Joyce: Phenol extraction

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Contents

Concept

  • To remove contaminating protein that could inhibit downstream protocols, or affect the quality of your DNA in the long-term.
  • Phenol completely denatures proteins and concentrates them in the nonpolar (phenol) phase, leaving DNA undamaged in the aqueous phase

Procedure

  1. In a fume hood and while wearing gloves, add an equal volume of TE-saturated phenol to your DNA sample WARNING: Wear gloves and do this in a fume hood; phenol is some nasty stuff
  2. Vortex for at least 1 minute
  3. Spin at 14K RPM for 3 minutes
  4. Pipette off 90% of the aqueous phase to a new tube (avoid the phenol:aqueous interface as it contains contaminating proteins)
  5. Add an equal volume of water-saturated ether to the DNA solution (fume hood, wear gloves)
  6. Vortex for at least 1 minute
  7. Spin at 14K RPM for 3 minutes
  8. Pipette off the ether phase (ether is on top), discard in a falcon tube containing activated charcoal in the fume hood
  9. Repeat the ether wash again
  10. Precipitate the DNA

Notes

  • Discard anything phenol-contaminated in a special-marked waste container (in fume hood)
  • The ether is to remove any residual phenol that would otherwise ruin downstream applications
  • Ether is a pain to pipette, it's recommended that you pipette the ether up and down a few times (to coat the pipette tip), so it doesn't flow out unintentionally. Have your ependorff open and ready to accept the ether to make sure you transfer the desired volume.

Links

Biosize Bio - How Phenol Extraction Works

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