RESTRICTION ENDONUCLEASES

Technical Guide 2009|10

 restriction enzymes FROM NEB

For over 35 years, New England Biolabs has led the industry in restriction enzyme Cloning & Mapping
innovation. As the first company to commercialize restriction enzymes, we are DNA AMPLIFICATION & PCR
committed to providing a wide selection of high quality reagents at exceptional value.
RNA ANALYSIS
Our dedication to research has led to key advances in the restriction enzyme field
that allow us to offer maximum performance and convenience under a broader range Protein Expression & Analysis
of reaction conditions. Gene Expression & Cellular Analysis

Selection
In 1975, New England Biolabs (NEB) became the first company to commercialize Restriction Enzymes –
restriction enzymes, thereby providing researchers with a key set of tools that proved to be Inside the Numbers*
invaluable for the early development of the biotechnology industry. Since then, NEB
has continued an aggressive program of cloning and overexpressing restriction enzymes.
231 – restriction enzymes sold by NEB
This is done biochemically, by the analysis of cell extracts, and computationally, by the 222 – enzyme specificities
analysis of sequenced genomes. As a result, we now supply more than 220 specificities, 173 – recombinant enzymes
the largest number commercially available. 162 – enzymes exhibiting
100% activity in a single
buffer (NEBuffer 4)
153 – Time-Saver enzymes that
Quality digest substrate DNA in
5 minutes

As a result of our active screening and cloning program, NEB supplies the largest
15 – High Fidelity (HF) enzymes
engineered for reduced
selection of recombinant enzymes available. Expression using a recombinant source star activity
improves product yield and removes the uncertainties of potential contaminants. Each 10 – 8-base cutters
enzyme is carefully quality controlled using a number of sensitive assays that specifically
address nuclease contamination. This results in a higher quality product with proven
10 – nicking enzymes that cleave
only one strand of duplex DNA
lot-to-lot consistency.
4 – homing endonucleases that
r Choose recombinant enzymes for a higher quality product with lot-to-lot consistency. have large asymmetric
recognition sites
52 – Type IIS enzymes

Performance
as of 03/26/09
*

High Fidelity enzymes offer reduced star activity

Cloning and sequencing restriction enzymes has enabled EcoRI-HF
our researchers to enhance screening efforts as well as NEB Supplier A Supplier B

alter enzyme properties through engineering. This has 10 30 50 100 M 10 30 50 100 M 10 30 50 100
led to the discovery of nicking enzymes, the generation
of new specificities, and most recently the introduction
of a new line of High Fidelity (HF) enzymes. These engi-
neered enzymes have the same specificity as their estab-
lished counterparts, and will maximize performance under
a wider range of conditions, including reaction volume,
incubation time and buffer compatibility.
E High Fidelity enzymes have been engineered
for maximum performance.
EcoRI from other suppliers produces the correct banding pattern when 10 units are used; however, star activity is observed with larger
amounts of enzyme. Star activity is not observed with EcoRI-HF™, even at higher enzyme amounts. Reactions were set up according to
recommended reaction conditions of each manufacturer. Reactions contained 1 µg Lambda DNA in a 50 µl volume and were incubated
overnight at 37°C. Marker M is the 1 kb DNA Ladder (NEB #N3232).
2
 Quality & Innovation

Restriction Enzyme Facts

• In nature, restriction enzymes are found
in bacteria and archaea and serve to
restrict viral growth by destroying
foreign DNA

Convenience
• Restriction enzymes usually occur in
combination with one or two modifying
enzymes (DNA methyltransferases) that
Our dedication to restriction enzyme research has led to improvements that simplify protect the cell’s own DNA from cleavage
reaction setup. NEB utilizes a 4 buffer system, with over 160 enzymes recommended for • Type I enzymes are multisubunit proteins
use in a single buffer (NEBuffer 4). This improves efficiency and ease-of-use, especially that cut DNA randomly at a distance far
when performing double digests. Over 150 of our enzymes are Time-Saver qualified, from their recognition sequence
and will digest substrate DNA in five minutes under recommended reaction conditions. • Type II enzymes cut DNA at defined
Time-Saver qualified enzymes are easy-to-use as there is no special formulation or change positions close to or within their recogni-
in concentration. These same enzymes can be used in overnight digests without sample tion sequence and are commonly used in
loss, providing additional flexibility to reaction setup. the laboratory. There are over ten subtypes
with different types of recognition sites,
4 Over 160 restriction enzymes are recommended for use in NEBuffer 4. cleavage sites and cofactor requirements.
C Over 150 enzymes are Time-Saver qualified and will digest substrate DNA in 5 minutes. • The most common Type II enzymes
cleave within their recognition site (e.g.,
Power and purity of Time-Saver qualified restriction enzymes BamHI, EcoRI); sites can be symmetric
HindIII is powerful enough to
Incubation time (minutes) digest 1 µg of DNA in 5 minutes,
or asymmetric
0 5 15 30 60 0/N M but can also be used in overnight
digests with no indication of
• Type IIS enzymes cleave outside their
nuclease contamination. recognition sequence (e.g., FokI, AlwI)
Marker (M) is the 1 kb DNA and are invaluable for emerging technolo-
Ladder (NEB #N3232).
gies in the biotechnology industry
• Type IIM enzymes recognize methylated
targets (e.g., DpnI)
• Type III enzymes are large combination
restriction-and-modification enzymes that
cleave outside their recognition sequences
and require 2 sequences in opposite
Value orientations to cleave one DNA molecule
• Type IV enzymes recognize modified
The development of recombinant enzymes allows NEB to offer products at a lower DNA (methylated, hydroxymethylated,
cost per unit, enabling substantial savings, improved purity and consistency of product. etc.). They require two sites and cleave
There is no added cost for the convenience of Time-Saver qualified enzymes. non-specifically.
Additionally, the scientific expertise of our research staff and extensive technical • Isoschizomers are restriction enzymes
information available through our catalog and website are invaluable resources for that recognize the same sequence as
questions regarding restriction enzymes and for support in experimental design. the prototype
• Neoschizomers are isoschizomers
with different cleavage sites

www.neb.com 3
 maximum performance

New England Biolabs is the only company

to engineer restriction enzymes to address the

High Fidelity (HF) Enzymes undesirable effects of star activity

The latest innovation in restriction enzyme technology

As part of our ongoing commitment to the study and improvement of restriction enzymes, FAQs
NEB is pleased to introduce a line of High Fidelity (HF) restriction enzymes. These
engineered enzymes have the same specificity as their established counterparts with the Q. Why does the HF version of the enzyme
benefit of reduced star activity. Star activity, or relaxed specificity, is an intrinsic property have a different recommended buffer than
of restriction enzymes. Most restriction enzymes will not exhibit star activity when used as the wild type enzyme?
recommended. However, for enzymes that have reported star activity, extra caution must A. In many cases, the mutation introduced
be taken to set up reactions according to the recommended conditions to avoid unwanted into the HF enzyme results in significant
cleavage. changes in buffer preference. For example,
wild type SalI has a strict requirement for
Many techniques such as cloning, genotyping, mutational analysis, mapping, probe NEBuffer 3, a high ionic strength buffer.
preparation, sequencing and methylation detection employ a wide range of reaction However, SalI-HF™ works well in NEBuffer
conditions and require the use of enzymes under suboptimal conditions. These new 4, which is a moderate ionic strength buffer.
products with reduced star activity offer increased flexibility to reaction setup and help
maximize results under a broader range of conditions. Q. What is the advantage of using NEBuffer 4?

Why choose an HF enzyme? A. All HF enzymes work optimally

In addition to reduced star activity, all of these engineered enzymes work optimally in in NEBuffer 4, which has the highest level
NEBuffer 4, which has the highest level of enzyme compatibility and will simplify double of enzyme compatibility in the NEBuffer
digest reactions. They are all Time-Saver qualified and will digest substrate DNA in five system. This can be advantageous when
minutes. To distinguish these engineered enzymes, the letters -HF™ have been added to designing double digests.
the restriction enzyme name and they are packaged in unique purple-capped tubes.

The following table indicates the number of units of HF-enzyme that can be used compared to the wild type counterparts
before significant star activity is detected. The HF Factor refers to the x-fold increase that is achieved by choosing an HF enzyme,
and clearly illustrates the flexibility that is offered by using an HF restriction enzyme.
Maximum Maximum
Product Product Units with no HF Product Product Units with no HF
Name Number Buffer †
Star Activity* factor Name Number Buffer †
Star Activity* factor
BamHI-HF™ #R3136 4 4,000 125 NotI-HF™ #R3189 4 + BSA 64,000 16
BamHI #R0136 3 + BSA 32 NotI #R0189 3 + BSA 4,000
EagI-HF™ #R3505 4 500 2 PvuII-HF™ #R3151 4 500 31
EagI #R0505 3 250 PvuII #R0151 2 16
EcoRI-HF™ #R3101 4 16,000 64 SacI-HF™ #R3156 4 + BSA 4,000 33
EcoRI #R0101 U 250 SacI #R0156 1 + BSA 120
EcoRV-HF™ #R3195 4 64,000 64 SalI-HF™ #R3138 4 2,000 500
EcoRV #R0195 3 + BSA 1,000 SalI #R0138 3 + BSA 4
MfeI-HF™ #R3589 4 500 15 SbfI-HF™ #R3642 4 250 31
MfeI #R0589 4 32 SbfI #R0642 4 8
NcoI-HF™ #R3193 4 16,000 133 ScaI-HF™ #R3122 4 250 62
NcoI #R0193 3 120 ScaI #R0122 3 4
NheI-HF™ #R3131 4 + BSA 32,000 266 SphI-HF™ #R3182 4 2,000 62
NheI #R0131 2 + BSA 120 SphI #R0182 2 32
SspI-HF™ #R3132 4 500
†
Wild type enzymes were tested in supplied buffer for comparisons.
SspI #R0132 U ND
* Wei, H. et al (2008) Nucleic Acids Reseach 36, e50.

4
 maximum performance

Avoiding Star Activity

Tips for preventing unwanted cleavage in
restriction enzyme digests
Under non-standard reaction conditions, some restriction enzymes are capable of cleav- FAQs
ing sequences that are similar but not identical to their defined recognition sequence. This
Q. I would like to digest DNA with EcoRI and
altered specificity has been termed “star” activity. It has been suggested that star activity is a
Xbal at the same time. The Double Digest Finder
general property of restriction endonucleases (1) and that any restriction endonuclease will
recommends a sequential digest, using each enzyme
cleave noncanonical sites under certain extreme conditions, some of which are listed below.
in its supplied NEBuffer. However, both of these
Although the propensity for star activity varies, the vast majority of enzymes from New
enzymes show 100% activity in NEBuffer 2.
England Biolabs will not exhibit star activity when used under recommended conditions in
Why is a double digest not recommended?
their supplied NEBuffers. If an enzyme has been reported to exhibit star activity, it will be
indicated in the product description found in the catalog or on our website. A. Although EcoRI shows 100% activity
in NEBuffer 2, it also exhibits significant
star activity in this buffer. This is observed
CONDITIONS THAT CONTRIBUTE STEPS THAT CAN BE TAKEN
TO STAR ACTIVITY TO INHIBIT STAR ACTIVITY when using NEBuffer 4 as well. For this
reason, a sequential digest is recommended.
Restriction enzymes are stored in 50% glycerol, so a good
rule of thumb is to limit the total amount of enzyme added
Alternatively, EcoRI-HF™ could be used,
High glycerol concentration (>5% v/v) to 10% of the total reaction volume. which has 100% activity in NEBuffer 4.
Use the standard 50 µl reaction volume to reduce evaporation during Since XbaI is also 100% active in NEBuffer
incubation. 4, a double digest could easily be set up.
High concentration of enzyme/µg of Use the fewest units possible to achieve digestion. This avoids
DNA ratio (varies with each enzyme, overdigestion and reduces the final glycerol concentration in the
usually >100 units/µg) reaction. Q. Why is BamHI now supplied with
Non-optimal buffer
Whenever possible, set up reactions in the recommended buffer. NEBuffer 3 rather than a unique buffer?
Buffers with differing ionic strength and pH may contribute to star
activity.
Why is BamHI not recommended for use in
NEBuffer 2 or 4, even though it is listed as being
Use the minimum reaction time required for complete digestion. 100% active in these buffers?
Prolonged reaction time
Prolonged incubation may result in increased star activity.
A. In an effort to simplify our buffer system,
Presence of organic solvents [DMSO,
ethanol (2), ethylene glycol, Make sure the reaction is free of any organic solvents, such as BamHI is now supplied with NEBuffer 3.
dimethylacetamide, dimethylformamide, alcohols, that might be present in the DNA preparation. BamHI has been carefully purified and
sulphalane (3)]
characterized so there is no loss of activity
Use Mg2+ as the divalent cation. Other divalent cations may not
Substitution of Mg2+ with other
fit correctly into the active site of the restriction enzyme, possibly
in this buffer. BamHI works in most of our
divalent cations (Mn2+, Cu2+, Co2+, Zn2+)
interfering with proper recognition. buffers but, like EcoRI, has significant star
activity, particularly in buffers with low salt
New England Biolabs recommends setting up restriction enzyme digests in a 50 µl reaction concentrations. Therefore, BamHI is not
volume. However, different methods may require smaller reaction volumes. When perform- recommended in NEBuffers 1, 2 and 4.
ing restriction enzyme digests in smaller reaction volumes, extra caution must be taken As an alternative, NEB offers BamHI-HF™
to avoid star activity. Alternatively, using our new line of HF restriction enzymes which has the same activity as BamHI,
allows greater flexibility in reaction setup. Please visit our website to learn about new but is 100% active in NEBuffer 4, which
additions to the HF restriction enzyme product line. will simplify double digest reactions.

References:
1. Nasri, M. and Thomas, D. (1986) Nucleic Acids Res. 14, 811.
2. Nasri, M. and Thomas, D. (1987) Nucleic Acids Res. 15, 7677.
3. Tikchonenko, T.I., et al. (1978) Gene, 4, 195.

www.neb.com 5
 additional convenience

Time-Saver Qualified Restriction Enzymes

Whether you are quickly screening large numbers of clones or setting up overnight Only NEB can offer enzymes with power and
digests, you will benefit from our high quality enzymes. Typically, a restriction digest purity – the power to digest in 5 minutes and the
involves the incubation of 1 µl of enzyme with 1 µg of purified DNA in a final vol- purity to withstand overnight digestions with no
ume of 50 µl for 1 hour. However, to speed up the screening process, choose one of loss of sample.
NEB’s enzymes that are Time-Saver qualified. These enzymes will digest 1 µg of DNA
in 5 minutes using 1 µl of enzyme under recommended reaction conditions. Unlike
other suppliers, there is no special formulation, change in concentration or need to buy
TIME- UNIT ASSAY PLASMID
more expensive new lines of enzymes to achieve digestion in 5 minutes. In fact, 63% of ENZYME SAVER SUBSTRATE SUBSTRATE
our enzymes will digest 1 µg of DNA in 5 minutes, while 83% will fully digest in 15 Bme1580I n s
minutes (see our website for a comprehensive list). That means >199 of our restriction BmgBI C l l
enzymes have the power to get the job done fast. BmrI n s

BpmI s
In an effort to provide as much information as possible, NEB has tested all of its enzymes n

on unit assay substrate as well as plasmid substrate. The rate of cutting for plasmid sub- BsaAI C l l

strate can be used as a guide as it is not definitive for all plasmids. Restriction enzymes BsaBI C l s

can often show site preference, presumably determined by the sequence flanking the BsaHI n n

recognition site. In addition, supercoiled DNA may have varying rates of cleavage. BsaWI n s

Information on site preferences and cleavage of supercoiled DNA is found in the BsaXI C l s
technical reference section of our catalog and on our website. BseRI C l l

BsgI C l l
Since all of our enzymes are rigorously tested for nuclease contamination, you can also
safely set up digests for long periods of time without sample degradation. BsiEI C l s

BsiHKAI C l n

BsiWI C l l
TIME- UNIT ASSAY PLASMID TIME- UNIT ASSAY PLASMID
ENZYME SAVER SUBSTRATE SUBSTRATE ENZYME SAVER SUBSTRATE SUBSTRATE BslI C l n

AatII n s AvaI C l s BsmI C l l

AccI n s AvaII C l l BsmAI C l s

Acc65I C l s AvrII C l s BsmBI n s

AciI C l l BaeI n l BsmFI C l l

AclI C l n BamHI C l l BsoBI C l n

AcuI n s BanII C l n Bsp1286I C l l

AflII C l l BbsI n s BspCNI n s

AgeI C l l BbvI C l s BspEI C l s

AhdI C l l BbvCI C l s BspHI n l

AluI C l s BccI n s BsrI C l n

AlwI C l l BceAI n n BsrBI C l n

AlwNI C l l BciVI C l n BsrDI C l n

ApaI C l l BclI C l NT BsrFI C l s

ApaLI C l l BfaI n s BsrGI n s

ApeKI C l n BfuAI C l l BssHII C l s

ApoI C l l BfuCI n s BssKI n s

AscI C l l BglI C l l BstBI C l l

AseI C l l BglII C l n BstEII C l l

AsiSI C l s BlpI C l l BstNI C l l

6
 additional convenience

Legend:
l digests in 5 minutes Over 150 of our enzymes are Time-Saver qualified
n digests in 15 minutes and will digest 1µg of substrate DNA in 5 minutes.
s not completely digested in 15 minutes Look for the Time-Saver icon C on our website.
C Time-Saver qualified - will digest substrate DNA in 5 minutes under recommended reaction conditions

NT not tested

TIME- UNIT ASSAY PLASMID TIME- UNIT ASSAY PLASMID TIME- UNIT ASSAY PLASMID
ENZYME SAVER SUBSTRATE SUBSTRATE ENZYME SAVER SUBSTRATE SUBSTRATE ENZYME SAVER SUBSTRATE SUBSTRATE
BstUI C l l HinfI C l l PmlI C l s

BstXI C l l HinP1I C l s PpuMI C l s

BstYI n l HpaI n l PshAI n n

BstZ17I C l s HpaII C l l PstI C l l

Bsu36I n s HphI C l s PvuI C l s

BtgI C l l Hpy188I n s PvuII C l l

BtsCI l n HpyAV C l l RsaI n l

BtsI C l l HpyCH4IV C l l SacI C l l

Cac8I n s HpyCH4V C l l SacII C l s

ClaI C l l KpnI C l l SalI C l n

CspCI C l l MboI C l s SapI n s

CviAII n l MboII C l l SbfI C l l

CviKI-1 n n MfeI C l l ScaI C l l

CviQI C l l MluI C l l ScrFI C l s

DdeI C l n MlyI C l s SfiI C l s

DpnI C l l MmeI C l l SfoI C l l

DpnII n s MnlI C l l SmaI C l n

DraI C l l MseI n n SnaBI C l l

DraIII C l l MslI C l l SpeI C l l

DrdI n l MspI C l l SphI C l l

EagI C l s MspA1I C l l SspI C l l

EarI n n MwoI n s StuI n s

EcoNI C l n NciI C l l StyI n s

EcoO109I C l s NcoI C l n StyD4I n s

EcoP15I n s NdeI C l l SwaI n s

EcoRI C l l NgoMIV n l TaqI C l l

EcoRV C l l NheI n n TfiI n l

Fnu4HI C l n NlaIII n s TseI n s

FokI C l l NotI C l l Tsp509I C l l

FseI C l l NruI C l n TspMI C l n

FspI n s NsiI C l l TspRI C l n

HaeII n s NspI C l n Tth111I n n

HaeIII C l l PacI C l l XbaI C l l

HgaI n s PaeR7I C l s XcmI n l

HhaI C l n PflfI C l n XhoI C l l

HincII n s PflMI C l s XmaI n s

HindIII C l n PmeI l n XmnI C l l

www.neb.com 7
 technical tips

Optimizing Restriction Enzyme Reactions

There are several key factors to consider when setting up a restriction endonuclease Typical Restriction Enzyme Digest
digest. Using the recommended amounts of DNA, enzyme and buffer components in COMPONENT SPECIFICATION
the correct reaction volume enables optimal digestion. By definition, 1 unit of restriction Restriction Enzyme 10 units is sufficient
enzyme will completely digest 1 µg of substrate DNA in a 50 µl reaction in 60 minutes. DNA 1 µg
This enzyme : DNA : reaction volume ratio can be used as a guide when designing 10X NEBuffer 5 µl (1X)
reactions. However, most researchers follow the “typical” reaction conditions listed, Add to a final concentration of
BSA
where a 5-10-fold overdigestion is recommended to overcome variability in DNA source, 100 µg/ml (1X) if necessary

quantity and purity. The following tips will help achieve maximum success in your Total Reaction Volume 50 µl
restriction endonuclease reactions. Incubation Time 1 hour*

Incubation Temperature Enzyme Dependent

Enzyme
• Keep on ice when not in the freezer
• Should be the last component added to
reaction
• Mix components after addition of enzyme
by pipetting the reaction mixture up and
down, or by “flicking” the reaction tube.
Follow with a quick (“touch”) spin-down
in a microcentrifuge. Do not vortex the
reaction.
DNA
• Should be free of contaminants such
as phenol, chloroform, alcohol, EDTA,
detergents, nucleases or excessive salts
• Methylation of DNA can inhibit digestion
with certain enzymes
Buffer
• Use at a 1X concentration
• If required, add BSA to a final concentra-
tion of 100 µg/ml (1:100 dilution)
• Restriction enzymes that do not require
Incubation Time
• Can often be decreased by using an
excess of enzyme, or by using one of our
Time-Saver qualified enzymes (see page 6)
• With many enzymes, it is possible to use
fewer units and digest for up to 16 hours.
For more information, visit www.neb.com.
Stopping a Reaction
If no further manipulation of DNA is
required:
• Terminate with a stop solution [50%
glycerol, 50 mM EDTA (pH 8.0), and
0.05% bromophenol blue]. Use 10 µl
per 50 µl reaction.
When further manipulation of DNA is
required:
• Heat inactivation can be used (buffer
chart indicates if the enzyme can be
heat inactivated)
• Remove enzyme by using a spin column 
or phenol/chloroform extraction
Control Reactions
* This can be decreased by using a Time-Saver qualified enzyme.
Reactions in Smaller Volumes
Many techniques such as cloning,
genotyping, mutational analysis, mapping,
probe preparation, sequencing and
methylation detection require the use
of enzymes under suboptimal conditions.
Additives in the restriction enzyme
storage buffer (e.g., glycerol, salt) as well
as contaminants found in the substrate
solution (e.g., salt, EDTA, or alcohol) can
be problematic in smaller reaction volumes.
NEB has introduced a line of High Fidelity
(HF) enzymes that provide added flexibility
to reaction setup. For more information on
HF enzymes, see page 4. The following
guidelines can be used for techniques that
require smaller reaction volumes.
Restriction digests in
smaller reaction volumes
10 µl † 25 µl 50 µl
REACTION REACTION REACTION

BSA for optimal activity are not adversely
affected if BSA is present in the reaction
Reaction Volume
• A 50 µl reaction volume is recommended
for digestion of 1 µg of substrate
• Keep glycerol concentration at less than
5% of total reaction volume to prevent star
activity
• The restriction enzyme (supplied in 50%
glycerol) should not exceed 10% of the
total reaction volume
8
For difficulty cleaving DNA substrate, Restriction
1 unit 5 units 10 units
Enzyme*
we recommend the following controls:
DNA 0.2 µg 0.5 µg 1 µg
• Experimental DNA without restriction
10X
enzyme to check for contamination in the NEBuffer
1 µl 2.5 µl 5 µl
DNA preparation or reaction buffer
100X BSA** 0.1 µl 0.25 µl 0.5 µl
• Control DNA (DNA with multiple known
sites for the enzyme) with restriction
enzyme to test enzyme viability
* Restriction enzyme can be diluted using the recommended diluent
buffer when smaller amounts are needed.
† 10 µl reactions should not be incubated for longer than
• If the control DNA is cleaved and the 1 hour to avoid evaporation.
experimental DNA resists cleavage, the ** BSA can be diluted in 1X buffer
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.
 troubleshooting

Troubleshooting Guide
Problem possible cause Solution FAQs
• Test enzyme on control DNA with known multiple sites Q. Do restriction enzymes cleave
Enzyme is inactive • Enzyme should be stored at –20°C. Enzymes stored at –70°C will
freeze, and repeated thaw/freeze cycles reduce enzyme activity. single-stranded DNA?
• Use recommended buffer supplied with restriction enzyme A. Although some restriction enzymes
Reaction conditions • Follow double digest recommendations, or try a sequential digest
are not optimal • Repeat with fresh buffer. Additives present in buffer have been reported to cleave ssDNA it
(e.g., DTT, SAM) may degrade over time. is unclear whether cleavage occurs on
Enzyme a ssDNA molecule or on two ssDNA
concentration is Some plasmids or genomic DNAs may require up to 10-20 units/µg
too low molecules which transiently anneal at a
Repeat reaction setup, being sure that enzyme and/or additives
region of partial homology (1-3). For this
Additive is missing
(e.g., BSA) are added reason, we hesitate to make unconstrained
DNA concentration NEB recommends 1 µg of DNA in a 50 µl reaction. claims about a restriction enzyme’s ability
is not optimal Excess DNA may result in incomplete cleavage. to cut ssDNA.
Incubation time is Some enzymes can exhibit slower cleavage towards specific sites.
too short In most cases, 1-2 hours are sufficient. Q. How stable are restriction enzymes?
Incomplete • Assay substrate DNA in the presence of a control DNA.
or no DNA is Control DNA will not cleave if there is an inhibitor is present. A. All restriction enzymes from NEB are
digestion contaminated with Miniprep DNA is particularly susceptible to contaminants. assayed for activity every 3-6 months.
an inhibitor • Clean DNA with a spin column, resin or drop dialysis,
or increase volume to dilute contaminant. Most are very stable when stored at
Recognition site is -20°C in the recommended storage buffer.
Confirm DNA sequence
not present Exposure to temperatures above -20°C
• DNA isolated from a bacterial source may be blocked by Dam and Dcm should be minimized whenever possible.
methylation. DNA should be passed through a dam-/dcm- strain
Cleavage is blocked (NEB #C2925).
by methylation
Q. Is extended digestion (incubation times
• Eukaryotic genomic DNA may be blocked by CpG methylation.
This can be overcome by cloning into a bacterial host. > 1 hour) recommended?
• PCR products are not methylated.
A. The unit definition of our restriction
DNA may be Restriction enzymes cleave supercoiled DNA with varying efficiency.
supercoiled Additional enzyme may be required. enzymes is based on a 1 hour incuba-
Recognition site tion. Incubation time may be shortened if
may be too close to As a general rule, add 6 base pairs on either side of the recognition site additional units of restriction enzyme are
the end of the DNA for efficient cleavage
fragment added to the reaction. Conversely, longer
Site preference Enzyme requires two recognition sites for efficient cleavage
incubation times are often used to allow
Run uncut substrate DNA alongside the digest. A partial digest will
a reaction to proceed to completion with
Determine nature of
pattern
show bands found in the uncut, whereas star activity will show bands of fewer units of enzyme. This is contingent
unexpected size. on how long a particular enzyme can
DNA sample is survive (maintain activity) in a reaction.
Unexpected Prepare a new DNA sample
contaminated
Cleavage Additional information on extended
Pattern Additional
recognition sites are Confirm DNA sequence digestion can be found at www.neb.com.
present in DNA
See tips for avoiding star activity (see page 5) and/or use a
Star Activity
High Fidelity Restriction Enzyme (see page 4)
References
Enzyme has a high
Add SDS to the gel loading dye/stop solution to a final concentration 1. Blakesley, R.W., Wells, R.D. (1975) Nature 257, 421-422.
binding affinity to
of 0.1–0.5% to help dissociate the enzyme from the DNA, or treat 2. Blakesley, R.W., et al. (1977) J. Biol. Chem. 252, 7300-7306.
DNA and will not
with protease before loading 3. Yoo, O.J., Agarwal, K.L, (1980) J. Biol. Chem. 255, 10559-
dissociate well
Smearing of 10562.
DNA on gel Nuclease Care should be taken to avoid cross contamination when
contamination setting up reactions
Agarose running
Use fresh running buffer and appropriate voltage to avoid overheating
conditions

www.neb.com 9
 technical reference

For help choosing double digest conditions,

Double Digestion try the Double Digest Finder (page 13).

Digesting DNA substrate with two restriction endonucleases simultaneously (double digestion)
is a common timesaving procedure. Selecting the best NEBuffer to provide reaction conditions
that optimize enzyme activity and avoid star activity associated with some enzymes is
an important consideration. Each enzyme is supplied with an NEBuffer that ensures 100%
activity. (For compositions, see page 15). The Activity Chart for Restriction Enzymes
(pages 15 - 19) rates the percentage activity of each restriction endonuclease in the four
standard NEBuffers and NEBuffer EcoRI.

Setting up a Double Digest • If two different incubation temperatures Additional double digest information with
are necessary, choose the optimal reac- unique buffers can be found on our website,
• Choose an NEBuffer that results in the www.neb.com.
tion buffer and set up reaction accordingly.
most activity for both enzymes, provided Add the first enzyme and incubate at the
that star activity is not a concern. desired temperature. Heat inactivate the first Setting up a Sequential Digest
• If BSA is required for either enzyme, enzyme, add the second enzyme and • Set up a reaction using the restriction
add it to the double digest reaction (BSA incubate at the recommended temperature. endonuclease that has the lowest salt
does not inhibit restriction enzymes). concentration in its recommended buffer
• Depending on an enzyme’s activity rating
• Set up reaction according to recom- and incubate to completion.
in a non-optimal NEBuffer, the number of
mended conditions (see p. 8). The final • Adjust the salt concentration of the
units or incubation time may be adjusted
concentration of glycerol in any reaction reaction (using a small volume of a
to compensate for slower rate of cleavage.
should be less than 5% to minimize the concentrated salt solution) to approxi-
possibility of star activity (see p. 5). Setting up a Double Digest mate the reaction conditions of the
For example, in a 50 µl reaction, the second restriction endonuclease.
total amount of enzyme added should with a unique buffer
• Add the second enzyme and incubate
not exceed 5 µl. • Our buffer system has been streamlined, to complete the second reaction.
• Incubate at recommended temperature. leaving three enzymes that have unique
• Alternatively, a spin column can be used
Overnight digests should be avoided due NEBuffers: EcoRI (included in activity
to isolate the DNA prior to the second
to the possibility of star activity. chart), SspI (same buffer composition as
reaction.
EcoRI) and DpnII. In most cases, DpnII
requires a sequential digest.
Double Digest Chart
ENZYME AatII AvrII BamHI BglII BsgI EagI EcoRI EcoRV HindIII KpnI MseI NcoI NdeI NheI NotI PstI PvuI SacI SacII SalI SmaI SpeI SphI XbaI XhoI
NEBuffer 4 4 3 3 4 3 U 3 2 1 4 3 4 2 3 3 3 1 4 3 4 4 2 4 4
AvrII 4 4
BamHI 3 seq 3
BglII 3 seq 2 3
BsgI 4 4 4 3 3 Enzymes in purple are also available in
EagI 3 seq 3 3 3 seq a High Fidelity (HF) format. HF enzymes
EcoRI U seq EcoRI EcoRI EcoRI seq EcoRI are 100% active in NEBuffer 4 and will
EcoRV 3 4 2 3 3 4 3 EcoRI simplify double digest reactions.
HindIII 2 4 2 seq 2 2 seq seq 2
KpnI 1 4 1 seq 2 seq seq 1 2 2
MseI 4 4 4 3 2 4 3 EcoRI 2 2 1
NcoI 3 4 4 3 3 4 3 EcoRI 3 2 1 4
NdeI 4 4 4 3 3 4 3 EcoRI 2 2 1 4 4
NheI 2 4 2 seq 2 4 seq 1 2 2 1 2 2 4
NotI 3 seq 3 3 3 3 3 EcoRI 3 2 2 2 3 3 2
PstI 3 4 3 3 3 3 3 EcoRI 3 2 1 3 3 3 2 3
PvuI 3 seq 2 3 3 3 3 EcoRI 3 2 2 3 3 3 2 3 3
SacI 1 4 1 seq 2 4 seq 1 2 2 1 4 1 4 1 2 1 2
SacII 4 4 4 seq seq 4 seq EcoRI 2 2 4 4 4 4 4 2 2 2 4
SalI 3 seq 3 3 3 3 3 EcoRI 3 seq seq 3 3 3 seq 3 3 3 seq seq
SmaI 4 4 4 seq seq 4 seq seq 4 4 seq 4 4 4 4 seq 4 seq 4 4 seq
SpeI 4 4 4 seq 2 4 seq EcoRI 2 2 1 4 4 4 2 2 2 2 1 4 seq 4
SphI 2 4 2 3 2 4 3 EcoRI 2 2 1 2 2 2 2 2 2 2 1 4 3 4 2
XbaI 4 4 4 3 2 4 3 seq 2 2 2 4 4 4 2 3 3 3 4 4 3 4 4 2
XhoI 4 4 4 3 3 4 3 EcoRI 3 2 1 4 4 4 2 3 3 3 4 4 3 4 4 2 4
XmaI 4 4 4 seq seq 4 seq seq 4 seq 4 4 4 4 4 2 4 seq 4 4 seq 4 4 4 4 4
10
 technical reference

DNA Methylation & Restriction Digests

Two common types of methylation that can block cutting at a restriction site are Dam and Dam/Dcm Sensitive
Dcm methylation. Both arise from replicating DNA in a strain of E. coli that has functional
Dam and Dcm methylation systems.
Restriction Enzymes
available from NEB
Site Blocked by Dam/Dcm Methylation The following enzymes are blocked or
Restriction by some enzymes can be inhibited due to methylation caused by the common impaired by Dam or Dcm methylation.
E. coli methyltransferases. Dam methyltransferase causes methylation of the adenine in In order to achieve cleavage with these
the sequence GATC while Dcm methyltransferase causes methylation of the first cytosine enzymes, the DNA should be passed
in the sequence CC(A/T)GG. Any restriction enzyme whose site contains either of these through a dam–/dcm– strain.
sequences may be affected by the relevant methylation. For example, the site for AlwI
Acc651 BspHI PflMI
(GGATC4/5) contains the recognition site for Dam methyltransferase, GATC. If the DNA
AlwI BssKI PhoI
is produced in a methylating E. coli strain, the adenine is methylated and cleavage by AlwI
AlwNI BstXI PpuMI
is blocked. In the case of AlwI or any other restriction enzyme blocked by Dam methy-
lation, blocked cleavage can be avoided by cloning the DNA into a dam– strain such as ApaI ClaI PspGI

dam–/dcm– Competent E. coli (NEB #C2925). AvaII DpnII PspOMI

BanI EaeI Sau96I
On the other hand BamHI is not Dam/Dcm sensitive; the BamHI site contains GATC BcgI EcoO1091 ScrFI
but cleavage by this enzyme is not blocked even when the adenine is methylated. Bc1I HphI SexAI
The same principles apply for Dcm methylation but the enzyme sites affected would
BsaI Hpy188I SfiI
contain the sequence CC(A/T)GG.
BsaBI Hpy1881II SfoI

dam/dcm Overlapping Sites BsaHI Mbol StuI

Bs1I MboII StyD41
Restriction sites can also be blocked if an overlapping site is present. In this case, BsmFI MscI TaqIa
part of the dam or dcm sequence is generated by the restriction enzyme sequence followed
BspDI NlaIV XbaI
by the flanking sequence. For example, the site for ClaI (AT/CGAT) contains GAT.
BspEI Nrul
If it is followed by a C, the A can be methylated, and cleavage will be blocked.
Methylation sensitivity information can
More information regarding methylation sensitivity can be found in the technical
also be found on REBASE (Restriction Enzyme
reference section of the NEB Catalog & Technical Reference, or www.neb.com.
Database), a comprehensive database of
information about restriction enzymes and
Key Points to Consider:
related proteins. For more information, see
• Genomic DNA directly isolated from a mammalian source is not Dcm or Dam methylated,
and is therefore not an issue when digesting mammalian DNA. page 13.

• Mammalian and plant DNA that has been cloned into a methylating E. coli strain will be
Dam/Dcm methylated. Most commonly used laboratory E. coli strains methylate DNA.
• Directly isolated mammalian and plant genomic DNA are CpG methylated. Some enzymes
are inhibited by CpG methylation. (See www.neb.com for more information).
• Most bacterial DNA (including E. coli DNA) is not CpG methylated. Inhibition of enzyme
activity by CpG methylation is not an issue for DNA prepared from E. coli strains.
• DNA amplified by PCR does not contain any methylated bases.
• To avoid Dam/Dcm methylation when subcloning in bacteria, NEB offers the
methyltransferase deficient cloning strain dam–/dcm– Competent E. coli (NEB #C2925)
for propagation.

www.neb.com 11
 web tools

Online Tools
As the leader in enzyme technology, NEB’s technical support is an invaluable resource.
Our scientists provide assistance via info@neb.com or 1-800-NEB-LABS (1-800-632-7799).
To aid in experimental design, NEB has created several user-friendly web-based online tools.
These tools and the technical reference section of www.neb.com can help you choose an enzyme,
design an experiment or troubleshoot an unexpected result.

Enzyme Finder
Enzyme Finder can be used to search for
restriction enzymes by name, sequence, overhang
or type. Search results include all enzyme matches,
with properties for NEB enzymes listed.
www.neb.com/nebecomm/EnzymeFinder.asp

NEBcutter
NEBcutter allows you to input sequence data and find large
ORFs, identify restriction sites and generate custom digests
and virtual gels. Sequences submitted are maintained locally
for 2 days and then discarded for your privacy.
tools.neb.com/NEBcutter2/index.php
Submit your sequence to find largest
ORF, identify restriction sites and
generate custom digests

Zoom in to view
cleavage overhangs

Identify enzymes affected

by methylation
Calculate GC Content

Introduce silent mutation sites

into ORF sequence with a
single mouse click Expand region of interest to
obtain sequence information

Generate a custom digest

and create a virtual gel
by choosing appropriate List enzymes by number of
markers and gel types sites produced, alphabetically
or by cut frequency

Choose linear or circular maps

Summarize ORF sequences
12
 Web tools

Double Digest Finder

Select optimal conditions for double digest including buffer,
incubation, temperature and supplement requirements.
www.neb.com/nebecomm/DoubleDigestCalculator.asp

REBASE
REBASE (The Restriction Enzyme Database) is a complete listing of all known restriction
endonucleases. It contains data such as recognition sequences, cleavage sites, methylation
sensitivity, isoschizomers and commercial availability of enzymes.
rebase.neb.com/rebase/

Lists all references

containing this enzyme,
with links to reference
details

Lists available suppliers

Summary of enzyme source,

status and properties Includes labeled map view,
links to sequence databases,
gene information and ability
to download DNA and
protein sequence files

View a complete list of

enzymes in the database
including recognition
sequences, isoschizomers Contains detailed methylation
and commercial suppliers sensitivity testing data

Click here for specialized infor- Access available tools (theoretical digests,
mation (star activity, methylation, BLAST, NEBcutter, REBpredictor)
cleavage type, etc.), REBASE
genomes and sequence collections

Additional web-based tools can be found on

our website, www.neb.com.

www.neb.com 13
 technical reference

Cleavage Close to the Ends of DNA Fragments

To simulate cloning reactions, a selection of NEB restriction enzymes have been tested
for their ability to cleave close to the end of a DNA fragment. Reaction conditions are
described below. Note that the data reported represents the minimum number of bases
that will work, and will not necessarily result in maximum cleavage. As a general rule,
6 base pairs should be added on either side of a restriction enzyme recognition site to
cleave efficiently.

Experimental: Linearized vectors were incubated with the indicated enzymes (10 units/µg) The technical reference section of our website
for 60 minutes at the recommended reaction conditions for each enzyme. Following ligation contains additional charts, protocols and
and transformation, cleavage efficiencies were determined by dividing the number of technical tips related to restriction enzymes.
transformants from the digestion reaction by the number obtained from religation of the
linearized DNA (typically 100–500 colonies) and subtracting from 100%. “Base Pairs
from End” refers to the number of double-stranded base pairs between the recognition
site and the terminus of the fragment; this number does not include the single-stranded
overhang from the initial cut.

BASE PAIRS % CLEAVAGE INITIAL BASE PAIRS % CLEAVAGE INITIAL

ENZYME FROM END EFFICIENCY VECTOR CUT ENZYME FROM END EFFICIENCY VECTOR CUT
AatII 3 88 LITMUS 29 NcoI MluI 2 99 LITMUS 39 EagI
2 100 LITMUS 28 NcoI MunI 2 100 LITMUS 39 NgoMIV
1 95 LITMUS 29 PinAI NcoI 2 100 LITMUS 28 HindIII
Acc65I 2 99 LITMUS 29 SpeI NgoMIV 2 100 LITMUS 39 MunI
1 75 pNEB193 SacI NheI 1 100 LITMUS 39 EcoRI
AflII 1 13 LITMUS 29 StuI 2 82 LITMUS 39 EagI
AgeI 1 100 LITMUS 29 XbaI NotI 7 100 Bluescript SK- SpeI
1 100 LITMUS 29 AatII 4 100 Bluescript SK- KspI
ApaI 2 100 LITMUS 38 SpeI 1 98 Bluescript SK- XbaI
AscI 1 97 pNEB193 BamHI NsiI 3 100 LITMUS 29 BssHII
AvrII 1 100 LITMUS 29 SacI 3 77 LITMUS 29 BglII
BamHI 1 97 LITMUS 29 HindIII 2 95 LITMUS 28 BssHII
BglII 3 100 LITMUS 29 NsiI PacI 1 76 pNEB193 BamHI
BsiWI 2 100 LITMUS 29 BssHII PmeI 1 94 pNEB193 PstI
BspEI 2 100 LITMUS 39 BsrGI PstI 3 98 LITMUS 29 EcoR V
1 8 LITMUS 38 BsrGI 2 50 LITMUS 39 HindIII
BsrGI 2 99 LITMUS 39 SphI 1 37 LITMUS 29 EcoRI
1 88 LITMUS 38 BspEI SacI 1 99 LITMUS 29 AvrII
BssHII 2 100 LITMUS 29 BsiWI SalI 3 89 LITMUS 39 SpeI
EagI 2 100 LITMUS 39 NheI 2 23 LITMUS 39 SphI
EcoRI 1 100 LITMUS 29 XhoI 1 61 LITMUS 38 SphI
1 88 LITMUS 29 PstI SfiI* 9 81 LITMUS 38 BamHI
1 100 LITMUS 39 NheI 4 97 LITMUS 38 MluI
EcoRV 1 100 LITMUS 29 PstI 1 93 LITMUS 38 EcoRI
HindIII 3 90 LITMUS 29 NcoI SpeI 2 100 LITMUS 29 Acc65I
2 91 LITMUS 28 NcoI 2 100 LITMUS 29 KpnI
1 0 LITMUS 29 BamHI SphI 2 99 LITMUS 39 SalI
KasI 2 97 LITMUS 38 NgoMIV 2 97 LITMUS 39 BsrGI
1 93 LITMUS 38 HindIII 1 92 LITMUS 38 SalI
KpnI 2 100 LITMUS 29 SpeI XbaI 1 99 LITMUS 29 AgeI
2 100 LITMUS 29 SacI 1 94 LITMUS 29 PinAI
1 99 pNEB193 SacI XhoI 1 97 LITMUS 29 EcoRI
XmaI 2 98 pNEB193 AscI
*
A modified version of LITMUS 38 with an introduced SfiI site was used for this test.
2 92 pNEB193 BssHII

14
 selection

Activity Chart for Restriction Enzymes

New England Biolabs utilizes a simple 4 buffer system. A color-coded 10X NEBuffer is Chart Legend
supplied with every restriction endonuclease, ensuring 100% activity. To help select the Supplied with a unique reaction buffer
best conditions for double digests, this chart shows the optimal (supplied) NEBuffer and that is different from the four standard
U NEBuffers. The compatibility with the
approximate activity in the four standard NEBuffers and NEBuffer EcoRI for each enzyme. four standard NEBuffers is indicated
in the chart.
If BSA is supplied with an enzyme, it is included in all NEBuffer activity reactions.
Additional double digest information can be found on page 10. Supplied with a separate vial of bovine
serum albumin (10 mg/ml). To obtain
BSA 100% activity, BSA should be added
Activity Chart Highlights to the 1X reaction mix to a final
concentration of 100 µg/ml.
• Over 160 restriction enzymes exhibit 100% activity in a single buffer (NEBuffer 4)
Supplied with a separate vial of
• Over 150 enzymes are Time-Saver qualified and will digest substrate DNA in 5 minutes S-adenosylmethionine (SAM).
SAM To obtain 100% activity, SAM should
be added to the 1X reaction mix as
• A selection of High Fidelity (HF) enzymes are available that have been engineered for specified on the product data card.
reduced star activity
This NEBuffer is recommended for
dd a double digest because it minimizes
star activity.

This buffer is not recommended for

HEAT NR
use with this enzyme.
SUPPLIED % ACTIVITY in NEBuffers INACTIVATION INCUBATION
ENZYME NEBuffer 1 2 3 4 EcoRI (TEMP) TEMPERATURE
r Produced from a recombinant source.
r AatII 4 0 50 50 100 25 65° 37°
r AccI 4 50 50 10 100 5 80° 37° Time-Saver qualified (digests 1 µg
r C Acc65I 3 + BSA 10 75 100 25 50 65° 37° C of substrate DNA in 5 minutes under
recommended reaction conditions).
C AciI 3 25 50 100 50 100 65° 37°
r C AclI 4 + BSA 10 10 0 100 5 No 37° Engineered enzyme for maximum
E performance.
r AcuI 4 + SAM 50 100 50 100 100 65° 37°
r AfeI 4 25 50 25 100 100 65° 37°
r C AflII 4 + BSA 50 100 25 100 0 65° 37°
NEBuffer Compositions (1X)
r AflIII 3 + BSA 25 75 100 50 100 80° 37°
r C AgeI 1 100 50 10 75 50 65° 37° 10 mM Bis Tris Propane-HCl,
NEBuffer 1
10 mM MgCl2, 1 mM DTT
r C AhdI 4 + BSA 25 75 0 100 10 65° 37° (yellow)
(pH 7.0 @ 25°C).
r AleI 4 10 25 10 100 0 65° 37°
10 mM Tris-HCl, 10 mM
r C AluI 4 100 100 75 100 100 65° 37° NEBuffer 2
MgCl2, 50 mM NaCl,
(blue)
r C AlwI 4 50 100 10 100 5 65° 37° 1 mM DTT (pH 7.9 @ 25°C).
C AlwNI 4 50 100 50 100 10 65° 37°
50 mM Tris-HCl, 10 mM
r C ApaI 4 + BSA 25 50 0 100 5 65° 25° NEBuffer 3
MgCl2, 100 mM NaCl,
(red)
r C ApaLI 4 + BSA 100 100 10 100 5 No 37° 1 mM DTT (pH 7.9 @ 25°C).
r C ApeKI 3 25 75 100 50 100 No 75°
 0 mM Tris-acetate, 10 mM
2
r C ApoI 3 + BSA 10 75 100 75 100 80° 50° NEBuffer 4 magnesium acetate, 50 mM 
(green) potassium acetate, 1 mM DTT
r C AscI 4 0 10 10 100 5 65° 37°
(pH 7.9 @ 25°C).
r C AseI 3 NR 75 dd
100 NR 50 65° 37°
50 mM NaCI, 100 mM
r C AsiSI 4 + BSA 50 100 100 100 0 80° 37° NEBuffer Tris-HCI, 10 mM MgCI2,
r C AvaI 4 10 75 10 100 50 80° 37° EcoRI 0.025% Triton X-100
(pH 7.5 @ 25°C).
r C AvaII 4 50 75 10 100 50 65° 37°
r C AvrII 4 100 100 50 100 50 80° 37°
Buffers should be thawed completely and
BaeI 4 + BSA + SAM 50 100 50 100 100 65° 25°
mixed thoroughly before using.
C BaeGI 1 100 75 10 50 100 65° 37°
r C BamHI 3 + BSA 75 100 dd
100 100 100 No 37°
r C E BamHI-HF 4 100 50 10 100 25 No 37°
r BanI 4 50 100 50 100 25 65° 37°

Note: The values listed in this table are approximate. Values were obtained using each enzyme’s specific unit assay substrate DNA. Updates to this chart can
be found on our web site, www.neb.com.

www.neb.com 15
 selection

HEAT
SUPPLIED % ACTIVITY in NEBuffers INACTIVATION INCUBATION
ENZYME NEBuffer 1 2 3 4 EcoRI (TEMP) TEMPERATURE

BbsI 2 100 100 25 75 50 65° 37°

r C BbvCI 4 50 100 10 100 100 80° 37°
r C BbvI 2 100 100 25 75 100 65° 37°
r BccI 1 + BSA 100 50 10 50 15 65° 37°
BceAI 3 + BSA 100 100 100 100 100 65° 37°
r BcgI 3 + SAM NR NR dd
100 NR 50 65° 37°
r C BciVI 4 100 50 0 100 25 65° 37°
r C BclI 3 50 100 dd
100 75 100 No 50°
BfaI 4 75 50 10 100 0 80° 37°
C BfuAI 3 0 75 100 10 100 65° 50°
BfuCI 4 + BSA 100 50 25 100 15 80° 37°
r C BglI 3 50 75 100 50 100 65° 37°
r C BglII 3 10 75 100 10 100 No 37°
r C BlpI 4 50 100 10 100 50 No 37°
C BmgBI 3 + BSA 0 25 100 10 50 65° 37°
r BmrI 2 75 100 75 100 50 65° 37°
r BmtI 2 25 100 25 50 100 65° 37°
BpmI 3 + BSA 75 100 100 100 100 65° 37°
r Bpu10I 3 10 25 100 25 100 80° 37°
BpuEI 4 + SAM 10 100 50 100 100 65° 25°
r BsaI 4 75 75 100 100 100 65° 50°
r C BsaAI 4 100 100 100 100 100 No 37°
C BsaBI 4 50 100 75 100 100 80° 60°
r BsaHI 4 + BSA 50 100 100 100 100 80° 37°
r BsaJI 4 100 100 100 100 100 80° 60°
BsaWI 4 + BSA 50 100 50 100 40 80° 60°
C BsaXI 4 75 100 10 100 15 No 37°
r C BseRI 4 100 100 75 100 100 65° 37°
r BseYI 3 10 50 100 50 100 65° 37°
C BsgI 4 + SAM 50 75 50 100 40 65° 37°
C BsiEI 4 + BSA 50 100 10 100 5 80° 60°
BsiHKAI 4 + BSA 50 100 100 100 25 No 65°
r C BsiWI 3 100 100 100 25 100 80° 55°
r C BslI 3 10 50 100 75 100 80° 55°
r C BsmI 4 75 100 75 100 5 80° 65°
r C BsmAI 4 50 100 100 100 100 80° 55°
r BsmBI 3 75 100 100 100 100 80° 55°
r C BsmFI 4 + BSA 25 50 50 100 15 80° 65°
r C BsoBI 2 10 100 100 50 100 No 37°
C Bsp1286I 4 + BSA 25 25 25 100 10 65° 37°
BspCNI 4 + BSA + SAM 100 75 10 100 15 80° 25°
BspDI 4 25 75 50 100 100 65° 37°
r C BspEI 3 0 10 100 0 100 80° 37°
r BspHI 4 10 100 50 100 5 65° 37°
r BspMI 3 NR NR 100 NR 100 65° 37°
r C BspQI 4 10 50 100 100 100 80° 50° New England Biolabs offers
C BsrI 3 0 50 100 10 100 80° 65°
over 220 enzyme specificities.
C BsrBI 4 50 100 100 100 100 80° 37°
C BsrDI 2 + BSA 50 100 50 75 50 80° 65°
r C BsrFI 4 10 100 100 100 100 No 37°
BsrGI 2 + BSA 25 100 10 100 50 80° 37°
r C BssHII 3 100 100 dd
100 100 100 80° 50°
r BssKI 3 + BSA 0 50 100 50 100 80° 60°
r BssSI 3 0 50 dd
100 10 100 80° 37°
r BstAPI 4 25 100 100 100 100 80° 60°

16
 selection

HEAT
SUPPLIED % ACTIVITY in NEBuffers INACTIVATION INCUBATION
ENZYME NEBuffer 1 2 3 4 EcoRI (TEMP) TEMPERATURE

r C BstBI 4 75 50 25 100 50 No 65°

r C BstEII 3 + BSA 50 75 100 75 100 No 60°
r C BstNI 2 + BSA 10 100 100 75 50 No 60°
BstUI 4
C 100 100 50 100 100 No 60° Chart Legend
r C BstXI 3 0 50 100 25 100 65° 37°
BstYI 2 Supplied with a unique reaction buffer
r 50 100 75 100 50 80° 60° that is different from the four standard
C BstZ17I 4 NR NR 100 100 100 No 37° U NEBuffers. The compatibility with the
Bsu36I 3 + BSA four standard NEBuffers is indicated
r 0 25 100 0 15 80° 37° in the chart.
r C BtgI 3 + BSA 25 50 100 100 100 80° 37°
BtgZI 4 + BSA Supplied with a separate vial of bovine
10 25 0 100 100 80° 60°
serum albumin (10 mg/ml). To obtain
r C BtsI 4 + BSA 100 50 50 100 25 80° 55° BSA 100% activity, BSA should be added
BtsCI 4 to the 1X reaction mix to a final
r C 50 100 50 100 25 No 50° concentration of 100 µg/ml.
Cac8I 4 50 75 100 100 50 65° 37°
r C ClaI 4 + BSA 10 50 50 100 50 65° 37° Supplied with a separate vial of
S-adenosylmethionine (SAM).
r C CspCI 4 + SAM 10 100 10 100 40 65° 37° SAM To obtain 100% activity, SAM should
r CviAII 4 75 25 10 100 100 65° 25° be added to the 1X reaction mix as
specified on the product data card.
r CviKI-1 4 10 50 25 100 100 80° 37°
r C CviQI 3 + BSA 75 100 100 75 0 No 25° This NEBuffer is recommended for
r C DdeI 3 75 100 100 75 100 65° 37° dd a double digest because it minimizes
DpnI 4 star activity.
r C 100 100 75 100 100 80° 37°
r DpnII U NR NR dd
100 NR 100 65° 37°
This buffer is not recommended for
r C DraI 4 NR
75 75 50 100 100 65° 37° use with this enzyme.
r C DraIII 3 + BSA 100 75 100 25 50 65° 37°
DrdI 4 r Produced from a recombinant source.
25 50 10 100 100 65° 37°
r EaeI 4 100 100 50 100 100 80° 37°
Time-Saver qualified (digests 1 µg
r C EagI 3 10 25 100 10 100 65° 37° C of substrate DNA in 5 minutes under
r C E EagI-HF 4 25 50 10 100 100 65° 37° recommended reaction conditions).

r EarI 4 100 100 50 100 100 65° 37°

EciI 2 + BSA Engineered enzyme for maximum
50 100 100 50 100 65° 37° E performance.
C EcoNI 4 100 100 75 100 100 65° 37°
r C EcoO109I 4 + BSA 100 100 75 100 15 65° 37°
r EcoP15I 3 + BSA + ATP 75 100 100 100 50 65° 37° NEBuffer Compositions (1X)
r C EcoRI Udd 100 100 100 100 100 65° 37° 10 mM Bis Tris Propane-HCl,
EcoRI-HF 4 NEBuffer 1
r C E 10 100 0 100 5 65° 37° (yellow)
10 mM MgCl2, 1 mM DTT
EcoRV 3 + BSA (pH 7.0 @ 25°C).
r C 50 75 100 50 50 80° 37°
r C E EcoRV-HF 4 25 100 100 100 100 65° 37° 10 mM Tris-HCl, 10 mM
NEBuffer 2
r FatI 2 10 100 50 50 100 65° 55° MgCl2, 50 mM NaCl,
(blue)
1 mM DTT (pH 7.9 @ 25°C).
r FauI 4 100 50 10 100 50 65° 55°
r C Fnu4HI 4 10 25 25 100 10 65° 37° 50 mM Tris-HCl, 10 mM
NEBuffer 3
FokI 4 MgCl2, 100 mM NaCl,
r C 100 100 75 100 25 65° 37° (red)
1 mM DTT (pH 7.9 @ 25°C).
r C FseI 4 + BSA 100 75 0 100 50 65° 37°
r FspI 4 10 75 10 100 50 65° 37°  0 mM Tris-acetate, 10 mM
2
NEBuffer 4 magnesium acetate, 50 mM 
r HaeII 4 + BSA 75 100 50 100 25 80° 37° (green) potassium acetate, 1 mM DTT
r C HaeIII 4 50 100 25 100 100 80° 37° (pH 7.9 @ 25°C).

r HgaI 1 100 75 50 100 100 65° 37° 50 mM NaCI, 100 mM

r C HhaI 4 + BSA NEBuffer Tris-HCI, 10 mM MgCI2,
75 100 100 100 100 65° 37°
EcoRI 0.025% Triton X-100
r HincII 3 + BSA 75 100 100 100 100 65° 37° (pH 7.5 @ 25°C).
r C HindIII 2 50 100 10 50 25 65° 37°
r C HinfI 4 75 100 75 100 100 80° 37° Buffers should be thawed completely and
r C HinP1I 4 100 100 100 100 100 65° 37° mixed thoroughly before using.
r HpaI 4 25 50 10 100 100 No 37°
r C HpaII 1 100 50 10 100 15 65° 37°
r C HphI 4 NR 75 0 100 10 65° 37°
r Hpy99I 4 + BSA 10 10 0 100 25 65° 37°
r C Hpy166II 4 100 100 50 100 40 65° 37°
r Hpy188I 4 50 25 10 100 50 65° 37°

www.neb.com 17
 selection

HEAT
SUPPLIED % ACTIVITY in NEBuffers INACTIVATION INCUBATION
ENZYME NEBuffer 1 2 3 4 EcoRI (TEMP) TEMPERATURE

r Hpy188III 4 + BSA 100 100 10 100 5 65° 37°

r C HpyAV 4 + BSA 100 100 25 100 25 65° 37°
r HpyCH4III 4 100 50 25 100 20 80° 37°
r C HpyCH4IV 1 100 25 10 25 100 65° 37°
r C HpyCH4V 4 50 50 25 100 50 65° 37°
r KasI 4 + BSA 25 100 0 100 100 65° 37°
r C KpnI 1 + BSA 100 75 0 50 25 No 37°
r C MboI 4 75 100 100 100 100 65° 37°
r C MboII 4 100 100 50 100 100 65° 37°
r C MfeI 4 75 50 10 100 5 65° 37°
r C E MfeI-HF 4 75 50 10 100 0 65° 37°
r C MluI 3 25 75 100 50 100 65° 37°
r C MlyI 4 + BSA 50 50 25 100 25 65° 37°
r C MmeI 4 + SAM NR NR NR dd
100 50 80° 37°
r C MnlI 4 + BSA 75 100 50 100 50 65° 37°
r MscI 4 75 75 75 100 100 65° 37°
r MseI 4 + BSA 75 100 75 100 50 65° 37°
r C MslI 4 100 100 25 100 10 80° 37°
r C MspI 4 75 100 50 100 50 65° 37°
r C MspA1I 4 + BSA 50 100 50 100 50 65° 37°
r MwoI 3 10 75 100 75 100 No 60°
r NaeI 4 100 75 10 100 25 65° 37°
r NarI 4 100 75 75 100 50 65° 37°
r Nb.BbvCI 2 50 100 10 100 N/A 80° 37°
r Nb.BsmI 3 25 100 100 25 N/A 80° 65°
r Nb.BsrDI 2 10 100 25 50 N/A 80° 65°
r Nb.BtsI 4 + BSA 75 100 75 100 N/A 80° 37°
r C NciI 4 100 25 10 100 25 No 37°
r C NcoI 3 100 100 100 100 100 65° 37°
r C E NcoI-HF 4 50 100 10 100 100 80° 37°
r C NdeI 4 75 100 75 100 100 65° 37°
r NgoMIV 4 100 50 10 100 0 80° 37°
r C NheI 2 + BSA 100 100 10 100 15 65° 37°
r C E NheI-HF 4 + BSA 100 10 0 100 0 80° 37°
r NlaIII 4 + BSA 25 25 25 100 5 65° 37°
r NlaIV 4 + BSA 10 10 10 100 100 65° 37°
r C NmeAIII 4 + SAM 10 10 0 100 0 80° 37°
r C NotI 3 + BSA 0 50 100 25 100 65° 37°
r C E NotI-HF 4 + BSA 25 100 25 100 50 65° 37°
r C NruI 3 0 10 100 10 100 65° 37°
r C NsiI 3 10 75 100 25 100 80° 37°
r NspI 2 + BSA 100 100 0 100 5 65° 37°
r Nt.AlwI 2 25 100 25 50 N/A 80° 37°
r Nt.BbvCI 4 50 100 10 100 N/A 80° 37°
r C Nt.BspQI 3 10 75 100 25 N/A 80° 50°
r Nt.BstNBI 3 0 10 100 10 N/A 80° 55° New England Biolabs offers
r Nt.CviPII 4 25 100 50 100 N/A 65° 37°
over 170 recombinant enzymes.
r C PacI 1 + BSA 100 75 10 100 5 65° 37°
r C PaeR7I 4 25 100 10 100 50 No 37°
r PciI 3 50 75 100 50 0 80° 37°
C PflFI 4 + BSA 0 25 25 100 5 65° 37°
r C PflMI 3 + BSA 0 100 100 50 100 65° 37°
r PhoI 3 50 50 100 75 100 No 75°
r PleI 4 100 100 75 100 50 65° 37°
r C PmeI 4 + BSA 0 50 10 100 5 65° 37°
C PmlI 1 + BSA 100 75 0 75 15 65° 37°
r C PpuMI 4 0 25 0 100 10 No 37°
18
 selection

HEAT
SUPPLIED % ACTIVITY in NEBuffers INACTIVATION INCUBATION
ENZYME NEBuffer 1 2 3 4 EcoRI (TEMP) TEMPERATURE

r PshAI 4 + BSA 50 75 10 100 5 65° 37°

r PsiI 4 10 100 10 100 10 65° 37°
r PspGI 4 75 100 50 100 100 No 75°
r PspOMI 4 25 25 10 100 15 65° 37° Chart Legend
r PspXI 4 0 100 10 100 100 80° 37°
Supplied with a unique reaction buffer
r C PstI 3 + BSA 75 75 100 50 50 80° 37° that is different from the four standard
C PvuI 3 + BSA 10 75 100 10 100 80° 37° U NEBuffers. The compatibility with the
four standard NEBuffers is indicated
r C PvuII 2 100 100 100 100 100 No 37° in the chart.
r C E PvuII-HF 4 0 25 0 100 0 80° 37°
Supplied with a separate vial of bovine
r C RsaI 4 100 100 50 100 15 65° 37° serum albumin (10 mg/ml). To obtain
r RsrII 4 25 75 10 100 25 65° 37° BSA 100% activity, BSA should be added
to the 1X reaction mix to a final
r C SacI 1 + BSA 100 50 10 100 15 65° 37° concentration of 100 µg/ml.
r C E SacI-HF 4 + BSA 100 50 10 100 15 65° 37°
r C SacII 4 25 75 10 100 100 65° 37° Supplied with a separate vial of
S-adenosylmethionine (SAM).
r C SalI 3 + BSA 0 0 100 0 50 65° 37° SAM To obtain 100% activity, SAM should
r C E SalI-HF 4 10 100 100 100 100 65° 37° be added to the 1X reaction mix as
specified on the product data card.
r SapI 4 75 50 0 100 50 65° 37°
r Sau3AI 1 + BSA 100 50 10 100 100 65° 37° This NEBuffer is recommended for
r Sau96I 4 50 100 100 100 50 80° 37° dd a double digest because it minimizes
star activity.
r C SbfI 4 75 50 0 100 25 65° 37°
r C E SbfI-HF 4 50 25 0 100 0 65° 37°
This buffer is not recommended for
NR
r C ScaI 3 NR NR dd
100 NR 50 80° 37° use with this enzyme.
r C E ScaI-HF 4 100 100 10 100 15 65° 37°
r Produced from a recombinant source.
C ScrFI 4 100 100 100 100 100 65° 37°
r SexAI 4 + BSA 100 75 50 100 50 65° 37°
Time-Saver qualified (digests 1 µg
r SfaNI 3 0 75 100 25 50 65° 37° C of substrate DNA in 5 minutes under
r SfcI 4 + BSA 75 50 10 100 25 65° 37° recommended reaction conditions).

r C SfiI 4 + BSA 0 100 10 100 15 No 50°

Engineered enzyme for maximum
r C SfoI 4 25 100 50 100 100 No 37° E performance.
r SgrAI 4 100 50 10 100 50 65° 37°
r C SmaI 4 0 0 0 100 0 65° 25°
r SmlI 4 + BSA 25 75 25 100 50 No 55° NEBuffer Compositions (1X)
r C SnaBI 4 + BSA 25 50 25 dd
100 5 80° 37° 10 mM Bis Tris Propane-HCl,
NEBuffer 1
r C SpeI 4 + BSA 75 100 25 100 50 80° 37° 10 mM MgCl2, 1 mM DTT
(yellow)
(pH 7.0 @ 25°C).
r C SphI 2 100 100 50 100 50 65° 37°
r C E SphI-HF 4 50 25 10 100 0 65° 37°
NEBuffer 2
10 mM Tris-HCl, 10 mM
MgCl2, 50 mM NaCl,
r C SspI U 50 100 50 50 100 65° 37° (blue)
1 mM DTT (pH 7.9 @ 25°C).
r C E SspI-HF 4 25 100 0 100 25 65° 37°
r C StuI 4 100 100 50 100 50 65° 37° NEBuffer 3
50 mM Tris-HCl, 10 mM
MgCl2, 100 mM NaCl,
r StyD4I 2 10 100 100 100 20 65° 37° (red)
1 mM DTT (pH 7.9 @ 25°C).
r StyI 3 + BSA 25 75 100 10 15 65° 37°
r  0 mM Tris-acetate, 10 mM
2
SwaI 3 + BSA 10 10 100 10 15 65° 25°
NEBuffer 4 magnesium acetate, 50 mM 
r C TaqI 4 + BSA 50 75 100 100 100 80° 65° (green) potassium acetate, 1 mM DTT
(pH 7.9 @ 25°C).
r TfiI 4 100 100 dd
100 100 100 No 65°
r TliI 3 + BSA 25 25 100 10 50 No 75° 50 mM NaCI, 100 mM
NEBuffer Tris-HCI, 10 mM MgCI2,
TseI 4 75 100 100 100 100 No 65° EcoRI 0.025% Triton X-100
Tsp45I 1 + BSA 100 25 0 75 50 No 65° (pH 7.5 @ 25°C).
r C Tsp509I 1 100 100 100 NR 100 No 65°
C TspMI 4 50 75 50 100 20 No 75° Buffers should be thawed completely and
r C TspRI 4 + BSA 25 50 25 100 5 No 65° mixed thoroughly before using.
r Tth111I 4 50 25 25 100 100 No 65°
r C XbaI 4 + BSA 0 100 75 100 15 65° 37°
r XcmI 2 10 100 50 50 20 65° 37°
r C XhoI 4 + BSA 75 100 100 100 100 65° 37°
r XmaI 4 + BSA 25 50 0 100 100 65° 37°
r C XmnI 4 + BSA 100 100 50 100 5 65° 37°
r ZraI 4 100 25 10 100 10 No 37°
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New England Biolabs (UK), Ltd.
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