- 10X restriction enzyme buffer (see manufacturer’s recommendation)
- sterile water
- restriction enzyme
- phenol:chloroform (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.)
- Add 1 to 2 µL (3 to 20 units) enzyme and mix gently. Spin for a few
seconds in the microfuge.
- Incubate at the appropriate temperature (usually 37°C) for 1 to 2 hours.
- Run a small aliquot on a gel to check for digestion.
- 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.