Restriction Enzyme Digestion of DNA
Materials:
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10X restriction enzyme buffer (see manufacturer's recommendation)
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DNA
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sterile water
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restriction enzyme
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phenol:chloroform (1:1) (optional)
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- Add the following to a microfuge tube:
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2 μl of appropriate 10X restriction enzyme buffer
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0.1 to 5 μg DNA
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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.
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- Add 1 to 2 μl (3 to 20 units) enzyme and mix gently. Spin for a few seconds in microfuge. Incubate at the appropriate temperature (usually 37 degrees 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 degrees C for 15 min, phenol/chloroform extract and ethanol precipitate, or purify on Qiagen DNA purification column (http://www.qiagen.com).
This Web page is maintained by Julie B. Wolf, UMBC;
Last updated on
3/2/2010
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