Restriction Enzyme Digestion of DNA

Materials:

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 microfuge.

3.Incubate at the appropriate temperature (usually 37E 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 E C for 15 min, phenol/chloroform extract and ethanol precipitate, or purify on Qiagen DNA purification column.