Ethanol precipitation of small DNA fragments

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(Procedure)
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=Procedure=
=Procedure=
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#Add 2 volumes absolute ethanol to sample.  <br> Generally the sample is in a 1.5 mL eppendorf tube.
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#Add 2 volumes ice cold absolute ethanol to sample.  <br> Generally the sample is in a 1.5 mL eppendorf tube.  I recommend storing the absolute ethanol at -20&deg;C.
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#Incubate 1 hr at -80&deg;C.  This step is critical for small fragments.
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#Incubate 1 hr at -80&deg;C.  <br> The long incubation time is critical for small fragments.
#Centrifuge for 30 minutes at 0&deg;C at maximum speed (generally >10000 g at least).
#Centrifuge for 30 minutes at 0&deg;C at maximum speed (generally >10000 g at least).
#Remove supernatant.
#Remove supernatant.
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#Wash with 750 &mu;L 95% ethanol.  Another critical step for small fragments.
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#Wash with 750-1000 &mu;L room-temperature 95% ethanol.  <br> Another critical step for small fragments under 200 base pairs.  Generally washing involves adding the ethanol and inverting several times.
#Centrifuge for 10 minutes at 4&deg;C at maximum speed (generally >10000 g at least).
#Centrifuge for 10 minutes at 4&deg;C at maximum speed (generally >10000 g at least).
#Let air dry on benchtop. <br> I generally let the pellet air dry completely such that it becomes white so that all residual ethanol is eliminated.
#Let air dry on benchtop. <br> I generally let the pellet air dry completely such that it becomes white so that all residual ethanol is eliminated.
#Resuspend in an appropriate volume of H<sub>2</sub>O. <br> 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.
#Resuspend in an appropriate volume of H<sub>2</sub>O. <br> 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.

Revision as of 13:55, 3 May 2005

This protocol is for a simple ethanol precipitation of small fragments. This protocol was used to (partially) purify a DNA fragment containing a ribosome binding site (~40 bp) during 3A assembly. The fragment was generated via restriction digest and it was used in a ligation reaction. Note that this protocol simply concentrates your sample and removes enough salts/enzymes for ligation to be successful. All DNA fragments from your digest will still be present in your pellet. These residual DNA fragments do not matter for 3A assembly which selects against incorrect ligation products.

Materials

  • Absolute Ethanol (200 proof)
  • 95% ethanol
  • Tabletop centrifuge
  • -80°C freezer

Procedure

  1. Add 2 volumes ice cold absolute ethanol to sample.
    Generally the sample is in a 1.5 mL eppendorf tube. I recommend storing the absolute ethanol at -20°C.
  2. Incubate 1 hr at -80°C.
    The long incubation time is critical for small fragments.
  3. Centrifuge for 30 minutes at 0°C at maximum speed (generally >10000 g at least).
  4. Remove supernatant.
  5. Wash with 750-1000 μL room-temperature 95% ethanol.
    Another critical step for small fragments under 200 base pairs. Generally washing involves adding the ethanol and inverting several times.
  6. Centrifuge for 10 minutes at 4°C at maximum speed (generally >10000 g at least).
  7. Let air dry on benchtop.
    I generally let the pellet air dry completely such that it becomes white so that all residual ethanol is eliminated.
  8. Resuspend in an appropriate volume of H2O.
    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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