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HOME > Protocols > Molecular Biology > DNA > Protocol for Phenol Purification of DNA from Low Melting Point Agarose

Phenol Purification of DNA From Low Melting Point Agarose

  1. Place phenol mix at 37oC for at least 1 hour
  2. Melt excised agarose fragment at 65oC. for 10 minutes or until agarose is molten, then place molten agarose at 37oC for 2 minutes
  3. Add 0.5X volume of phenol mix and shake vigorously for 45 seconds
  4. Centrifuge for 5 minutes at room temperature and transfer aqueous layer to clean tube
  5. Repeat phenol extraction and centrifugation step
  6. Transfer aqueous layer to a clean tube and add 1/50 volume of 5M NaCl, shake for 45 seconds, and centrifuge for 5 minutes at room temperature
  7. Transfer aqueous material to a clean tube, add 2X volume of EtOH and precipitate (either 15 minutes at -70oC. or at least 1 hour at -20oC)
  8. Centrifuge for 15 minutes and aspirate off aqueous material
  9. Wash pellet in 1X volume of 70% EtOH and repeat centrifugation
  10. Aspirate off aqueous material and air dry pellet for 5 minutes (or dry in speedvac for ~2 minutes)
  11. Resuspend pellet in 20Ál TE buffer

Phenol mix: 25ml pure phenol, 15ml H2O, 400ml 1M Tris-HCl, pH to 8.0 with 2 drops 5M NaCl

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