Restriction Enzyme Digestion of DNA

• 10X restriction enzyme buffer (see manufacturer’s recommendation)
• DNA
• sterile water
• restriction enzyme
• phenol:chloroform (1:1)

1. Add the following to a microfuge tube:
   2 µL of appropriate 10X restriction enzyme buffer. 0.1 to 5 µg DNA 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.)
2. Add 1 to 2 µL (3 to 20 units) enzyme and mix gently. Spin for a few seconds in the microfuge.
3. Incubate at the appropriate temperature (usually 37°C) for 1 to 2 hours.
4. Run a small aliquot on a gel to check for digestion.
5. If the DNA is to be used for another manipulation, heat-inactivate the enzyme (if it is heat-labile) at 70°C for 15 min, phenol/chloroform extract, and ethanol precipitate, or purify on Qiagen DNA purification column.