  6. Dry the pellet in air.
    I generally let the pellet air dry completely such that it becomes white so that all residual ethanol is eliminated.
  7. Add water to pellet.
    Use appropriate volume of water.
    Resuspend the pellet by vortexing/by shaking vigorously.
    Many protocols recommend resuspending in 10 mM Tris-HCl or TE. The advantage of TE is that EDTA chelates magnesium ions which makes it more difficult for residual DNases to degrade the DNA. I generally prefer H2O and don't seem to experience problems of this sort. If you plan to ultimately use electroporation to transform your DNA then resuspending in H2O has the advantage of keeping the salt content of your ligation reaction down.

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