Protocol: Restriction Enzyme Digestion of DNA

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Tony Rook
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Protocol: Restriction Enzyme Digestion of DNA

Restriction Enzyme Digestion of DNA

Materials:

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

Methods:

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 37
degrees 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 degrees C
for 15 min, phenol/chloroform extract and ethanol
precipitate, or purify on Qiagen DNA purification column
(http://www.qiagen.com).

http://www.research.umbc.edu/%7Ejwolf/m3.htm#restriction%20enzyme