Shreffler:DNA Cleanup

From OpenWetWare

Revision as of 10:43, 2 April 2012 by Alex Ma (Talk | contribs)
(diff) ←Older revision | Current revision (diff) | Newer revision→ (diff)
Jump to: navigation, search

Contents

Overview

DNA Cleanup protocol adapted from http://www-ufk.med.uni-rostock.de/lablinks/protocols/e_protocols/phenolch.htm removes protein debris and resuspends DNA in appropriate final buffer.

Materials

  • 1.5 ml microcentrifuge tubes (eppies)
  • DNA of interest
  • Phenol
  • Chloroform
  • Ethanol
  • 3 M sodium acetate
  • 10mM tris pH 7.5

Procedure

Phenol Step (removes protein)

  1. Pipette 500 µl DNA suspension into eppendorf tube (can vary volume). Add an equal volume of phenol (tris-saturated Phenol-Chloroform-Isoamyethanol).
  2. Vortex, then centrifuge for 2 min @ 12000 rpm 4°C.
  3. Transfer supernatant into new tube, avoiding drawing the interlayer or organic phase.

Chloroform Step (removes phenol)

  1. Add an equal volume of chloroform to the supernatant from previous step.
  2. Vortex, then centrifuge for 2 min @ 12000 rpm 4°C.
  3. Transfer supernatant into new tube, avoiding drawing the interlayer or organic phase.

100% Ethanol Step (precipitates DNA)

  1. Add 0.1 volume 3 M sodium acetate.
  2. Add 2.5 volumes 100% ethanol.
  3. Vortex and precipitate at -20°C overnight. Can also precipitate at -80°C for 1 hr or dry ice for 15 min, but precipitation won't be as complete as -20°C overnight.
  4. Centrifuge for 20 min at 12000 rpm 4°C.
  5. Carefully pour out or aspirate supernatant, taking care not to lose DNA pellet.

70% Ethanol Step (washes out salt)

  1. Carefully add 1 ml cold 70% ethanol. DO NOT VORTEX.
  2. Centrifuge for 10 min at 12000 rpm 4°C.
  3. Carefully pour out or aspirate supernatant, taking care not to lose DNA pellet.
  4. Air dry for 10 min at RT, but don't overdry (DNA becomes hard to dissolve).
  5. Dissolve in 10 mM tris pH 7.5.
    • TE-Buffer will work also, but EDTA present may inhibit downstream enzymatic reactions.
    • dH2O can be used but must be frozen immediately at -20°C, since unbuffered DNA undergoes degradation.
Personal tools