Joyce: DNA precipitation

From OpenWetWare

Jump to: navigation, search

Contents

Concept

  • DNA needs to be precipitated to remove residual salts that may inhibit other reactions (like restriction digestion, PCR, and so on)

Procedure

  1. Add enough 3M NaOAc pH 5.2 to make the final volume 0.33 M, vortex
  2. Add 2 volumes of 99% EtOH, vortex
  3. Place at -70°C for at least 30 minutes
  4. Spin at 14K (?) at 4°C for 30 minutes
  5. Pipette off supernatant (don't disturb the pellet)
  6. Wash (gently invert tube 3-4 times) with 300 ul 80% EtOH
  7. Spin at 14K for 2 minutes
  8. Pipette off supernant (don't disturb the pellet)
  9. Spin down residual EtOH and remove by pipetting
  10. Air dry for 20 minutes at 37°C, dessicate or speed vac it
  11. Resuspend in sterile water or TE

Notes

  • To make the final volume of NaOAc 0.33 M, use 1/10th of the final volume. For instance, if I have 90 ul of a DNA solution, use 10 ul of the 3 M NaOAc to make a final concentration of 0.33 M.
  • NaCl apparently helps precipitate SDS better than NaOAc, so use NaCl (final concentration of 0.2 M) when you have a DNA solution that's dirty with SDS. Also check out the Bitesize Bio link to see what salts are better suited to precipitate what DNA.
  • Placing the solution at -70°C for a longer time will help precipitate smaller (less than 100 bp) DNA fragments
  • The pellet will stick strongly to the ependorff wall after treatment with 99% EtOH, but will stick less when using 80% EtOH (because DNA is more soluble in water), so be careful when pipetting off the 80% EtOH not to disturb the pellet
  • Organic solvents like EtOH can inhibit downstream reactions (some REs are more sensitive to ethanol than others, for instance), so be sure to pipette off as much as possible using a P10-type tip, and allow the ethanol adequate time to evaporate
  • Most people seem to use 70% EtOH and 2 volumes of this (although it has been commented that the volume of the wash isn't so important, so 300 ul is probably sufficient)

Links

Protocol-online thread

Biosize Bio - How Ethanol Precipitation Works

Personal tools