Restriction Digests
Single Enzymes
Volume (µL)
X
1/10V
1/100V
89/100V-X-Y
Y
V
Component
Source DNA (½ - 5 µg)
Working Buffer (10X)
BSA (if needed)
H2O
Restriction Enzyme (Y ≤ 1/10V)
Total (15-30 µL)
Incubate at 25/37/50/55/60/65 ° C for 30 minutes to 3 hours as appropriate for enzyme activity.
Notes:
Choose working buffer best suited for enzyme.
Adhere to enzyme volume limits, as some are sensitive to total glycerol concentrations
For easy visualization, only ~0.5 µg dsDNA is needed using Ethidium Bromide and ~3 µg
using Crystal Violet.
Use as little DNA as practical for the application to prevent smearing in the gel.
General rule is 1 Unit of enzyme digests 1 µg of dsDNA in 1 hour.
Enzyme volume should be kept to one tenth or less of the reaction total to minimize glycerol
content.
Multiple Enzymes
Notes:
As for single enzyme digests, except total volume of restriction enzyme (Y) should account
for all used.
Working buffer must be chosen to maximize activity of all enzymes used together.
Ensure reaction temperatures are compatible.
Partial Digests
Notes:
Not all enzymes will cut all of their designated recognition sites all the time or even at the
same time. Short incubations may yield a mixture of products representing the various
sites as either cut or uncut. These can be useful for interpreting diagnostic digest gels and
even for extracting fragments that may not be available through a regular complete
digestion. Keep in mind the sum of all the fragment lengths, when visualized, will be
greater that the length of the source DNA product. Also, if attempting to utilize partially
digested DNA fragments for ligations, keep in mind that mixtures of fragments may run
close to or on top of one another and supercoiled or nicked DNA will also be present in the
field.