Restriction endonuclease digestion
Lenzmeier Research Laboratory
General guidelines for carrying out restriction digests:
Restriction Enzyme unit definition:
1 unit of enzyme digests 1 ug of DNA in 1 hour at 37oC.
Enzymes come in concentrations of 5-20 units per ul.
Specific information about restriction enzymes can be found at the following New England Biolabs web site:
https://www.neb.com/products/restriction-endonucleases
Restriction Enzymes have different activities in different buffers. New England Biolabs supplies different
buffers with their enzymes (as well as special buffers for some enzymes like BamHI and EcoRI). Use a buffer
that gives you maximal digestion (100%). Consult the New England Biolabs web site for a chart of activity of
each enzyme in the different buffers available: https://www.neb.com/~/media/NebUs/Files/nebufferperformance-chart-with-restriction-enzymes.pdf
When doing digests with two different enzymes or more, consult the New England Biolabs double digest
chart at https://www.neb.com/tools-and-resources/interactive-tools/double-digest-finder to determine if the
reaction can be done with several enzymes simultaneously (buffer to use will be noted) or if the reactions must
be done sequentially (noted “seq”)
BSA is the protein bovine serum albumin. Addition of BSA to some restriction digestion reactions is required.
You should check the information for each enzyme (https://www.neb.com/~/media/NebUs/Files/nebufferperformance-chart-with-restriction-enzymes.pdf) to determine if BSA should be added to your reaction.
A typical restriction digest reaction, added in this order to the tube:
10-20 ul of DNA (mini prep or PCR product)
3.0 ul
of 10X NEB Buffer #____ (need to choose based on enzyme(s) being used)
0.3 ul of 100X BSA (if required—if you have doubts, add it because it can not harm a reaction)
___ul of water to bring the final reaction volume up to 30 ul after enzyme addition
1-3 ul of Restriction Enzyme*
30 ul TOTAL VOLUME
*typically 10 units of enzyme is sufficient
*volume of enzyme(s) used should NEVER exceed 10% of reaction volume (3 ul max)
*when digesting cloning vectors, addition of Calf Intestinal Phosphatase (CIP) will remove phosphates and
prevent re-annealing of the vector when you are trying to ligate in new DNA molecules.
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Restriction endonuclease digestion
Lenzmeier Research Laboratory
Other notes:
Reactions are usually carried out at 37oC, although some enzymes require different temperatures (see each
enzyme’s information from New England Biolabs).
Reactions typically last 1-2 hours, although in some cases overnight digestion might (or might not) be prudent.
Check with Dr. Lenzmeier regarding this issue.
Reactions can be scaled up or down (simply adjust volumes of buffer and BSA and enzyme amounts
accordingly).
The activity of many (but not all) enzymes can be destroyed by heating the enzyme to 65oC for 20 minutes.
You will want to do this 65oC enzyme inactivation if you are ligating DNA directly into a vector that has just
undergone a restriction digest. Check the New England Biolabs web page for specific information on this issue.
When analyzing restriction digests by agarose gel electrophoresis:
Typically, 5 ul of the restriction digest is added to 2-3 ul of agarose gel loading dyes and the entire amount of
DNA + dyes is loaded. More or less DNA may need to be loaded, depending on concentration of DNA in the
reaction.
Be sure to run out “uncut” DNA from the same source to ensure that your restriction digestion reaction worked.
Run your digests in the gel alongside one lane of a DNA size ladder so as to identify the approximate sizes of
your restriction fragments. The ladder routinely used in the Lenzmeier laboratory is the KB+ ladder.
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