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 37EC) 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 70EC for 15 min, phenol/chloroform extract and ethanol precipitate, or purify on Qiagen DNA purification column.