Ethanol precipitation of nucleic acids protocol

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Revision as of 01:31, 20 November 2009 by Vaishnavi Ananth (Talk | contribs)
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Solutions/reagents:

  • sample
  • 100% ethanol
  • 3M sodium acetate, pH 5.2
  • water

Equipment:

  • Centrifuge

Steps:

  1. Option 1: Measure out 2 volumes 100% ethanol into sample.
    (or)
    Option 2: Measure out 3 volumes 100% ethanol into sample.

    Measure out 0.1 volume 3M sodium acetate, pH 5.2 into sample.

  2. Mix solution by pipetting up and down several times.
    Store at -20°C for 12 hrs(overnight).
    --This step some say is unnecessary but others swear by it. If you are in a rush you can also put it in the -80 for ten minutes to a few hours. Dry ice for 10-15 minutes also works.
    --In general, the time you need to incubate in the freezer depends on how much nucleic acid you have, how big it is and the volume it is in. The general protocol is to freeze for 20 min to 1 hr at -80 °C. This seems to work well for most things, but you may want to freeze longer if you have only a small concentration of nucleic acid or if it is small in size(<15 nucleotides).
    --If you are in a hurry, you can also dip you epi shortly into liquid nitrogen. If you added enough ethanol, the mix won't freeze. Careful with isopropanol - it freezes more quickly. This works well and saves a lengthy incubation in the fridge.
  3. Centrifuge at maximum speed for 30 mins at 4°C, gently aspirate out the supernatant and discard it.
    --Make sure to mark the outermost edge of the tube so you can find the pellet easily (or just put the hinge portion of the tube to the outside). It is clear and usually looks like a little smudge on the tube.
  4. Option 1: Dry the pellet in air for ~15 mins.
    (or)
    Option 2: Dry the pellet in a speedvac for ~15 mins.

    --DNA and RNA (if you don't have RNases in your sample) are typically hearty enough for you to air dry at 37 ?C, if desired.
    --Overdrying can make DNA hard to re-dissolve.

  5. Measure out water into sterile 1.5-ml microcentrifuge tube (1).
    Resuspend pellet by vortexing/by shaking vigorously.
    --Beware of using water unless you are sure of what you are getting in to. The pH of water can vary widely (I've seen from pH 5 to pH 8.5), and depurination of DNA at low pH or degradation of RNA at high pH are possibilities. Water also typically contains trace metals, which can accelerate these reactions. I typically recommend resuspension in TE (10 mM Tris-HCl, pH 7.5, 1 mM EDTA). This makes sure your nucleic acid is at a neutral pH and the EDTA will chelate any trace metals. Since they are in such small amounts, neither the buffer nor the EDTA will affect most downstream reactions.

TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :~ 45 mins

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