ETHANOL PRECIPITATION
REAGENTS
  • 3 M sodium acetate (or 4 M lithium chloride), filter sterilise and store at room temperature.
  • 96% ethanol at 20 °C
  • 70% ethanol at 20 °C
METHODS
  • For a volume of 50 (or 20) m l from DNA labeling reactions or 100 l from RNA probes labeled by in vitro translation.
  • For DNA probes, add 5 (or 2) m l of 3M sodium acetate (or 4 M LiCl) and 150 (or 60) m l cold 96% ethanol. For RNA probes, add 5 l 10% acetic acid to neutralize the carbonate buffer, and 10 l 3M sodium acetate and 250 l cold 96% ethanol. It is convenient to transport DNA between laboratories in this state
  • Precipitate the DNA or RNA in the freezer (-20 ° C) for 1-2 h or overnight
  • Spin the tubes in a microcentrifuge for 30 min, 12,000 g (ideally at 10 °C, but a centrifuge in a cold room is sufficient)
  • Discard the supernatant and then wash the pellet by carefully adding 0.5 ml of ice cold 70% ethanol and then centrifuging for 5 min, 12,000g.
  • Discard the supernatant by inverting the tube onto an absorbent tissue and leave the pellet until dry (be careful it does not fall out or blow away)
  • Inspect the pellet and check the size and color (pale brown for pure DNA or after labeling with biotin or digoxigenin, or the color of the fluorochrome label).
  • Dissolve the labeled DNA or RNA in 10-20 m l water
  • Labeled DNA probes are stable and can be stored for long periods at -20°C. RNA probes should be used immediately or can be stored for short periods at -80°C

NOTES

  • 100% ethanol can only be produced by chemical drying, and absorbs water from the atmosphere to give 96% ethanol. 96% ethanol is suitable for all protocols given here except where stated, but 100% can also be used
  • Formerly colder and longer periods were used, but this is no longer considered necessary
  • It is helpful to have a standard position to place microcentrifuge tubes: with the lid hinge to the upper outside. Then you know to look (and not wipe with tissue etc.) on the hinge site for a pellet. A typical pellet from labeled DNA will be visible as a 1x 2 mm oval smudge, cream or the color of a fluorophore direct-label
  • Shake the tube and centrifuge briefly to bring the solution to the bottom of the tube. Leave overnight at room temperature. Do not use a vortex mixer as this will break long DNA molecules. DNA can also be dissolved in TE buffer (10 mM Tris-HCl pH 7.6 and 1 mM EDTA pH 8; note the EDTA may inhibit subsequent enzyme reactions) or in 100% formamide which may reduce background during hybridization