Setting up a Reaction
A "Typical" Restriction Digest
Most researchers follow the general rules that 10 units of restriction enzyme
is sufficient to overcome variability in DNA source, quantity and purity. Generally,
1ul of enzyme is added to 1 ug of purified DNA in a final volume of 50 ul of
the appropriate 1X NEBuffer followed by incubation for 1 hour at the recommended
temperature. If an excess of enzyme is used, the length of incubation can often
be decreased to save time. Alternatively, you can productively digest with fewer
units of enzyme for up to 16 hours with many restriction enzymes.
Choosing the Right Enzyme
Obviously the DNA to be digested must contain a recognition sequence for the
restriction enzymes chosen. Restriction enzymes with shorter recognition
sequences cut DNA more frequently than those with longer recognition sequences.
Assuming a 50% G-C content, a restriction enzyme with a 4-base recognition
sequence will cleave, on average, every 44 (256) bases compared to every
46 (4096) bases for a restriction enzyme with a 6-base recognition sequence.
Cleavage by a restriction enzyme produces either cohesive (having either
a 5´ or 3´ single-stranded protrusion) or blunt-ended (no single-stranded
protrusion) fragments. Cohesive fragments can be subsequently ligated to
other restriction fragments if their single-stranded protrusions or "overhangs" are
compatible. All blunt-ended fragments can be ligated to each other. See Compatible
Cohesive Ends and Recleavable Blunt
Ends.
Enzyme
Restriction enzymes should be kept on ice when they are not in the freezer.
The enzyme should always be the last component added to a reaction (reaction
components should be mixed prior to addition of enzyme). The number of units
added to a reaction must be adjusted to the varying cleavage rates of DNA
substrates. For example, supercoiled plasmids and agarose-embedded DNAs generally
require more than 1 unit/µg to be cleaved completely. More information
about cleavage of plasmid DNA and agarose-embedded
DNA.
DNA
The preparation of DNA to be cleaved should be free of contaminants such
as phenol, chloroform, alcohol, EDTA, detergents, or excessive salts, all
of which can interfere with restriction enzyme activity. DNA methylation
is also an important element of a restriction digest. More information about
Effect of CpG Methylation on Restriction
Enzyme Cleavage and Dam and
Dcm Methylases of E.coli.
Reaction Buffer
New England Biolabs provides a color-coded 10X NEBuffer with each restriction
enzyme to ensure optimal (100%) activity. The buffer should be used at 1X
concentration in the reaction. Some restriction enzymes require bovine serum
albumin (BSA) at a final concentration of 100 µg/ml for optimal activity.
When required, BSA is supplied as a 10 mg/ml (100X) stock and should be added
to the reaction mixture. Restriction enzymes that do not require BSA for
optimal activity are not adversely affected if BSA is present in the reaction.
More information about NEBuffers.
Reaction Volume
By definition, 1 unit of restriction enzyme will completely digest 1 µg
of substrate DNA in a 50 µl reaction in 60 minutes. This enzyme : DNA
: reaction volume ratio can be used as a guide when designing reactions. Smaller
reaction volumes are more susceptible to pipetting errors. To keep glycerol
concentration at less than 5% in a reaction, the restriction enzyme, which
is supplied in 50% glycerol, should not exceed 10% of the total reaction volume.
Mixing
An extremely important, yet often overlooked, element of a successful restriction
digest is mixing. The reaction must be thoroughly mixed to achieve complete
digestion. We recommend gently pipetting the reaction mixture up and down
or “flicking” the reaction tube. Follow with a quick (“touch”)
spin-down in a microcentrifuge. Do not vortex the reaction.
Incubation Temperature
The recommended incubation temperature for most restriction enzymes is 37°C.
Restriction enzymes isolated from thermophilic bacteria require higher incubation
temperatures ranging from 50°C to 65°C. More information on the activity
of thermophiles at 37°C.
Incubation Time
The unit definition of our restriction enzymes is based on a 1 hour incubation.
Incubation time may be shortened if additional units of restriction enzyme
are added to the reaction. Conversely, longer incubation times are often
used to allow a reaction to proceed to completion with fewer units of enzyme.
Refer to the specific information about enzyme
survival times in a reaction.
Stopping a Reaction
If no further manipulations of the digested DNA are planned, the reaction can
be terminated by adding a stop solution. At NEB we use the following stop
solution: 50% Glycerol, 50 mM EDTA (pH 8.0), and 0.05% bromophenol blue (10 µl/50 µl
reaction). If further manipulations of the digested DNA are required, heat
inactivation (raising the temperature to 65 or 80°C for 20 minutes) is
the simplest method of stopping a reaction. Since this method does not work
for all restriction enzymes, refer to the heat
inactivation chart. Phenol/chloroform
extraction is another means of inactivating a restriction enzyme.
Storage
We recommend storage at –20°C for most restriction enzymes. For a
few enzymes, storage at –70°C is recommended for periods longer than
30 days. Please refer to the enzyme's technical data sheet or catalogue entry
for storage information. 10X NEBuffers and concentrated BSA should also be
stored at –20°C. BSA should not be mixed directly into NEBuffers
and then frozen because the BSA may precipitate..
Stability
All enzymes are assayed for activity every 1-2 months; the most recent assay
date is given on the label attached to each vial of enzyme. After thirty
years of experience with restriction enzymes, we have found that most are
very stable when stored at –20°C in the recommended storage buffer.
Exposure to temperatures above –20°C should be minimized whenever
possible.
Control Reactions
If you are having difficulty cleaving your DNA substrate, we recommend the
following control reactions. Incubate experimental DNA without restriction
enzyme (degradation of DNA indicates contamination in the DNA preparation
or reaction buffer) and control DNA (DNA with multiple known sites for the
enzyme, e.g. lambda or adenovirus-2 DNA) with restriction enzyme to more
accurately judge whether or not the reaction went to completion. If the control
DNA is cleaved and the experimental DNA resists cleavage, the two DNAs can
be mixed to determine if an inhibitor is present in the experimental sample.
If an inhibitor (often salt, EDTA or phenol) is present, the control DNA
will not cut after mixing.