Ethanol precipitation of small DNA fragments protocol

From OpenWetWare

Revision as of 02:35, 20 November 2009 by Vaishnavi Ananth (Talk | contribs)
(diff) ←Older revision | Current revision (diff) | Newer revision→ (diff)
Jump to: navigation, search

Solutions/reagents:

  • absolute ethanol stored at -20°C
  • DNA sample
  • 95% ethanol stored at room temperature
  • water

Equipment:

  • Incubator
  • Centrifuge

Steps:

  1. Measure out 2 volumes absolute ethanol into DNA sample.
    Generally the sample is in a 1.5 mL eppendorf tube. I recommend storing the absolute ethanol at -20°C.
  2. Incubate at -80°C for 1 hr.
    The long incubation time is critical for small fragments.
  3. Centrifuge at a speed of at least 10000 Xg for 30 mins at 0°C, gently aspirate out the supernatant and discard it.
  4. Measure out 750 - 1000 µl of 95% ethanol into Eppendorf tube (1).
    Another critical step for small fragments under 200 base pairs. Generally washing involves adding the ethanol and inverting several times.
  5. Centrifuge at a speed of at least 10000 Xg for 10 mins at 4°C, gently aspirate out the supernatant and discard it.
  6. Dry the pellet in air.
    I generally let the pellet air dry completely such that it becomes white so that all residual ethanol is eliminated.
  7. Add water to pellet.
    Use appropriate volume of water.
    Resuspend pellet by vortexing/by shaking vigorously.
    Many protocols recommend resuspending in 10 mM Tris-HCl or TE. The advantage of TE is that EDTA chelates magnesium ions which makes it more difficult for residual DNases to degrade the DNA. I generally prefer H2O and don't seem to experience problems of this sort. If you plan to ultimately use electroporation to transform your DNA then resuspending in H2O has the advantage of keeping the salt content of your ligation reaction down.

TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :~ 1 hr, 40 mins

Personal tools