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DNA purification

Typically, we purify DNA using DNA/plasmid/gel purification kits. However, in specific situations you may want to use ethanol precipitation or phenol/chloroform extraction.

Ethanol precipitation

Overview

Nucleic acids are polar due to a PO4- group along the sugar backbone and thus are hydrophilic. Positively charged salt ions neutralize PO4-, making nucleic acids much less hydrophilic, resulting in precipitation. Moreover, ethanol increases electrostatic interactions between PO4- and salt ions (due to a lower dielectric constant).

Salt choices: - DNA: - Regular and large: 3 M Na acetate - Small and low concentration: also add MgCl2 (final concentration: .01 M) - With SDS: NaCl because it keeps SDS soluble - After restriction digestion: also add 1/20 of DNA sample volume of .5 M EDTA - RNA: - LiCl – note that Cl- inhibits DNA polymerase and protein synthesis - 5 M NH4 acetate to remove NTPs/dNTPs (e.g., after in vitro transcription), but won't work with T4 polynucleotide kinase reactions - Adding 20 ng of glycogen can help for small amounts of RNA

Procedure

  1. Mix the following:
Component Vol. formula Vol. example (uL) Notes
DNA / RNA V 45
Salt V / 10 4.5 Typically Na acetate
100% ethanol 2 x V 90
Glycogen 1 uL 1 Helps visualize pellet
  1. Chill at -20C overnight.
    • Larger DNA (e.g, a plasmid) will precipitate faster (30 min might suffice), but short fragments will benefit for an overnight or even over the weekend cooling.
  2. Precool a microcentrifuge to 4C and pellet for 15-30 min at top speed.
  3. Transfer to ice and discard nearly all supernatant with a pipette, angled at the opposite wall to the pellet.
    • If the amount small, you may not see anything pelleting. Glycogen helps visualize the pellet.
    • Be extra careful not to get any of the pellet into pipette. Best practice is to keep tilting the tube while withdrawing the supernatant, such that by the end the tube is lying horizontally and all little droplets are collected.
    • Alway keep the tube cold. Hold it at the top so that the pellet doesn't dissolve by warming up from your hands.
  4. Washing: Add ~100 uL of 70% cold ethanol to wash it, do not vortex, centrifuge it at top speed for 10-20 min, transfer to ice, and discard most of supernatant.
    • Optionally, repeat washing again.
  5. Work with pipette to aspire as much of the remaining liquid as possible while gradually tilting the tube. Then lie the tube sideways and leave it open for a few minutes for ethanol to evaporate but not too long for the pellet to completely dry.
  6. Resuspend in dH2O or TE buffer to the desired volume.

Resources for further optimization

  • Ethanol Precipitation of DNA and RNA: An Authoritative Guide
  • Crouse & Amorese, 1987 – Ethanol Precipitation: Ammonium Acetate as an Alternative to Sodium Acetate:
    • Longer chilling is in all case better, with overnight being the best for recovery (90% vs 30%)
    • This is especially true for lower sample concentrations.
    • Higher volume is worse: 90% to 40% when going from 20 uL to 200 uL
    • Centrifugation at room temperature for 30 min is better than when cooling and using a shorter duration
    • To remove proteins, first precipitate them with 2.5M (final) NH4 acetate, centrifuge, then add ethanol (2.5 volume) to DNA to precipitate, and wash with 70% ethanol.
    • General recipe:
      • Add 2.5 volume of ethanol and .5 volume of NH4 acetate (7.5M stock)
      • Keep at room temperature overnight
      • Centrifuge for 30 min
      • Wash with 70% ethanol and centrifuge for 15 min

Phenol/chloroform extraction

Overview

  • Purpose: Remove proteins from samples in order to have samples ready for ethanol precipitations
  • Caveats:
    • The sample will contain traces of phenol that inhibits many enzymatic reactions.
    • Nanodrop is sensitive to phenol, so you should measure the resulting concentration with Qubit instead.

Procedure

  1. Mix sample with an equal volume of phenol/chlorofom mixture (pH 8), vortex and centrifuge at max speed for 10 min.
    • Recommended sample volume: at least 100 uL. If you have less, dilute your sample first.
    • Centrifugation could be as short as 1 min, unless you have a high amount of proteins.
  2. Carefully aspirate the top (aqueous) phase, and repeat the previous step.
    • High salt concentrations might invert the phases. The organic phase (the one you don't need) should be yellow.
    • For small DNA amounts (less than 1 ug), you may treat the organic phase with 100 uL of TE buffer (pH 8).
  3. Carefully aspirate the top (aqueous) phase and proceed to the typical ethanol extraction.