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Restriction Enzyme Digestion of DNA


bullet 10X restriction enzyme buffer (see manufacturer's recommendation)
bullet DNA
bullet sterile water
bullet restriction enzyme
bullet phenol:chloroform (1:1) (optional)
  1. Add the following to a microfuge tube:
  2. bullet 2 μl of appropriate 10X restriction enzyme buffer
    bullet 0.1 to 5 μg DNA
    bullet sterile water to a final volume of 19 μl (Note: These volumes are for analytical digests only. Larger volumes may be necessary for preparative digests or for chromosomal DNA digests.
  3. Add 1 to 2 μl (3 to 20 units) enzyme and mix gently. Spin for a few seconds in microfuge. Incubate at the appropriate temperature (usually 37 degrees C) for 1 to 2 hours. Run a small aliquot on a gel to check for digestion.
  4. If the DNA is to be used for another manipulation, heat inactivate the enzyme (if it is heat labile) at 70 degrees C for 15 min, phenol/chloroform extract and ethanol precipitate, or purify on Qiagen DNA purification column (

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Last updated on 3/2/2010

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