SECTION IV ENZYMATIC CLEAVAGE AND

LABELING OF DNA

UNIT 10.8 Digestion of DNA with Restriction Endonucleases

Restriction endonucleases recognize short DNA sequences and catalyze the cleavage of
double-stranded DNA at specific sites within or adjacent to the recognition sequences.
This unit describes how to cleave DNA (basic protocol) and refers the investigator to
frequently updated manuals by endonuclease suppliers for the precise optimized condi-
tions. Alternate procedures are given for digesting a DNA sample with several different
enzymes and digesting multiple DNA samples with the same endonuclease.

BASIC STANDARD CLEAVAGE REACTION

PROTOCOL
Restriction endonuclease cleavage is accomplished simply by incubating the enzyme(s)
with the DNA in appropriate reaction conditions. The amounts of enzyme and DNA, the
buffer and ionic concentrations, and the temperature and duration of the reaction will vary
depending upon the specific application and enzyme.

Materials
DNA sample: 0.1 to 4 µg DNA in H2O or TE buffer
10× restriction endonuclease buffer
Restriction endonuclease
Loading buffer (UNIT 10.4)
0.5 M EDTA (optional)
1. Pipet the following into a clean microcentrifuge tube:
x µl DNA sample
2 µl 10× restriction buffer (see commentary)
18 − x µl H2O.
A 20-µl reaction is convenient for analysis by electrophoresis in polyacrylamide or agarose
gels. The amount of DNA to be cleaved and/or the reaction volume can be increased or
decreased provided the proportions of the components remain constant.
Most manufacturers provide 5× or 10× buffers optimized for the particular enzyme
purchased. It is best to use these, although the standard recipes provided in the reagents
and solutions section of this unit may be substituted.
2. Add restriction endonuclease (1 to 5 U/µg DNA) and incubate the reaction mixture
1 hr at the recommended temperature (in general, 37°C).
In principle, 1 U restriction endonuclease completely digests 1 µg of purified DNA in 60
min using the recommended assay conditions. However, crude DNA preparations, such as
those made by rapid procedures (UNIT 10.3), often require more enzyme and/or more time
for complete digestion (see critical parameters). The volume of restriction endonuclease
added should be < 1 ⁄1⁄⁄⁄0 the volume of the final reaction mixture, because glycerol in the
enzyme storage buffer may interfere with the reaction.
3. Stop the reaction and prepare it for agarose or acrylamide gel electrophoresis by
adding 5 µl (20% of reaction vol) loading buffer.
The reaction can also be stopped by chelating Mg++ with 0.5 µl of 0.5 M EDTA (final
concentration 12.5 mM). If the digested DNA is to be used in subsequent enzymatic
Digestion of DNA reactions (e.g., ligation or “filling-in” reactions), addition of EDTA should be avoided.
with Restriction DNA may be purified from the reaction mixture by extraction with phenol and precipitation
Endonucleases in ethanol (UNIT 10.1) or with GeneClean (Bio101).

10.8.1
Supplement 2 CPI Copyright © 1991 by Current Protocols
CLEAVAGE WITH MULTIPLE RESTRICTION ENDONUCLEASES ALTERNATE
PROTOCOL
It is often desired to cleave a DNA sample with more than one endonuclease. Two or more
enzymes may be added to the same reaction mixture if all are relatively active in the same
buffer and at the same temperature. Many enzymes are active over a wide range of salt
concentrations, so it is frequently possible to choose a buffer in which two or more
enzymes will retain activity (see Table 10.8.1 and commentary). However, if the reaction
conditions are too dissimilar, follow the procedure below. Alternatively, most restriction
endonucleases and some DNA-modifying enzymes are active to some extent in potassium
glutamate– and potassium acetate–based buffers (see reagents and solutions). Hence,
these buffers may be useful for digesting DNA with multiple enzymes.

1. Determine the composition of the manufacturer’s reaction buffer for the two enzymes
to be used. Digest the DNA with the enzyme(s) that is active at the lower NaCl
concentration.
If optimal digestion conditions differ only in incubation temperature, cleave the DNA with
one enzyme, then shift the temperature and add the second enzyme (the order of cleavage
does not matter). However, many enzymes with optimal activity at high temperatures are
also active at 37°C. In these cases, the enzymes can be added simultaneously and the
reaction mixture incubated at 37°C (it may be necessary to add more of the “high-tem-
perature” enzyme than usual).
2. For enzymes active at higher salt concentrations, add 1 M NaCl (1 to 3 µl for a 20-µl
reaction) so that the final concentration is appropriate for digestion by the next
enzyme(s).
Purification of the DNA fragments between digestions is the most reliable method to ensure
complete, multiple digestions. However, it is much more laborious and is rarely necessary.

CLEAVAGE OF MULTIPLE DNA SAMPLES ALTERNATE

PROTOCOL
This procedure minimizes the number of pipetting steps when multiple samples are to be
digested with the same enzyme(s) and hence, saves time. More importantly, by minimiz-
ing the number of times of going into the tube containing the restriction enzyme, the
potential for contamination of the enzyme is reduced.

1. For each sample to be tested, add a constant volume of DNA to a separate microcen-
trifuge tube.
It is critical to use a different pipet tip for each DNA sample in order to prevent
cross-contamination.
2. Prepare a “premix solution” containing sufficient 10× restriction endonuclease buffer
and water for digesting all the samples. Place solution on ice.
For example, if ten 3-µl samples of DNA are each to be digested in a 20-µl reaction mixture,
the premix will contain 20 µl of 10× restriction buffer and 150 µl water. It is prudent to
make up a solution for at least one more sample than is to be tested.
3. Add sufficient restriction endonuclease(s) for digesting all the samples. Mix quickly
by flicking the tube and replace on ice.
The solution to which the enzyme is added should not be more concentrated than 3×
buffer.
4. Add the appropriate amount of solution containing the restriction endonuclease (17
µl for above example) to each tube of DNA and incubate the reactions at the
appropriate temperature.
Molecular Biology

10.8.2
Current Protocols in Immunology Supplement 8
 Table 10.8.1 Effect of NaCl Concentration on Restriction Endonuclease Activitya
100 150 100 150
0 mM 50 mM 0 mM 50 mM
Enzyme mM mM Enzyme mM mM
NaCl NaCl NaCl NaCl
NaCl NaCl NaCl NaCl
AatII + ++ ++ + BstXI ++ +++ +++ +++
AccI +++ +++ + + BstYI +++ +++ ++ ++
AciI + +++ +++ +++ Bsu36I + ++ +++ ++
AflII + +++ ++ ++ ClaI +++ +++ +++ ++
AhaII + ++ +++ +++ DdeI ++ +++ +++ +++
AluI + +++ +++ ++ DpnI + ++ +++ +++
AlwI +++ +++ ++ + DraI ++ +++ + +
AlwNI ++ +++ +++ + DraIII ++ +++ +++ ++
ApaI +++ +++ ++ + EaeI ++ +++ ++ +
ApaLI +++ +++ ++ + EagI ++ +++ +++ +++
AscI + ++ ++ + Eam1105I + ++ + +
AseI + +++ +++ +++ EcoNI +++ +++ +++ +++
AvaI +++ +++ +++ +++ EcoO109 +++ +++ +++ +++
AvaII +++ +++ ++ + Eco57I +++ +++ +++ ++
AvrII +++ +++ +++ ++ EcoRI b +++ +++ +++
BalI +++ ++ ++ + EcoRV + + + +++
BamHI + ++ +++ +++ Fnu4HI +++ +++ ++ +
BanI +++ +++ ++ ++ FokI +++ +++ +++ +++
BanII +++ +++ +++ +++ FspI + +++ ++ ++
BbvI +++ +++ +++ +++ HaeII +++ +++ +++ ++
BcgI +++ +++ +++ +++ HaeIII +++ +++ +++ +++
BclI + +++ +++ + HgaI +++ +++ + +
BglI + +++ +++ +++ HgiAI + + ++ +++
BglII ++ +++ +++ +++ HhaI + + + ++
BsaHI ++ +++ +++ + HincII ++ +++ +++ +++
BsmI +++ +++ +++ +++ HindIII ++ +++ +++ ++
Bsp1286 +++ +++ ++ + HinfI ++ +++ +++ +++
BspHI ++ +++ +++ +++ HinPI +++ +++ +++ +++
BspMI ++ ++ +++ +++ HpaI + +++ +++ +
BspMII ++ ++ +++ +++ HpaII +++ +++ ++ +
BssHII +++ +++ +++ +++ HphI +++ +++ +++ +
BstBI +++ +++ ++ + KasI + +++ + +
BstEII + ++ +++ +++ KpnI +++ + + +
BstNI ++ ++ +++ +++ MboI ++ +++ +++ +++
BstUI +++ +++ ++ + MboII +++ +++ +++ +++

continued

For most analytical purposes, the same pipet tip can be used to dispense the restriction
endonuclease solution provided that care is taken to avoid direct contact with the DNA at
the bottom of the tubes. For preparative purposes, it is advisable to use a different pipet tip
for each sample.

REAGENTS AND SOLUTIONS

10× restriction endonuclease buffer
100 mM Tris⋅Cl, pH 7.5
100 mM MgCl2
10 mM dithiothreitol (DTT)
1 mg/ml bovine serum albumin (BSA)
0, 0.5, 1.0, or 1.5 M NaCl
The concentration of NaCl depends upon the restriction endonuclease (see manufacturer’s
instructions and Table 10.8.1). These four 10× buffers are sufficient to cover the range for
Digestion of DNA essentially all commercially available enzymes except SmaI buffer, which requires KCl (see
with Restriction recipe below). Autoclaved gelatin (at 1 mg/ml) can be used instead of BSA (see critical
Endonucleases parameters).

10.8.3

Supplement 8 Current Protocols in Immunology

Table 10.8.1 Effect of NaCl Concentration on Restriction Endonuclease Activitya, continued
0 mM 50 mM 100 mM 150 mM 0 mM 50 mM 100 mM 150 mM
Enzyme Enzyme
NaCl NaCl NaCl NaCl NaCl NaCl NaCl NaCl

MluI ++ +++ +++ ++ RsrII +++ ++ + +

MnlI +++ +++ +++ ++ SacI +++ ++ ++ +
MseI +++ +++ ++ + SacII +++ + + +
MspI +++ +++ +++ +++ SalI + + ++ +++
NaeI +++ +++ +++ + Sau3AI +++ +++ +++ +++
NarI +++ +++ + + Sau96I +++ +++ +++ +++
NciI +++ +++ ++ + ScaI + +++ +++ ++
NcoI + ++ +++ +++ ScrFI ++ +++ +++ +++
NdeI + + ++ +++ SfaNI + + +++ +++
NheI +++ +++ +++ ++ SmaI + + + +
NlaIII + + + + SnaBI +++ +++ ++ +
NlaIV + + + + SpeI ++ +++ +++ ++
NotI + +++ +++ +++ SphI + + +++ +++
NruI + +++ +++ +++ SspI ++ +++ +++ ++
NsiI ++ ++ ++ ++ StuI +++ +++ +++ +++
PaeR7I +++ +++ +++ + StyI + ++ +++ +++
PflMI ++ +++ +++ ++ TaqI +++ +++ +++ ++
PleI +++ + + + Tth111I +++ +++ +++ +
PpuMI +++ +++ ++ + XbaI + +++ +++ +++
PstI +++ +++ +++ +++ XcaI +++ + + +
PvuI + ++ +++ +++ XhoI ++ +++ +++ +++
PvuII +++ +++ +++ +++ XmaI +++ +++ ++ +
RsaI +++ +++ +++ +++ XmnI +++ +++ + +
aReprinted by permission of New England Biolabs. The activity of each enzyme listed is compared at specified

NaCl concentrations to its activity in recommended assay bufffer. Recommended assay buffers in some cases
differ widely in terms of pH and specific ion requirements. The conditions here varied only the NaCl
concentration. All buffers contained 10 mM Tris⋅Cl (pH 7.5) and 100 µg/ml bovine serum albumin. All
incubations were done for 60 min at the optimum temperature for each enzyme. Scoring is as follows:
+ <10% of the activity can be obtained using these conditions compared to the recommended conditions.
++ between 100% and 20% of the activity can be obtained using these conditions compared to the recommended
conditions.
+++ between 30% and 100% of the activity can be obtained using these conditions compared to the recommended
conditions.
bNot recommended because of star activity, which refers to cleavage at sites other than the usual recognition

sequence. Star activity occurs under nonoptimal reaction conditions, such as low ionic strength, high
endonuclease concentrations, high glycerol concentrations, high pH, and when Mn++ is used in place of Mg++.

10× potassium acetate–based buffer

660 mM potassium acetate
330 mM Tris⋅acetate, pH 7.9
100 mM magnesium acetate
5 mM DTT
1 mg/ml bovine serum albumin (optional)
2× potassium glutamate–based buffer
200 mM potassium glutamate
50 mM Tris⋅acetate, pH 7.5
100 µg/ml bovine serum albumin
1 mM β-mercaptoethanol
10× SmaI buffer
60 mM Tris⋅Cl, pH 8.0
60 mM MgCl2
200 mM KCl
60 mM β-mercaptoethanol
1 mg/ml BSA Molecular Biology

10.8.4
Current Protocols in Immunology Supplement 8
 COMMENTARY
Background Information
Restriction endonucleases are enzymes that
cleave DNA in a sequence-dependent manner.
Such cleavage is used for a wide number of
applications in molecular biology, including (a)
establishment of an endonuclease map of a
plasmid or bacteriophage clone; (b) fragmen-
tation of genomic DNA prior to electrophoretic
separation and Southern blotting; (c) genera-
tion of fragments that can be subcloned in
appropriate vectors; and (d) generation of frag-
ments to be used as labeled probes in both
Southern (UNIT 10.6) and northern (UNIT 10.12)
blotting, and in nuclease protection analysis
(UNIT 10.13).
Members of a large subgroup of these en-
zymes (type II restriction endonucleases) rec-
ognize short nucleotide sequences and cleave
double-stranded DNA at specific sites within
or adjacent to the sequences. The recognition
sequences are generally, but not always, 4 to 6
nucleotides in length and are usually charac-
terized by dyad symmetry (the 5′ to 3′ nucleo-
tide sequence of one DNA strand is identical to
that of the complementary strand). Two en-
zymes, NotI and SfiI, recognize an 8-bp se-
quence found infrequently in genomic DNA
and hence are extremely useful for cleaving
DNA into large fragments. More than 600 type
II restriction endonucleases have been isolated.
Some type II restriction endonucleases
cleave at the axis of symmetry, yielding “flush”
or “blunt” ends. Others make staggered cleav-
ages, yielding overhanging single-stranded 3′
or 5′ ends known as cohesive termini.
BamHI cleavage generates cohesive 5′ over-
hanging ends:
5′ G↓G-A-T-C-C 3′
3′ C-C-T-A-G↑G 5′
KpnI cleavage generates cohesive 3′ over-
hanging ends:
5′ G-G-T-A-C↓C 3′
3′ C↑C-A-T-G-G 5′
DraI cleavage generates blunt ends:
5′ T-T-T↓A-A-A 3′
3′ A-A-A↑T-T-T 5′
Endonucleases with the same recognition
sequences are called isoschizomers. Isoschizo-
mers may or may not cleave DNA identically
to produce the same ends. Conversely, two or
more restriction endonucleases recognizing
Digestion of DNA
with Restriction
Endonucleases
10.8.5
Supplement 8
identical or different sequences may generate
identical DNA fragment termini. These en-
donucleases are said to produce compatible
ends. DNA fragments with compatible termini
may be ligated with DNA ligases to produce
hybrid DNA molecules.
For example, BglII recognizes a different
six-nucleotide sequence from BamHI, but gen-
erates cohesive termini compatible with those
of BamHI. BglII cleaves:
5′ A↓G-A-T-C-T 3′
3′ T-C-T-A-G↑A 5′
The hybrid sites generated by joining
BamHI and BglII cohesive ends cannot be
cleaved by either enzyme.
Restriction endonucleases, generally found
in prokaryotic organisms, are probably impor-
tant for degrading foreign DNA (particularly
bacteriophage DNA). Organisms that produce
restriction endonucleases protect their own
genomes by methylating nucleotides within the
endonuclease recognition sequences. A spe-
cific methylase covalently links methyl groups
to adenine or cytosine nucleotides within target
sequences, thus rendering them resistant to
cleavage by the restriction enzyme.
Several sources provide comprehensive and
up-to-date listings of restriction endonucleases,
including their restriction sites and reaction
conditions. One source is the catalogs of com-
mercial suppliers of enzymes. Another
source—updated annually and additionally
listing commercial suppliers of each enzyme—
is a special supplement to Nucleic Acids Re-
search (Roberts, 1991). The database repre-
sented in this table is also available online
through the GenBank computer resource. Fi-
nally, an annually updated list of restriction
enzymes and their suppliers is provided free of
charge in Biotech Buyers’ Guide (American
Chemical Society, 1991).
Critical Parameters
Purity of DNA. The efficiency of the
restriction endonuclease reaction is very de-
pendent upon the purity of DNA. Contami-
nants found in some preparations of DNA
(e.g., protein, phenol, chloroform, ethanol,
EDTA, SDS, high salt concentration) may
inhibit restriction endonuclease activity. Such
impurities are often present in DNA samples
prepared by miniprep procedures (UNIT 10.3).
Current Protocols in Immunology
The decreased reaction efficiency associated
with impure DNA preparations may be over-
come by increasing the number of enzyme units
added to the reaction mixture (up to 10 to 20 U
per microgram DNA), increasing the reaction
volume to dilute potential inhibitors, or increas-
ing the duration of incubation. Some prepara-
tions of DNA (particularly minipreps) are con-
taminated by DNases. Because DNases require
Mg++ for enzyme activity, DNA in such prepa-
rations is stable in its storage buffer (which
contains EDTA), but is rapidly degraded upon
addition of restriction endonuclease buffer.
This problem can be overcome only by repuri-
fying the DNA.
Digestion of genomic DNA (prepared as in
UNIT 10.2) can be facilitated by the addition of
the polycation spermidine (final concentration
1 to 2.5 mM), which acts by binding negatively
charged contaminants. However, as spermidine
will precipitate DNA at 4°C, it should be added
after the other components of the reaction mix-
ture have been incubated at the appropriate
temperature for a few minutes. Finally, some
preparations of DNA require repurification
(phenol and chloroform extractions and ethanol
precipitation) prior to digestion with restriction
enzymes.
Degree of methylation. Some restriction
endonucleases are inhibited by methylation of
nucleotides within their recognition sequences.
In general, Escherichia coli host strains from
which plasmids are harvested contain two nu-
cleotide sequence-specific methylases: dam,
which methylates adenine in the sequence
GATC, and dcm, which methylates the internal
cytosine residue in the sequences CC(A/T)GG.
Thus, plasmid DNA from normal strains may
be cleaved partially or not at all by restriction
endonucleases that are sensitive to methylation.
This problem can be avoided by preparing plas-
mid DNA from strains that lack these methy-
lases.
Mammalian DNA contains occasional 5-
methylcytosine residues, usually at the 5′ side
of guanosine residues. The degree of methyla-
tion varies from site to site, and is strongly
influenced by the cell type from which the DNA
is isolated. Methylation patterns in eukaryotic
genomic DNA can be investigated by using the
different methylation sensitivities of isoschi-
zomers. For example, MspI cleaves CCGG
when the internal cytosine is methylated,
whereas HpaII, which also cleaves CCGG, is
very sensitive to such methylation.
In some situations, it is useful to take advan-
Current Protocols in Immunology
tage of a restriction enzyme’s inability to cleave
methylated nucleotide sequences. Methylases
recognizing sequences close to some of a re-
striction enzyme’s recognition sequences can
inhibit cleavage at those sites, thereby altering
the enzyme’s apparent sequence specificity. Al-
ternatively, when using synthetic linkers to
modify the termini of a DNA fragment, it may
be important to protect internal restriction en-
zyme sites by methylation prior to enzyme
cleavage of linkers.
Other factors influencing DNA cleavage.
Larger amounts (up to 20-fold more) of some
enzymes are necessary to cleave supercoiled
plasmid or viral DNA as compared to the
amount needed to cleave linear DNA (Fuchs
and Blakesley, 1983). In addition, some en-
zymes cleave their defined sites with different
efficiency, presumably due to differences in
flanking nucleotides. In general, cleavage rates
for different sites recognized by a given enzyme
differ by less than a factor of 10. Although such
variability is usually irrelevant, it can be sig-
nificant in experiments involving partial diges-
tion. It may be difficult to cleave DNA at a
particular site without extensive cleavage at
other sites. A few restriction endonucleases
such as NarI, NaeI, SacII, and XmaIII show
extreme variability such that some sites are very
difficult to cleave.
Buffer conditions. The typical restriction
endonuclease buffer contains magnesium chlo-
ride, sodium or potassium chloride, Tris⋅Cl,
β-mercaptoethanol or dithiothreitol, and bo-
vine serum albumin. A divalent cation, usually
Mg++, is an absolute requirement for enzyme
activity. Buffer, typically Tris⋅Cl, is necessary
to maintain the optimal pH for enzyme func-
tion. Sulfhydryl reagents may be useful for
stabilization of some restriction enzymes, but
may also stabilize potential contaminants.
Some restriction endonucleases are very sensi-
tive to the concentration of sodium or potas-
sium ion, while others are active over a wide
range of ionic strengths.
For each restriction endonuclease, optimal
reaction conditions are recommended by the
manufacturer. However, strict adherence to
these recommendations would require the
investigator to stock a large number of buf-
fers. Because many enzymes retain most of
their activity over a wide range of reaction
conditions (Table 10.8.1), it is more con-
venient to prepare four buffers that are ade-
quate for essentially all digestions. These buf-
fers contain a common core of ingredients;
Molecular Biology
10.8.6
Supplement 2
 they differ by the amount of NaCl. Restriction
enzyme buffers may be stored at −20°C, as 10×
concentrates, for more than a year.
As an alternative to the four buffer system
described above, many laboratories use potas-
sium glutamate–based (McClelland et al.,
1988) and potassium acetate–based (O’Farrell
et al., 1980) buffers (known as “universal”
buffers) for digestion of DNA with restriction
endonucleases. The key features of these buff-
ers is that they include potassium glutamate or
potassium acetate instead of sodium chloride
and Tris⋅acetate instead of Tris⋅Cl. Most restric-
tion endonucleases are active in these buffers,
although some enzymes are less active (some-
times only 20%) than under optimal conditions
specified by the manufacturer. Several DNA-
modifying enzymes are also active in these
buffers, including T4 DNA polymerase and T4
DNA ligase. “Universal” buffers are useful for
digestion of DNA with multiple restriction en-
donucleases, particularly when the endonu-
cleases are incompatible in any one of the
standard buffers.
Some restriction endonucleases “relax”
their recognition sequence specificity in
“nonoptimal” reaction conditions (including
high endonuclease concentrations, high glyc-
erol concentrations, low ionic strength, Mn++
instead of Mg++, and high pH). This “star”
activity cleaves DNA at other sites besides
those containing the “correct” sequence. For
example, EcoRI star activity cleaves some, but
not all sequences of the form AATT (usually
sites with a 5 out of 6 match to GAATTC are
cleaved better than sites with a 4 out of 6 match).
The cleavage products all contain the identical
cohesive ends as products generated by the true
EcoRI activity.
Additional comments. Enzymes should be
stored at −20°C. While in use, enzymes should
be carefully maintained on ice.
Be extremely careful to avoid contaminating
enzyme solutions, particularly with plasmid
DNA, other restriction endonucleases, or
DNase I.
Digestion of DNA
with Restriction
Endonucleases
10.8.7
Supplement 2
Some restriction enzymes are expensive,
others relatively inexpensive. Their use is dic-
tated in part by their cost. Ironically, expensive
enzymes are often of lower quality.
Anticipated Results
Complete cleavage of the DNA by a restric-
tion endonuclease should generate a set of dis-
crete DNA fragments that are bounded by the
restriction sites. Upon analysis by gel electro-
phoresis (UNIT 10.5), the cleavage products
should be visualized as sharp bands.
Time Considerations
In principle, if 1 U of restriction endonu-
clease digests 1 µg of DNA in 1 hr, then incu-
bations for longer durations might be expected
to permit conservation of expensive enzymes.
In practice, however, some restriction endonu-
cleases have only limited stability in the reac-
tion mixture. Thus, enzyme reactions are usu-
ally carried out for 30 min to 2 hr unless very
large amounts of DNA or expensive enzymes
are involved. In addition, beware that extended
incubations often reveal low levels of contami-
nating nuclease activities, which may confound
experimental results.
Literature Cited
American Chemical Society. 1991. Biotech Buyers’
Guide 1991. ACS, Washington, D.C.
Fuchs, R., and Blakesley, R. 1983. Guide to the use
of type II restriction endonucleases. Methods
Enzymol. 100:3-38.
McClelland, M., Hanish, J., Nelson, M., and Patel,
Y. 1988. KGB: A single buffer for all restriction
endonucleases. Nucl. Acids Res. 16:364.
O’Farrell, P.H., Kutter, E., Nakanishe, M. 1980.
Mol. & Gen. Genet. 179:411-435.
Roberts, R.J. and Macelis, D. 1991. Restriction en-
zymes and their isoschizomers. Nucl. Acids Res.
19 (Supp.#1):2077-2109.
Contributed by Kenneth D. Bloch
Massachusetts General Hospital
Boston, Massachusetts
Current Protocols in Immunology