Restriction Endonucleases
TECHNICAL GUIDE
RESTRICTION ENZYMES FROM NEB
Simplify Reaction Setup and Double
Digestion with CutSmart®
Buffer
Over 205 restriction enzymes are 100% active in a single buffer,
CutSmart Buffer, making it significantly easier to set up your
double digest reactions. Since CutSmart Buffer includes BSA, there
are fewer tubes and pipetting steps to worry about. Additionally,
many DNA modifying enzymes are 100% active in CutSmart
Buffer, eliminating the need for subsequent purification.
For more information, visit www.NEBCutSmart.com
Cut Smarter with Restriction Enzymes from NEB®
Looking to bring CONVENIENCE to your workflow?
Speed up Digestions with Time-Saver™
Qualified Restriction Enzymes
190 of our restriction enzymes are able to digest DNA in
5–15 minutes, and can safely be used overnight with no loss of
sample. For added convenience and flexibility, most of these are
supplied with our new CutSmart Buffer.
For more information, visit www.neb.com/timesaver
Bring Flexibility to your Workflow
NEB offers the largest selection of restriction enzymes commercially available. With an
evergrowing list to choose from, currently at 280 enzymes – including traditional restriction
enzymes, nicking endonucleases, homing endonucleases and methylation-sensitive enzymes
for epigenetics studies – there is no need to look anywhere else.
pXba DNA was digested with
EcoRV-HF RE-Mix according
to the recommended protocol.
Lane L is the TriDye™
2-Log
DNA Ladder (NEB #N3270).
Complete digestion, free of
unwanted star activity, is seen
whether incubated for 5–15
minutes, 1 hour or overnight.
L	 0	 5 min	 15 min	 1 hr	 o/n
Keep it Simple with our RE-Mix®
Restriction Enzyme Master Mixes
RE-Mix Restriction Enzyme Master Mixes are pre-mixed solutions
that contain enzyme, buffer, BSA and loading dye. Just add
your DNA and water; it's that simple! RE-Mix master mixes are
Time-Saver qualified so you can trust your reaction to digest to
completion in 15 minutes, or leave it to digest overnight, with no
degradation of your final product.
For more information, visit www.NEBREMix.com
ENZYME
ACTIVITY
IN CUTSMART
REQUIRED
SUPPLEMENTS
Alkaline Phosphatase (CIP) + + +
Antarctic Phosphatase + + + Requires Zn2+
Bst DNA Polymerase + + +
CpG Methyltransferase (M. SssI) + + +
DNA Polymerase I + + +
DNA Polymerase I, Large (Klenow) Fragment + + +
DNA Polymerase Klenow Exo–
+ + +
DNase I (RNase free) + + + Requires Ca2+
E. coli DNA Ligase + + + Requires NAD
Endonuclease III (Nth), recombinant + + +
Endonuclease VIII + + +
Exonuclease III + + +
GpC Methyltransferase (M. CviPI) + Requires DTT
McrBC + + +
Micrococcal Nuclease + + +
Nuclease BAL-31 + + +
phi29 DNA Polymerase + + +
RecJf
+ + +
Shrimp Alkaline Phosphatase (rSAP) + + +
T3 DNA Ligase + + + Requires ATP + PEG
T4 DNA Ligase + + + Requires ATP
T4 DNA Polymerase + + +
T4 Phage β-glucosyltransferase (T4-BGT) + + +
T4 Polynucleotide Kinase + + + Requires ATP + DTT
T4 PNK (3´ phosphatase minus) + + + Requires ATP + DTT
T7 DNA Ligase + + + Requires ATP + PEG
T7 DNA Polymerase (unmodified) + + +
T7 Exonuclease + + +
USER Enzyme, recombinant + + +
	+ + + full functional activity 	+ + 50–100% functional activity 	+ 0–50% functional activity
Activity of DNA Modifying Enzymes in CutSmart Buffer
2
www.neb.com
Looking to optimize PERFORMANCE in your reaction?
Choose a High-Fidelity (HF®
)
Restriction Enzyme
As part of our ongoing commitment to the
advancement and improvement of enzymes for
the cloning and manipulation of DNA, NEB
has developed a line of High-Fidelity (HF)
restriction enzymes. These engineered enzymes
have the same specificity as the native enzyme,
with the added benefit of reduced star activity,
rapid digestion (5-15 minutes), and 100%
activity in CutSmart Buffer. Enjoy the improved
performance of NEB's engineered enzymes at the
same price as the native enzymes!
For more information, visit www.neb.com/HF
Benefit from Industry-leading Quality
NEB's reputation as a leader in enzyme technologies stems from the quality and reliability
of our restriction enzymes. All of our restriction enzymes undergo stringent quality control
testing, ensuring the highest levels of purity and lot-to-lot consistency.
EcoRI-HF (NEB #R3101)
shows no star activity in
overnight digests, even
when used at higher
concentrations. 50 μl
reactions were set up using
1 μg of Lambda DNA, the
indicated amount of enzyme
and the recommended
reaction buffer. Reactions
were incubated overnight at
37°C. Marker M is the 1 kb
DNA Ladder (NEB# N3232).
Restriction Enzyme
Competitor Study:
Nuclease Contamination
EcoRI, NotI, and BamHI
from multiple suppliers
were tested in reactions
containing a fluorescent
labeled single stranded,
double stranded blunt,
3’overhang or 5’
overhang containing
oligonucleotides. The
percent degradation is
determined by capillary
electrophoresis and peak
analysis. The resolution
is at the single nucleotide
level.
	0	1	3	5	10	M	0	1	3	5	10	µl
NEB EcoRI-HF
Competitor
FastDigest®
EcoRI
Unwanted
Cleavage
Visit NEBCutSmart.com for information
on the smarter choice of restriction enzymes.
HF®
, REBASE®
, RE-MIX®
, NEW ENGLAND BIOLABS®
, NEB®
, NEBCLONER®
,andNEBioCalculator®
are registered trademarks of New England Biolabs, Inc.
CUTSMART®
, NEBCUTTER®
, TIME-SAVER™
and TRIDYE™
are trademarks of New England Biolabs, Inc
FASTDIGEST®
is a registered trademark of Thermo Fisher Scientific.
IPAD®
and IPHONE®
are registered trademarks of Apple, Inc.
ANDROID™
is a trademark of Google, Inc.
3
CUT SMARTER
HIGHLIGHTS
• Industry-leading product quality
• State-of-the-art production and purification
• Over 40 years of experience
• Stringent quality control testing
• Lot-to-lot consistency
• ISO 9001- and 13485-certified
Single-stranded OverhangDouble-stranded blunt top
Double-stranded blunt bottom
3´ Overhang top
3´ Overhang bottom
5´ Overhang top
5´ Overhang bottom
0
20
40
60
80
100
120
Degradation(%)
EcoRI EcoRI-HF
NEB EcoRI
A B C D
Competitor EcoRI
0
NotI NotI-HF
NEB NotI
A B C D
Competitor NotI
2
4
6
8
10
12
14
16
Degradation(%)
0
BamHI BamHI-HF
NEB BamHI
A B C D
Competitor BamHI
20
40
60
Degradation(%)
80
100
120
TOOLS & RESOURCES
Visit NEBRestrictionEnzymes.com to find:
• The full list of HF restriction enzymes available
• Online tutorials on how to avoid star activity and
setting up digests using the Time-Saver protocol
High Fidelity (HF) Enzymes
High-Fidelity (HF) restriction enzymes are engineered enzymes that have the same specificity
as the native enzymes, are all active in CutSmart Buffer and have reduced star activity. Star
activity, or off-target cleavage, is an intrinsic property of restriction enzymes. Most restriction
enzymes will not exhibit star activity under recommended reaction conditions. However,
for enzymes that have reported star activity, extra caution must be taken to set up reactions
according to the recommended conditions to avoid unwanted cleavage. HF enzymes should
be used in these cases.
In addition to reduced star activity, HF enzymes work optimally in CutSmart Buffer, which has
the highest level of enzyme compatibility and will simplify double digest reactions. They are
all Time-Saver qualified and digest substrate DNA in 5–15 minutes and are flexible enough to
digest overnight. HF enzymes are supplied with our purple gel loading dye, which sharpens
bands and eliminates UV shadow. Lastly, they are available at the same price as the native enzymes.
The following table indicates the number of units of HF enzyme that can be used compared to the native enzyme before any significant
star activity is detected. The HF Factor refers to the X-fold increase in fidelity that is achieved by choosing an HF enzyme. This data
clearly illustrates the flexibility that is offered by using an HF restriction enzyme.
HF ENZYMES
4
WHAT IS A HIGH-FIDELITY ENZYME?
PRODUCT
NAME
PRODUCT
NUMBER BUFFER†
MAXIMUM
UNITS WITH
NO STAR
ACTIVITY*
HF
FACTOR
AgeI-HF #R3552 CutSmart ≥ 250 ≥ 8
AgeI #R0552 1.1 32
BamHI-HF #R3136 CutSmart ≥ 4,000 ≥ 125
BamHI #R0136 3.1 32
BmtI-HF #R3658 CutSmart 1,000,000 31,250
BmtI #R0658 3.1 32
BsaI-HF #R3535 CutSmart ≥ 8,000 ≥ 250
BsaI #R0535 CutSmart 32
BsrGI-HF #R3575 CutSmart ≥ 1,000 ≥ 62
BsrGI #R0575 2.1 16
BstEII-HF #R3162 CutSmart ≥ 2,000 ≥ 125
BstEII #R0162 3.1 16
DraIII-HF #R3510 CutSmart ≥ 2,000 ≥ 1,000
DraIII** N/A 3.1 2
EagI-HF #R3505 CutSmart 500 2
EagI #R0505 3.1 250
EcoRI-HF #R3101 CutSmart 16,000 64
EcoRI #R0101 U 250
EcoRV-HF #R3195 CutSmart ≥ 64,000 ≥ 64
EcoRV #R0195 3.1 1,000
HindIII-HF #R3104 CutSmart ≥ 500,000 ≥ 2,000
HindIII #R0104 2.1 250
KpnI-HF #R3142 CutSmart ≥ 1,000,000 ≥ 62,500
KpnI #R0142 1.1 16
MfeI-HF #R3589 CutSmart ≥ 500 ≥ 16
MfeI #R0589 CutSmart 32
MluI-HF #R3198 CutSmart ≥ 4,000 2
MluI #R0198 3.1 ≥ 2,000
NcoI-HF #R3193 CutSmart ≥ 64,000 ≥ 530
NcoI #R0193 3.1 120
PRODUCT
NAME
PRODUCT
NUMBER BUFFER†
MAXIMUM
UNITS WITH
NO STAR
ACTIVITY*
HF
FACTOR
NheI-HF #R3131 CutSmart ≥ 32,000 ≥ 266
NheI #R0131 2.1 120
NotI-HF #R3189 CutSmart ≥ 64,000 ≥ 16
NotI #R0189 3.1 4,000
NruI-HF #R3192 CutSmart ≥ 32,000 64
NruI #R0192 3.1 ≥ 500
NsiI-HF #R3127 CutSmart ≥ 8000 2
NsiI #R0127 3.1 ≥ 4,000
PstI-HF #R3140 CutSmart 4,000 33
PstI #R0140 3.1 120
PvuI-HF #R3150 CutSmart ≥ 16,000 ≥ 32
PvuI #R0150 3.1 500
PvuII-HF #R3151 CutSmart 500 32
PvuII #R0151 2.1 16
SacI-HF #R3156 CutSmart ≥ 32,000 ≥ 266
SacI #R0156 1.1 120
SalI-HF #R3138 CutSmart ≥ 32,000 ≥ 8,000
SalI #R0138 3.1 4
SbfI-HF #R3642 CutSmart 250 32
SbfI #R0642 CutSmart 8
ScaI-HF #R3122 CutSmart 250 62
ScaI** #R0122 3.1 4
SpeI-HF #R3133 CutSmart ≥ 8,000 ≥ 16
SpeI #R0133 CutSmart 500
SphI-HF #R3182 CutSmart 8,000 250
SphI #R0182 2.1 32
SspI-HF #R3132 CutSmart 500 16
SspI #R0132 U 32
StyI-HF #R3500 CutSmart 4,000 125
StyI #R0500 3.1 32
†
	 Wild type enzymes were tested in supplied buffer for comparisons. *	 Wei, H. et al (2008) Nucleic Acids Reseach 36, e50. **	 No longer available.
TOOLS & RESOURCES
Visit NEBRestrictionEnzymes.com to find:
• Video tutorials on how to avoid star activity,
and for setting up restriction enzyme digests
• The full list of HF enzymes available
• Troubleshooting guides
Tips for preventing unwanted cleavage in
restriction enzyme digests
Under non-standard reaction conditions, some restriction enzymes are capable of cleaving
sequences which are similar but not identical to their defined recognition sequence. This
altered specificity has been termed “star activity”. It has been suggested that star activity
is a general property of restriction endonucleases (1) and that any restriction endonuclease
will cleave noncanonical sites under certain extreme conditions, some of which are listed
below. Although the propensity for star activity varies, the vast majority of enzymes from
New England Biolabs will not exhibit star activity when used under recommended conditions
in their supplied NEBuffers. If an enzyme has been reported to exhibit star activity,
it will be indicated in the product entry found in the catalog, on the supplied card and on
our website.
Avoiding Star Activity
CONDITIONS THAT CONTRIBUTE
TO STAR ACTIVITY STEPS THAT CAN BE TAKEN TO INHIBIT STAR ACTIVITY
High glycerol concentration (> 5% v/v)
Restriction enzymes are stored in 50% glycerol, therefore the amount of
enzyme added should not exceed 10% of the total reaction volume.
Use the standard 50 µl reaction volume to reduce evaporation during
incubation.
High concentration of enzyme/µg of
DNA ratio (varies with each enzyme,
usually 100 units/µg)
Use the fewest units possible to achieve digestion. This avoids
overdigestion and reduces the final glycerol concentration in the reaction.
Non-optimal buffer Whenever possible, set up reactions in the recommended buffer. Buffers
with differing ionic strengths and pHs may contribute to star activity.
Prolonged reaction time
Use the minimum reaction time required for complete digestion. Prolonged
incubation may result in increased star activity, as well as evaporation.
Presence of organic solvents [DMSO,
ethanol (4), ethylene glycol,
dimethylacetamide, dimethylformamide,
sulphalane (5)]
Make sure the reaction is free of any organic solvents, such as alcohols,
that might be present in the DNA preparation.
Substitution of Mg2+
with other
divalent cations (Mn2+
, Cu2+
, Co2+
, Zn2+
)
Use Mg2+
as the divalent cation. Other divalent cations may not fit correctly
into the active site of the restriction enzyme, possibly interfering with
proper recognition.
New England Biolabs recommends setting up restriction enzyme digests in a 50 µl reaction
volume. However, different methods may require smaller reaction volumes. When performing
restriction enzyme digests in smaller reaction volumes, extra care must be taken to follow
the steps listed above to avoid star activity. Alternatively, using our line of High Fidelity
(HF) restriction enzymes will allow greater flexibility in reaction setup. Please visit
www.neb.com/HF frequently to learn about new additions to the HF restriction
enzyme product line.
Note: The relative significance of each of these altered conditions will vary from enzyme to enzyme.
HF ENZYMES
5
RESTRICTION ENZYME TYPES
• Type I enzymes are multisubunit proteins that cut
DNA randomly at a distance from their recognition
sequence.
• Type II enzymes cut DNA at defined positions
close to or within their recognition sequence
and are commonly used in the laboratory. There
are over ten subtypes with different types of
recognition sites, cleavage sites and cofactor
requirements.
• The most common Type II enzymes cleave within
their recognition site (e.g., BamHI, EcoRI); sites
can be symmetric or asymmetric.
• Type IIS enzymes cleave outside their recognition
sequence (e.g., FokI, AlwI) and are invaluable
for emerging technologies 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
two sequences in opposite orientations to cleave
one DNA molecule.
• Type IV enzymes recognize modified DNA
(methylated, hydroxymethylated, etc.). They
require two sites and cleave non-specifically.
• Isoschizomers are restriction enzymes that
recognize the same sequence as the prototype.
• Neoschizomers are isoschizomers
with different cleavage sites.
References:
1. Nasri, M. and Thomas, D. (1986) Nucleic Acids Res. 14, 811.
2. Barany, F. (1988) Gene, 68, 149.
3. Bitinaite, J. and Schildkraut, I. (2002) Proc. Natl. Acad. Sci. USA, 99, 1164–1169.
4. Nasri, M. and Thomas, D. (1987) Nucleic Acids Res. 15, 7677.
5. Tikchonenko, T.I., et al. (1978) Gene, 4, 195–212.
RESTRICTION ENZYME STAR ACTIVITY
Learn more about
restriction enzyme types
in our online tutorials.
TIME-SAVER ENZYMES
Time-Saver Qualified Restriction Enzymes
Whether you are quickly screening large numbers of clones or setting up overnight
digests, you will benefit from the high quality of our enzymes. Typically, a restriction
digest involves the incubation of 1 µl of enzyme with 1 µg of purified DNA in a final
volume of 50 µl for 1 hour. However, to speed up the screening process, choose one
of NEB’s enzymes that are Time-Saver qualified. 190 of our enzymes will digest 1
µg of substrate DNA in 5-15 minutes using 1 µl of enzyme under recommended
reaction conditions, and can also be used safely in overnight digestions. Unlike other
suppliers, there is no special formulation, change in concentration or need to buy more
expensive, new lines of enzymes to achieve digestion in 5-15 minutes. Nor do you have
to worry if you incubate too long.
In an effort to provide you with as much information as possible, NEB has tested all of its
enzymes on unit assay substrate, as well as plasmid substrate and PCR fragments.
We recommend that this data be used as a guide, as it is not definitive for all plasmids.
Restriction enzymes can often show site preference, presumably determined by the
sequence flanking the recognition site. In addition, supercoiled DNA may have varying
rates of cleavage. For more information, visit www.neb.com/TimeSaver. Note that there
are some enzymes indicated below that can cut in 5-15 minutes, but cannot be incubated
overnight. These are not Time-Saver qualified.
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. Only NEB
Time-Saver qualified enzymes offer power and flexibility – the power to digest in 5-15
minutes and the flexibility to withstand overnight digestions with no loss of substrate.
6
ENZYME
UNIT
ASSAY PLASMID PCR
AatII n s l
AccI n s s
Acc65I l s l
AciI l l l
AclI l n s
AcuI n s s
AflII l l l
AgeI-HF l l l
AgeI-HF RE-Mix l l n
AhdI l l n
AluI l s l
AlwNI l l s
ApaI l l l
ApaLI l l s
ApeKI l n s
ApoI l l l
AscI l l NT
AscI RE-Mix l l NT
AseI l l NT
AvaI l s s
AvaII l l l
AvrII l s NT
BaeI n l s
ENZYME
UNIT
ASSAY PLASMID PCR
BaeGI l s s
BamHI l l s
BamHI-HF l l l
BbsI n s s
BbvI l s s
BccI n s s
BceAI n n s
BciVI l n s
BclI l s s
BcoDI l l s
BfuAI l l s
BfuCI n s l
BglI l l s
BglII l n s
BlpI l l l
BmgBI l l s
BmrI n s n
BmtI-HF l l s
BpuEI l l s
BsaI l l s
BsaI-HF l l s
BsaAI l l n
BsaHI n n l
SUBSTRATESUBSTRATE
ENZYME
UNIT
ASSAY PLASMID PCR
BsaWI n s s
BsaXI l s s
BseRI l l n
BsgI l l s
BsiEI l s s
BsiWI l l s
BslI l n n
BsmI l l s
BsmAI l s l
BsmBI n s s
BsmFI l l s
BsoBI l n l
Bsp1286I l l s
BspCNI n s s
BspEI l s s
BspHI n l l
BspQI l l s
BsrI l n s
BsrBI l n s
BsrDI l n s
BsrGI n s s
BsrGI-HF l l s
BssHII l s s
BssKI n s l
BssSa
I l s s
BstBI l l s
BstEII l l s
BstEII-HF l l l
BstEII-HF RE-Mix l l n
BstNI l l s
BstUI l l s
BstXI l l s
BstYI n l s
BstZ17I l s s
Bsu36I n s n
Btsa
l l l n
BtsCI l n s
Cac8I n s s
ClaI l l s
CspCI l l s
CviAII n l l
CviQI l l l
DdeI l n n
SUBSTRATE
Chart Legend
l digests in 5 minutes
n digests in 15 minutes
s not completely digested in 15 minutes
NT not tested
TIME-SAVER ENZYMES
7
ENZYME
UNIT
ASSAY PLASMID PCR
DpnI l l s
DpnII n s l
DraI l l n
DraIII-HF l l s
DrdI n l l
EagI l s s
EagI-HF l l s
EarI n n s
Eco53KI l l n
EcoNI l n l
EcoO109I l s s
EcoP15I n s s
EcoRI l l s
EcoRI-HF l l l
EcoRI-HF RE-Mix l l s
EcoRV l l s
EcoRV-HF l l s
EcoRV-HF RE-Mix l l s
Fnu4HI l n n
FokI l l l
FseI l l s
FspI n s n
HaeII n s s
HaeIII l l l
HgaI n s s
HhaI l n s
HincII n s l
HindIII-HF l l l
HinfI l l l
HinP1I l s l
HpaII l l s
HphI l s s
Hpy166II l l l
HpyAV l l NT
HpyCH4IV l l l
HpyCH4V l l l
KpnI-HF l l l
KpnI-HF RE-Mix l l s
MboI l s l
MboII l l l
MfeI l l l
MfeI-HF l l l
MfeI-HF RE-Mix l l l
MluI l l l
MluI-HF l l s
MluCI l l s
MlyI l s l
MmeI l l s
MnlI l l n
MseI n n l
ENZYME
UNIT
ASSAY PLASMID PCR
MslI l l l
MspI l l l
MspA1I l l l
MwoI n s s
NciI l l l
NcoI l n s
NcoI-HF l l l
NcoI-HF RE-Mix l l l
NdeI l l s
NgoMIV n l s
NheI l n s
NheI-HF l l n
NheI-HF RE-Mix l l s
NlaIII n s n
NmeAIII l s s
NotI l l s
NotI-HF l l l
NotI-HF RE-Mix l l s
NruI l n s
NruI-HF l n s
NsiI l l l
NsiI-HF l l n
NspI l n s
PacI l l l
PacI-RE-Mix l l NT
PaeR7I l s s
PflfI l n s
PflMI l s s
PmeI l n NT
PmlI l s s
PpuMI l s s
PshAI n n n
PstI l l l
PstI-HF l l l
PvuI l s l
PvuI-HF l l l
PvuII l l s
PvuII-HF l l s
RsaI l l l
SacI l l s
SacI-HF l l l
SacII l s s
SalI l n s
SalI-HF l l s
SalI-HF RE-Mix l l s
SapI n s s
SbfI l l s
SbfI-HF l l s
ScaI-HF l l s
ScaI-HF RE-Mix l l s
SUBSTRATE SUBSTRATE
ENZYME
UNIT
ASSAY PLASMID PCR
SfiI l s s
SfoI l l l
SmaI l n n
SpeI l l l
SpeI RE-Mix l l l
SphI l l s
SspI l l s
SspI-HF l l s
StuI n s s
StyI n s s
StyI-HF l l s
StyD4I n s s
SwaI n s s
TaqI l l s
TfiI n l s
TseI n s s
TspMI l n s
TspRI l n s
Tth111I n n s
XbaI l l s
XbaI-RE-Mix l l n
XhoI l l s
XhoI RE-Mix l l NT
XmaI n s n
XmnI l l s
SUBSTRATE
TOOLS & RESOURCES
Visit www.neb.com/TimeSaver to find:
• The full list of Time-Saver qualified restriction
enzymes available
• Video tutorials on how Time-Saver qualified
enzymes speed up restriction enzyme digests
www.neb.com
Optimizing Restriction Enzyme Reactions
There are several key factors to consider when setting up a restriction enzyme digest. Using the proper amounts of DNA, enzyme and
buffer components in the correct reaction volume will allow you to achieve optimal digestion. 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. However, most researchers follow the “typical” reaction conditions listed, where a 5–10 fold overdigestion
is recommended to overcome variability in DNA source, quantity and purity. The Time-Saver protocol can be used for enzymes
that are Time-Saver qualified and will digest DNA in 5–15 minutes (see page 6–7 for the full list). For additional convenience, a RE-Mix
Restriction Enzyme Master Mix can also be used. NEB offers the following tips to help you to achieve maximal success in your restriction
enzyme reactions.
Enzyme
•• Keep on ice when not in the freezer
•• Should be the last component added to
reaction
•• Mix components 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.
•• In general, we recommend 5–10 units of
enzyme per µg DNA, and 10–20 units for
genomic DNA
•• NEB has introduced a line of HighFidelity
(HF) enzymes that provide added
flexibility to reaction setup.
•• If using a RE-Mix restriction enzyme master
mix, see page 9 for protocol.
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
•• BSA is included in NEBuffer 1.1, 2.1, 3.1
and CutSmart Buffer. No additional BSA
is needed.
Reaction Volume
•• A 50 µl reaction volume is recommended
for digestion of 1 µg of substrate
•• Enzyme volume should not exceed 10% of
the total reaction volume to prevent star
activity due to excess glycerol
•• 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. The following guidelines can be
used for techniques that require smaller
reaction volumes.
Standard Protocol
Restriction Enzyme 1 µl (or 10 units)*
DNA 1 µg
10X NEBuffer 5 µl (1X)
Total Reaction Volume 50 µl
Incubation Temperature Enzyme Dependent
Incubation Time 60 minutes
*Sufficient to digest all types of DNAs.
Restriction Enzyme 1 µl
DNA 1 µg
10X NEBuffer 5 µl (1X)
Total Reaction Volume 50 µl
Incubation Temperature Enzyme Dependent
Incubation Time 5–15 minutes*
Time-Saver Protocol:
*Time-Saver qualified enzymes can also be incubated overnight with no
star activity.
Alternative Volumes for Restriction Digests
RESTRICTION
ENZYME* DNA
10X
NEBUFFER
10 µl rxn**
1 unit 0.1 µg 1 µl
25 µl rxn 5 units 0.5 µg 2.5 µl
50 µl rxn 10 units 1 µg 5 µl
*	Restriction Enzymes can be diluted using the recommended
diluent buffer when smaller amounts are needed
**	10 µl rxns should not be incubated for longer than 1 hour to
avoid evaporation
OPTIMIZING REACTIONS
8
www.neb.com
Incubation Time
•• Incubation time for Standard Protocol is
1 hour. Incubation for Time-Saver
Protocol is 5–15 minutes.
•• 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 (10 µl per
50 µl rxn) [50% glycerol, 50 mM EDTA
(pH 8.0), and 0.05% bromophenol blue]
(e.g., NEB #B7021) or Gel Loading Dye,
Purple (6X) (NEB #B7024).
When further manipulation of DNA is
required:
•• Heat inactivation can be used (buffer
chart indicates if the enzyme can be
heat inactivated)
•• If enzyme cannot be heat inactivated,
remove by using a spin column or
phenol/chloroform extraction
Storage
•• Storage at –20°C is recommended for
most restriction enzymes. For a few
enzymes, storage at –70°C is recommended
for periods longer than 30 days. Visit
www.neb.com for storage information.
•• 10X NEBuffers should also be
stored at –20°C
Stability
•• All enzymes are assayed for activity every
3–6 months. The expiration date is found
on the label.
•• Exposure to temperatures above –20°C
should be minimized whenever possible
Control Reactions
For difficulty cleaving DNA substrate,
we recommend the following controls:
•• Control DNA (DNA with multiple known
sites for the enzyme) with restriction
enzyme to test enzyme viability
•• 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.
Star Activity
•• Can occur when enzyme is used under
sub-optimal conditions
•• Star activity can be reduced by using a
High-Fidelity (HF) enzyme, by reducing
incubation time, by using a Time-Saver
enzyme or by increasing reaction volume
•• RE-Mix master mixes include enzyme, buffer, BSA and loading dye. All that is required is the addition of DNA and
water. For the full list of RE-Mix master mixes, visit NEBREmix.com.
Optimizing Restriction Enzyme Reactions Using
RE-Mix Master Mixes
Many of the optimization tips for restriction
enzymes apply to RE-Mix. Additional tips
include:
•• RE-Mix Master Mixes should be used at
1X concentration
•• A 20 µl reaction volume is recommended
for digestion with a RE-Mix Master Mix
•• The recommended incubation time with a
RE-Mix Master Mix is 15 minutes
•• The RE-Master Mix includes a density
agent and dye, and does not require addition
of stop solution
•• RE-Mix Master Mixes should be stored at
–20°C
DNA X µl (up to 1 µg)
dH2
O 18 µl–X
10X RE-Mix 2 µl
Reaction Volume 20 µl
Incubation Temperature 37°C
Incubation Time 15 minutes
RE-Mix Protocol
OPTIMIZING REACTIONS
9
TOOLS & RESOURCES
Visit NEBCutSmart.com to find:
• Video tutorials on setting up restriction enzyme
reactions from NEB scientists
CUTSMART®
BUFFER FROM NEB
Troubleshooting Guide
References
1.	 Blakesley, R.W., Wells, R.D. (1975) Nature 257, 421–422.
2.	 Blakesley, R.W., et al. (1977) J. Biol. Chem. 252, 7300–7306.
3.	 Yoo, O.J., Agarwal, K.L, (1980) J. Biol. Chem. 255, 10559–10562.
TROUBLESHOOTING
10
PROBLEM CAUSE SOLUTION
Few or no
transformants
Restriction enzyme(s)
didn’t cleave completely
Check the methylation sensitivity of the enzyme(s) to determine if the enzyme is
blocked by methylation of the recognition sequence
Use the recommended buffer supplied with the restriction enzyme
Clean up the DNA to remove any contaminants that may inhibit the enzyme
When digesting a PCR fragment, make sure to have at least 6 nucleotides between the
recognition site and the end of the DNA molecule
The digested
DNA ran as a
smear on an
agarose gel
The restriction enzyme(s) is
bound to the substrate DNA
Lower the number of units
Add SDS (0.1–0.5%) to the loading buffer to dissociate the enzyme from the DNA
Nuclease contamination
Use fresh, clean running buffer and a fresh agarose gel
Clean up the DNA
Incomplete
restriction
enzyme
digestion
Cleavage is blocked
by methylation
DNA isolated from a bacterial source may be blocked by Dam and Dcm methylation
DNA isolated from eukaryotic source may be blocked by CpG methylation
Check the methylation sensitivity of the enzyme(s) to determine if the enzyme is
blocked by methylation of the recognition sequence
If the enzyme is inhibited by Dam or Dcm methylation, grow the plasmid in a dam-/
dcm- strain (NEB #C2925)
Salt inhibition
Enzymes that have low activity in salt-containing buffers (NEBuffer 3.1) may be salt
sensitive, so clean up the DNA prior to digestion
DNA purification procedures that use spin columns can result in high salt levels, which
inhibit enzyme activity. To prevent this, DNA solution should be no more than 25% of
total reaction volume.
Inhibition by PCR components Clean up the PCR fragment prior to restriction digest
Using the wrong buffer Use the recommended buffer supplied with the restriction enzyme
Too few units of enzyme used Use at least 3–5 units of enzyme per μg of DNA
Incubation time was too short Increase the incubation time
Digesting supercoiled DNA
Some enzymes have a lower activity on supercolied DNA. Increase the number of
enzyme units in the reaction.
Incomplete
restriction
enzyme
digestion
Presence of slow sites
Some enzymes can exhibit slower cleavage towards specific sites. Increase the
incubation time, 1–2 hours is typically sufficient.
Two sites required Some enzymes require the presence of two recognition sites to cut efficiently
DNA is contaminated with
an inhibitor
Assay substrate DNA in the presence of a control DNA. Control DNA will not cleave if
there is an inhibitor present. Mini prep DNA is particularly susceptible to contaminants.
Clean DNA with a spin column, resin or drop dialysis, or increase volume to dilute
contaminant
Extra bands
in the gel
If larger bands than expected
are seen in the gel, this
may indicate binding of the
enzyme(s) to the substrate
Lower the number of units in the reaction
Add SDS (0.1–0.5%) to the loading buffer to dissociate the enzyme from the substrate
Star activity
Use the recommended buffer supplied with the restriction enzyme
Decrease the number of enzyme units in the reaction
Make sure the amount of enzyme added does not exceed 10% of the total reaction
volume. This ensures that the total glycerol concentration does not exceed 5% v/v.
Decrease the incubation time. Using the minimum reaction time required for complete
digestion will help prevent star activity.
Try using a High-Fidelity (HF) restriction enzyme. HF enzymes have been engineered
for reduced star activity.
Partial restriction
enzyme digest
Enzymes that have low activity in salt-containing buffers (e.g., NEBuffer 3.1) may be
salt sensitive. Make sure to clean up the DNA prior to digestion.
DNA purification procedures that use spin columns can result in high salt levels, which
inhibit enzyme activity. To prevent this, DNA solution should be no more than 25% of
total reaction volume
Clean-up the PCR fragment prior to restriction digest
Use the recommended buffer supplied with the restriction enzyme
Use at least 3–5 units of enzyme per μg of DNA and digest the DNA for 1–2 hours
Q. Do restriction enzymes cleave singlestranded
DNA?
A. Although some restriction enzymes have been
reported to cleave ssDNA, it is unclear whether
cleavage occurs on a ssDNA molecule or on
two ssDNA molecules which transiently anneal
at a region of partial homology (1–3). For
this reason, we hesitate to make unreserved
claims about a restriction enzyme’s ability to
cut ssDNA.
Q. How stable are restriction enzymes?
A. All restriction enzymes from NEB are assayed
for activity every 3–6 months. 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.
Q. Is extended digestion (incubation times
> 1 hour) recommended?
A. 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 or if a
Time-Saver qualified restriction enzyme is used
(5–15 minutes). Conversely, longer incubation
times are often used to allow a reaction to proceed
to completion with fewer units of enzyme.
This is contingent on how
long a particular enzyme can survive
(maintain activity) in a reaction. Additional
information on extended digestion can be
found at www.neb.com.
FAQS
www.neb.com
TOOLS & RESOURCES
Visit www.neb.com/nebtools for:
• Help choosing double digest conditions using
NEB’s Double Digest Finder or NEBCloner®
Setting up a Double Digest
•• Double digests with CutSmart restriction
enzymes can be set up in CutSmart Buffer.
Otherwise, choose an NEBuffer that results
in the most activity for both enzymes. If star
activity is a concern, consider using one of
our High-Fidelity (HF) enzymes.
•• Set up reaction according to recommended
protocol. The final concentration of glycerol
in any reaction should be less than 5%
to minimize the possibility of star activity.
For example, in a 50 µl reaction, the total
amount of enzyme added should not exceed
5 µl.
•• If two different incubation temperatures are
necessary, choose the optimal reaction buffer
and set up reaction accordingly. Add the first
enzyme and incubate at the desired temperature.
Then, heat inactivate the first enzyme,
add the second enzyme and incubate at the
recommended temperature.
•• Depending on an enzyme’s activity rating
in a non-optimal NEBuffer, the number of
units or incubation time may be adjusted to
compensate for the slower rate of cleavage.
Setting up a Double Digest
with a unique buffer
•• NEB currently supplies three enzymes with
unique buffers: EcoRI, SspI and DpnII.
In most cases, DpnII requires a sequential
digest. Note that EcoRI has an HF version
which is supplied with CutSmart Buffer.
Setting up a Sequential Digest
•• If there is no buffer in which the two
enzymes both exhibit > 50% activity, a
sequential digest can be performed.
•• Set up a reaction using the restriction
endonuclease that has the lowest salt
concentration in its recommended buffer
and incubate to completion.
•• Adjust the salt concentration of
the reaction (using a small volume
of a concentrated salt solution) to
approximate the reaction conditions of
the second restriction endonuclease.
•• Add the second enzyme and incubate to
complete the second reaction.
•• Alternatively, a spin column can be used
to isolate the DNA prior to the second
reaction.
Digesting a DNA substrate with two restriction enzymes simultaneously (double digestion) is
a common timesaving procedure. Over 205 restriction enzymes are 100% active in CutSmart
Buffer, making double digestion simple. If you are using an enzyme that is not supplied with
CutSmart Buffer, the Performance Chart for Restriction Enzymes rates the percentage activity
of each restriction endonuclease in the four standard NEBuffers.
Double Digestion
DOUBLE DIGESTION
11
TIPS FOR SETTING UP DOUBLE DIGESTS
Double Digest Protocol using two
RE-Mix Enzymes:
DNA X µl (up to 1 µg)
dH2
O 36 µl–X
RE-Mix 1 2 µl
RE-Mix 2 2 µl
Total Volume 40 µl
Incubation Temperature 37°C
Incubation Time 15 minutes
Double Digest Protocol using One RE-Mix
and One Standard Restriction Enzyme.*
DNA X µl (up to 1 µg)
dH2
O 17 µl–X
RE-Mix 2 µl
Standard Enzyme 1 µl
Total Volume 20 µl
Incubation Temperature 37°C
Incubation Time
15 minutes
(Time-Saver Enzymes)
1 Hour
(Standard Enzymes)
*	Use only with standard restriction enzymes with 37°C
incubation temperature.
Setting up a Double
Digestion with RE-Mix
Master Mixes
•• RE-Mix master mixes can also be used
in double digest reactions.
KEY POINTS TO CONSIDER
DNA Methylation & Restriction Digests
DNA methyltransferases (MTases) that transfer a methyl group from S-adenosylmethionine to
either adenine or cytosine residues are found in a wide variety of prokaryotes and eukaryotes.
Methylation should be considered when digesting DNA with restriction endonucleases
because cleavage can be blocked or impaired when a particular base in the recognition site
is methylated.
Prokaryotic Methylation
In prokaryotes, MTases have most often been identified as elements of restriction/
modification systems that act to protect host DNA from cleavage by the corresponding
restriction endonuclease. Most laboratory strains of E. coli contain three site-specific
DNA methyltransferases.
•• Dam methyltransferases–methylation at the N6
position of the adenine in the sequence
GATC (1,2).
•• Dcm methyltransferases–methylation at the C5
position of cytosine in the sequences
CCAGG and CCTGG (1,3).
•• EcoKI methylase–methylation of adenine in the sequences AAC(N6
A)GTGC and
GCAC(N6
A)GTT.
Some or all of the sites for a restriction endonuclease may be resistant to cleavage when
isolated from strains expressing the Dam or Dcm MTase if the methylase recognition site
overlaps the endonuclease recognition site. For example, plasmid DNA isolated from dam+
E. coli is completely resistant to cleavage by MboI, which cleaves at GATC sites.
Not all DNA isolated from E. coli is methylated to the same extent. While pBR322 DNA
is fully modified (and is therefore completely resistant to MboI digestion), only about 50%
of λ DNA Dam sites are methylated, presumably because the methylase does not have the
opportunity to methylate the DNA fully before it is packaged into the phage head. As a
result, enzymes blocked by Dam or Dcm modification will yield partial digestion patterns
with λ DNA.
Restriction sites that are blocked by Dam or Dcm methylation can be un-methylated by
cloning your DNA into a dam–
, dcm–
strain of E. coli, such as dam–
/dcm–
Competent E. coli
(NEB #C2925).
Restriction sites can also be blocked if an overlapping site is present. In this case, part of
the Dam or Dcm sequence is generated by the restriction enzyme sequence, followed by
the flanking sequence. This situation should also be considered when designing restriction
enzyme digests.
Eukaryotic Methylation
CpG MTases, found in higher eukaryotes (e.g., Dnmt1), transfer a methyl group to the C5
position of cytosine residues. Patterns of CpG methylation are heritable, tissue specific and
correlate with gene expression. Consequently, CpG methylation has been postulated to play a
role in differentiation and gene expression (4).
Note: The effects of CpG methylation are mainly a concern when digesting eukaryotic
genomic DNA. CpG methylation patterns are not retained once the DNA is cloned into a
bacterial host.
Methylation Sensitivity
The table below summarizes methylation sensitivity for NEB restriction enzymes, indicating
whether or not cleavage is blocked or impaired by Dam, Dcm or CpG methylation if or
when it overlaps each recognition site. This table should be viewed as a guide to the
behavior of the enzymes listed rather than an absolute indicator. Consult REBASE
(http://rebase.neb.com/rebase/), the restriction enzyme database, for more detailed
information and specific examples upon which these guidelines are based.
References
1.	 Marinus, M.G. and Morris, N.R. (1973) J. Bacteriol., 114,
1143–1150.
2.	 Geier, G.E. and Modrich, P. (1979) J. Biol. Chem., 254,
1408–1413.
3.	 May, M.S. and Hattman, S. (1975) J. Bacteriol., 123, 768–770.
4.	 Siegfried, Z. and Cedar, H. (1997) Curr. Biol., 7, r305–307.
METHYLATION SENSITIVITY
12
• Genomic DNA directly isolated from a
mammalian source is not Dcm or Dam
methylated, and is therefore not an issue
when digesting mammalian DNA.
• 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
Legend:
1 not sensitive
2 blocked
3 ol blocked by overlapping
3 scol blocked by some combinations of overlapping
4 impaired
5 ol impaired by overlapping
5 scol impaired by some combinations of overlapping
R = A or G
K = G or T
H = A or C or T
D = A or G or T
Y = C or T
S = C or G
B = C or G or T
N = A or C or G or T
M = A or C
W = A or T
V = A or C or G
Methylation Sensitivity – Dam, Dcm and CpG Methylation
Single Letter Code:
METHYLATION SENSITIVITY
13
AatII GACGT/C 1 1 2
AccI GT/MKAC 1 1 3 ol
Acc65I G/GTACC 1 3 scol 3 scol
AciI CCGC(-3/-1) 1 1 2
AclI AA/CGTT 1 1 2
AcuI CTGAAG(16/14) 1 1 1
AfeI AGC/GCT 1 1 2
AflII C/TTAAG 1 1 1
AflIII A/CRYGT 1 1 1
AgeI A/CCGGT 1 1 2
AgeI-HF A/CCGGT 1 1 2
AgeI-HF RE-Mix A/CCGGT 1 1 2
AhdI GACNNN/NNGTC 1 1 5 scol
AleI CACNN/NNGTG 1 1 5 scol
AluI AG/CT 1 1 1
AlwI GGATC(4/5) 2 1 1
AlwNI CAGNNN/CTG 1 3 ol 1
ApaI GGGCC/C 1 3 ol 3 ol
ApaLI G/TGCAC 1 1 3 ol
ApeKI G/CWGC 1 1 3 ol
ApoI R/AATTY 1 1 1
AscI GG/CGCGCC 1 1 2
AscI RE-Mix GG/CGCGCC 1 1 2
AseI AT/TAAT 1 1 1
AsiSI GCGAT/CGC 1 1 2
AvaI C/YCGRG 1 1 2
AvaII G/GWCC 1 3 ol 3 ol
AvrII C/CTAGG 1 1 1
BaeI (10/15)ACNNNNGTAYC(12/7) 1 1 3 scol
BaeGI GKGCM/C 1 1 1
BamHI G/GATCC 1 1 1
BamHI-HF G/GATCC 1 1 1
BanI G/GYRCC 1 3 scol 3 scol
BanII GRGCY/C 1 1 1
BbsI GAAGAC(2/6) 1 1 1
BbvI GCAGC(8/12) 1 1 1
BbvCI CCTCAGC(-2/-5) 1 1 5 ol
BccI CCATC(4/5) 1 1 1
BceAI ACGGC(12/14) 1 1 2
BcgI (10/12)CGANNNNNNTGC(12/10) 5 ol 1 3 scol
BcoDI GTCTC(1/5) 1 1 3 scol
BciVI GTATCC(6/5) 1 1 1
BclI T/GATCA 2 1 1
BfaI C/TAG 1 1 1
BfuAI ACCTGC(4/8) 1 1 5 ol
BfuCI /GATC 1 1 3 ol
BglI GCCNNNN/NGGC 1 1 3 scol
BglII A/GATCT 1 1 1
BlpI GC/TNAGC 1 1 1
BmgBI CACGTC(-3/-3) 1 1 2
BmrI ACTGGG(5/4) 1 1 1
BmtI GCTAG/C 1 1 1
BmtI-HF GCTAG/C 1 1 1
BpmI CTGGAG(16/14) 1 1 1
Bpu10I CCTNAGC(-5/-2) 1 1 1
BpuEI CTTGAG(16/14) 1 1 1
BsaI GGTCTC(1/5) 1 5 scol 3 scol
BsaI-HF GGTCTC(1/5) 1 3 ol 3 scol
BsaAI YAC/GTR 1 1 2
BsaBI GATNN/NNATC 3 ol 1 3 scol
BsaHI GR/CGYC 1 3 scol 2
BsaJI C/CNNGG 1 1 1
BsaWI W/CCGGW 1 1 1
BsaXI (9/12)ACNNNNNCTCC(10/7) 1 1 1
BseRI GAGGAG(10/8) 1 1 1
BseYI CCCAGC(-5/-1) 1 1 3 ol
BsgI GTGCAG(16/14) 1 1 1
BsiEI CGRY/CG 1 1 2
BsiHKAI GWGCW/C 1 1 1
BsiWI C/GTACG 1 1 2
BslI CCNNNNN/NNGG 1 3 scol 3 scol
BsmI GAATGC(1/-1) 1 1 1
BsmAI GTCTC (1/5) 1 1 3 scol
BsmBI CGTCTC(1/5) 1 1 2
BsmFI GGGAC(10/14) 1 3 ol 3 ol
BsoBI C/YCGRG 1 1 1
Bsp1286I GDGCH/C 1 1 1
BspCNI CTCAG(9/7) 1 1 1
BspDI AT/CGAT 3 ol 1 2
BspEI T/CCGGA 3 ol 1 4
BspHI T/CATGA 5 ol 1 1
BspMI ACCTGC(4/8) 1 1 1
ENZYME	SEQUENCE	 Dam	 Dcm	CpG ENZYME	SEQUENCE	 Dam	 Dcm	CpG
METHYLATION SENSITIVITY
14
BspQI GCTCTTC(1/4) 1 1 1
BsrI ACTGG(1/-1) 1 1 1
BsrBI CCGCTC(-3/-3) 1 1 3 scol
BsrDI GCAATG(2/0) 1 1 1
BsrFI R/CCGGY 1 1 2
BsrGI T/GTACA 1 1 1
BsrGI-HF T/GTACA 1 1 1
BssHII G/CGCGC 1 1 2
BssKI /CCNGG 1 3 ol 3 ol
BssSa
I CACGAG(-5/-1) 1 1 1
BstAPI GCANNNN/NTGC 1 1 3 scol
BstBI TT/CGAA 1 1 2
BstEII G/GTNACC 1 1 1
BstEII-HF G/GTNACC 1 1 1
BstEII-HF RE-Mix G/GTNACC 1 1 1
BstNI CC/WGG 1 1 1
BstUI CG/CG 1 1 2
BstXI CCANNNNN/NTGG 1 3 scol 1
BstYI R/GATCY 1 1 1
BstZ17I GTA/TAC 1 1 3 scol
Bsu36I CC/TNAGG 1 1 1
BtgI C/CRYGG 1 1 1
BtgZI GCGATG(10/14) 1 1 4
Btsa
I GCAGTG(2/0) 1 1 1
BtsIMutI CAGTG(2/0) 1 1 1
BtsCI GGATG(2/0) 1 1 1
Cac8I GCN/NGC 1 1 3 scol
ClaI AT/CGAT 3 ol 1 2
CspCI (11/13)CAANNNNNGTGG(12/10) 1 1 1
CviAII C/ATG 1 1 1
CviKI-1 RG/CY 1 1 1
CviQI G/TAC 1 1 1
DdeI C/TNAG 1 1 1
DpnI GA/TC 1 1 3 ol
DpnII /GATC 2 1 1
DraI TTT/AAA 1 1 1
DraIII-HF CACNNN/GTG 1 1 5 scol
DrdI GACNNNN/NNGTC 1 1 3 scol
EaeI Y/GGCCR 1 3 ol 3 ol
EagI C/GGCCG 1 1 2
EagI-HF C/GGCCG 1 1 2
EarI CTCTTC(1/4) 1 1 5 ol
EciI GGCGGA(11/9) 1 1 3 scol
Eco53kI GAG/CTC 1 1 3 scol
EcoNI CCTNN/NNNAGG 1 1 1
EcoO109I RG/GNCCY 1 3 ol 1
EcoP15I CAGCAG(25/27) 1 1 1
EcoRI G/AATTC 1 1 3 scol
EcoRI-HF G/AATTC 1 1 3 scol
EcoRI-HF RE-Mix G/AATTC 1 1 3 scol
EcoRV GAT/ATC 1 1 5 scol
EcoRV-HF GAT/ATC 1 1 5 scol
EcoRV-HF RE-Mix GAT/ATC 1 1 5 scol
FatI /CATG 1 1 1
FauI CCCGC(4/6) 1 1 2
Fnu4HI GC/NGC 1 1 3 ol
FokI GGATG(9/13) 1 5 ol 5 ol
FseI GGCCGG/CC 1 5 scol 2
FspI TGC/GCA 1 1 2
FspEI C5mCNNNNNNNNNNNN 1 1 1
HaeII RGCGC/Y 1 1 2
HaeIII GG/CC 1 1 1
HgaI GACGC(5/10) 1 1 2
HhaI GCG/C 1 1 2
HincII GTY/RAC 1 1 3 scol
HindIII A/AGCTT 1 1 1
HindIII-HF A/AGCTT 1 1 1
HinfI G/ANTC 1 1 3 scol
HinP1I G/CGC 1 1 2
HpaI GTT/AAC 1 1 3 scol
HpaII C/CGG 1 1 2
HphI GGTGA(8/7) 2 2 1
Hpy99I CGWCG/ 1 1 2
Hpy166II GTN/NAC 1 1 3 ol
Hpy188I TCN/GA 3 ol 1 1
Hpy188III TC/NNGA 3 ol 1 3 ol
HpyAV CCTTC(6/5) 1 1 5 ol
HpyCH4III ACN/GT 1 1 1
HpyCH4IV A/CGT 1 1 2
HpyCH4V TG/CA 1 1 1
KasI G/GCGCC 1 1 2
KpnI GGTAC/C 1 1 1
KpnI-HF GGTAC/C 1 1 1
KpnI-HF RE-Mix GGTAC/C 1 1 1
LpnPI C5mCDGNNNNNNNNNN 1 1 1
MboI /GATC 2 1 5 ol
MboII GAAGA(8/7) 3 ol 1 1
MfeI C/AATTG 1 1 1
MfeI-HF C/AATTG 1 1 1
MfeI-HF RE-Mix C/AATTG 1 1 1
MluI A/CGCGT 1 1 2
MluI-HF A/CGCGT 1 1 2
MluCI /AATT 1 1 1
MlyI GAGTC(5/5) 1 1 1
MmeI TCCRAC(20/18) 1 1 3 ol
MnlI CCTC(7/6) 1 1 1
MscI TGG/CCA 1 3 ol 1
MseI T/TAA 1 1 1
MslI CAYNN/NNRTG 1 1 1
MspI C/CGG 1 1 1
MspA1I CMG/CKG 1 1 3 ol
MspJI 5mCNNRNNNNNNNNN 1 1 1
MwoI GCNNNNN/NNGC 1 1 3 scol
NaeI GCC/GGC 1 1 2
NarI GG/CGCC 1 1 2
Nb.BbvCI CCTCAGC (none/-2) 1 1 1
ENZYME	SEQUENCE	 Dam	 Dcm	CpG ENZYME	SEQUENCE	 Dam	 Dcm	CpG
www.neb.com
METHYLATION SENSITIVITY
15
Nb.BsmI GAATGC (none/-2) 1 1 1
Nb.BsrdI GCAATG (none/0) 1 1 1
Nb.BtsI GCAGTG 1 1 1
NciI CC/SGG 1 1 5 ol
NcoI C/CATGG 1 1 1
NcoI-HF C/CATGG 1 1 1
NcoI-HF RE-Mix C/CATGG 1 1 1
NdeI CA/TATG 1 1 1
NgoMIV G/CCGGC 1 1 2
NheI G/CTAGC 1 1 3 scol
NheI-HF G/CTAGC 1 1 3 scol
NheI-HF RE-Mix G/CTAGC 1 1 3 scol
NlaIII CATG/ 1 1 1
NlaIV GGN/NCC 1 3 ol 3 ol
NmeAIII GCCGAG(21/19) 1 1 1
NotI GC/GGCCGC 1 1 2
NotI-HF GC/GGCCGC 1 1 2
NotI-HF RE-Mix GC/GGCCGC 1 1 2
NruI TCG/CGA 3 ol 1 2
NruI-HF TCG/CGA 3 ol 1 2
NsiI ATGCA/T 1 1 1
NsiI-HF ATGCA/T 1 1 1
NspI RCATG/Y 1 1 1
Nt.AlwI GGATC(4/-5) 2 1 1
Nt.BbvCI CCTCAGC(-5/none) 1 1 3 scol
Nt.BsmAI GTCTC(1/none) 1 1 2
Nt.BspQI GCTCTTC(1/none) 1 1 1
Nt.BstNBI GAGTC(4/none) 1 1 1
Nt.CviPII (0/-1)CCD 1 1 2
PacI TTAAT/TAA 1 1 1
PacI RE-Mix TTAAT/TAA 1 1 1
PaeR7I C/TCGAG 1 1 2
PciI A/CATGT 1 1 1
PflFI GACN/NNGTC 1 1 1
PflMI CCANNNN/NTGG 1 3 ol 1
PhoI GG/CC 1 5 scol 5 scol
PleI GAGTC(4/5) 1 1 3 scol
PluTI GGCGC/C 1 1 2
PmeI GTTT/AAAC 1 1 3 scol
PmlI CAC/GTG 1 1 2
PpuMI RG/GWCCY 1 3 ol 1
PshAI GACNN/NNGTC 1 1 3 scol
PsiI TTA/TAA 1 1 1
PspGI /CCWGG 1 2 1
PspOMI G/GGCCC 1 5 scol 3 ol
PspXI VC/TCGAGB 1 1 4
PstI CTGCA/G 1 1 1
PstI-HF CTGCA/G 1 1 1
PvuI CGAT/CG 1 1 2
PvuI-HF CGAT/CG 1 1 2
PvuII CAG/CTG 1 1 1
PvuII-HF CAG/CTG 1 1 1
RsaI GT/AC 1 1 3 scol
RsrII CG/GWCCG 1 1 2
SacI GAGCT/C 1 1 1
SacI-HF GAGCT/C 1 1 1
SacII CCGC/GG 1 1 2
SalI G/TCGAC 1 1 2
SalI-HF G/TCGAC 1 1 2
SalI-HF RE-Mix G/TCGAC 1 1 2
SapI GCTCTTC(1/4) 1 1 1
Sau3AI /GATC 1 1 3 ol
Sau96I G/GNCC 1 3 ol 3 ol
SbfI CCTGCA/GG 1 1 1
SbfI-HF CCTGCA/GG 1 1 1
ScaI-HF AGT/ACT 1 1 1
ScaI-HF RE-Mix AGT/ACT 1 1 1
ScrFI CC/NGG 1 3 ol 3 ol
SexAI A/CCWGGT 1 2 1
SfaNI GCATC(5/9) 1 1 5 scol
SfcI C/TRYAG 1 1 1
SfiI GGCCNNNN/NGGCC 1 5 ol 3 scol
SfoI GGC/GCC 1 3 scol 2
SgrAI CR/CCGGYG 1 1 2
SmaI CCC/GGG 1 1 2
SmlI C/TYRAG 1 1 1
SnaBI TAC/GTA 1 1 2
SpeI A/CTAGT 1 1 1
SpeI RE-Mix A/CTAGT 1 1 1
SpeI-HF A/CTAGT 1 1 1
SphI GCATG/C 1 1 1
SphI-HF GCATG/C 1 1 1
SspI AAT/ATT 1 1 1
SspI-HF AAT/ATT 1 1 1
StuI AGG/CCT 1 3 ol 1
StyI C/CWWGG 1 1 1
StyI-HF C/CWWGG 1 1 1
StyD4I /CCNGG 1 3 ol 5 ol
SwaI ATTT/AAAT 1 1 1
TaqI T/CGA 3 ol 1 1
TfiI G/AWTC 1 1 3 scol
TseI G/CWGC 1 1 3 scol
Tsp45I /GTSAC 1 1 1
TspMI C/CCGGG 1 1 2
TspRI NNCASTGNN/ 1 1 1
Tth111I GACN/NNGTC 1 1 1
XbaI T/CTAGA 3 ol 1 1
XbaI RE-Mix T/CTAGA 3 ol 1 1
XcmI CCANNNNN/NNNNTGG 1 1 1
XhoI C/TCGAG 1 1 4
XhoI RE-Mix C/TCGAG 1 1 4
XmaI C/CCGGG 1 1 4
XmnI GAANN/NNTTC 1 1 1
ZraI GAC/GTC 1 1 2
ENZYME	SEQUENCE	 Dam	 Dcm	CpG ENZYME	SEQUENCE	 Dam	 Dcm	CpG
ONLINE TOOLS
Online Tools
The Tools & Resources tab, accessible on our homepage, contains a selection of interactive technical tools for use with
restriction enzymes. These tools can also be accessed directly in the footer of every web page.
Enzyme Finder
Use this tool to select
restriction enzymes by name,
sequence, overhang or type.
Enter your sequence using
single letter code, and Enzyme
Finder will identify the right
enzyme for the job.
DNA Sequences and Maps Tool
With the DNA Sequences and
Maps Tool, find the nucleotide
sequence files for commonly
used molecular biology tools,
including plasmid, viral and
bacteriophage vectors.
Double Digest Finder
Use this tool to guide your
reaction buffer selection when
setting up double-digests, a
common timesaving procedure.
Choosing the right buffers will
help you to avoid star activity
and loss of product.
NEB Tools for Restriction Enzymes
REBASE®
NEBioCalculator®
NEBcloner®
NEBcutter®
V2.0
Identify restriction sites within
your DNA sequence using
NEBcutter. Choose between
Type II and commercially
available Type III enzymes to
digest your DNA. NEBcutter
V2.0 indicates cut frequency
and methylation sensitivity.
Use this tool as a guide to the
ever-changing landscape of
restriction enzymes. REBASE,
the Restriction Enzyme
DataBASE, is a dynamic, curated
database of restriction enzymes
and related proteins.
NEBioCalculator is a
collection of calculators and
converters that are useful in
planning bench experiments in
molecular biology laboratories.
Use this tool to find the right
products and protocols for
each step (digestion, end
modification, ligation and
transformation) of your next
traditional cloning experiment.
It is also very helpful with
double digests! While you are
there, you can also, find other
relevant tools and resources to
enable protocol optimization.
Mobile Apps
NEB Tools for iPhone®
, iPad®
or Android™
NEB Tools brings New
England Biolabs’ most popular
web tools to your iPhone, iPad
or Android devices.
•	 Use Enzyme Finder to select a restriction
enzyme by category or recognition
sequence, or search by name to find
information on any NEB enzyme.
Sort your results so they make sense to
you, then email them to your inbox or
connect directly to www.neb.com.
•	 Use Double Digest Finder or NEBcloner
to determine buffer and reaction
conditions for experiments requiring two
restriction enzymes.
When using either of these tools, look for
CutSmart, HF and Time-Saver enzymes for
the ultimate in convenience. NEB Tools
enables quick and easy access to the most
requested restriction enzyme information,
and allows you to plan your experiments
from anywhere.
16
TOOLS & RESOURCES
Visit www.neb.com for:
• Technical information including additional
charts, protocols and technical tips related to
restriction enzymes
CLEAVAGE CLOSE TO THE ENDS
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) for
60 minutes at the recommended reaction conditions for each enzyme. Following ligation and
transformation, cleavage efficiencies were determined by dividing the number of 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.
ENZYME
BASE PAIRS
FROM END
% CLEAVAGE
EFFICIENCY VECTOR
INITIAL
CUT
AatII 3 88 LITMUS 29 NcoI
2 100 LITMUS 28 NcoI
1 95 LITMUS 29 PinAI
Acc65I 2 99 LITMUS 29 SpeI
1 75 pNEB193 SacI
AflII 1 13 LITMUS 29 StuI
AgeI 1 100 LITMUS 29 XbaI
1 100 LITMUS 29 AatII
ApaI 2 100 LITMUS 38 SpeI
AscI 1 97 pNEB193 BamHI
AvrII 1 100 LITMUS 29 SacI
BamHI 1 97 LITMUS 29 HindIII
BglII 3 100 LITMUS 29 NsiI
BsiWI 2 100 LITMUS 29 BssHII
BspEI 2 100 LITMUS 39 BsrGI
1 8 LITMUS 38 BsrGI
BsrGI 2 99 LITMUS 39 SphI
1 88 LITMUS 38 BspEI
BssHII 2 100 LITMUS 29 BsiWI
EagI 2 100 LITMUS 39 NheI
EcoRI 1 100 LITMUS 29 XhoI
1 88 LITMUS 29 PstI
1 100 LITMUS 39 NheI
EcoRV 1 100 LITMUS 29 PstI
HindIII 3 90 LITMUS 29 NcoI
2 91 LITMUS 28 NcoI
1 0 LITMUS 29 BamHI
KasI 2 97 LITMUS 38 NgoMIV
1 93 LITMUS 38 HindIII
KpnI 2 100 LITMUS 29 SpeI
2 100 LITMUS 29 SacI
1 99 pNEB193 SacI
MluI 2 99 LITMUS 39 EagI
ENZYME
BASE PAIRS
FROM END
% CLEAVAGE
EFFICIENCY VECTOR
INITIAL
CUT
MunI 2 100 LITMUS 39 NgoMIV
NcoI 2 100 LITMUS 28 HindIII
NgoMIV 2 100 LITMUS 39 MunI
NheI 1 100 LITMUS 39 EcoRI
2 82 LITMUS 39 EagI
NotI 7 100 Bluescript SK- SpeI
4 100 Bluescript SK- KspI
1 98 Bluescript SK- XbaI
NsiI 3 100 LITMUS 29 BssHII
3 77 LITMUS 29 BglII
2 95 LITMUS 28 BssHII
PacI 1 76 pNEB193 BamHI
PmeI 1 94 pNEB193 PstI
PstI 3 98 LITMUS 29 EcoR V
2 50 LITMUS 39 HindIII
1 37 LITMUS 29 EcoRI
SacI 1 99 LITMUS 29 AvrII
SalI 3 89 LITMUS 39 SpeI
2 23 LITMUS 39 SphI
1 61 LITMUS 38 SphI
SfiI*
9 81 LITMUS 38 BamHI
4 97 LITMUS 38 MluI
1 93 LITMUS 38 EcoRI
SpeI 2 100 LITMUS 29 Acc65I
2 100 LITMUS 29 KpnI
SphI 2 99 LITMUS 39 SalI
2 97 LITMUS 39 BsrGI
1 92 LITMUS 38 SalI
XbaI 1 99 LITMUS 29 AgeI
1 94 LITMUS 29 PinAI
XhoI 1 97 LITMUS 29 EcoRI
XmaI 2 98 pNEB193 AscI
2 92 pNEB193 BssHII
*
A modified version of LITMUS 38 with an introduced SfiI site was used for this test.
17www.neb.com
PERFORMANCE CHART
Performance Chart for Restriction Enzymes
New England Biolabs supplies > 200 restriction enzymes that are 100% active in a single buffer,
CutSmart. This results in increased efficiency, flexibility and ease-of-use, especially when performing
double digests.
This performance chart summarizes the activity information of NEB restriction enzymes. To help
select the best conditions for double digests, this chart shows the optimal (supplied) NEBuffer and
approximate activity in the four standard NEBuffers for each enzyme. Note that BSA is now included
in all NEBuffers, and is no longer provided as a separate tube. In addition, this performance chart
shows recommended reaction temperature, heat-inactivation temperature, recommended diluent
buffer, methylation sensitivity and whether the enzyme is Time-Saver™
qualified (i.e., cleaves
substrate in 5–15 minutes under recommended conditions, and can be used overnight without
degradation of DNA).
18
	 	 INCUB.	INACTIV.
	 SUPPLIED	 % ACTIVITY IN NEBUFFERS	 TEMP.	 TEMP.	 METHYLATION
ENZYME	 NEBUFFER	 1.1	2.1	3.1	CUTSMART	 (°C)	(°C)	DIL.	SUBSTRATE	SENSITIVITY	NOTE(S)
NEBuffer Compositions (1X)
NEBuffer 1.1 10 mM Bis Tris Propane-HCl, 10 mM MgCl2
, 100 µg/ml BSA (pH 7.0 @ 25°C).
NEBuffer 2.1 10 mM Tris-HCl, 10 mM MgCl2
, 50 mM NaCl, 100 µg/ml BSA (pH 7.9 @ 25°C).
NEBuffer 3.1 50 mM Tris-HCl, 10 mM MgCl2
, 100 mM NaCl, 100 µg/ml BSA (pH 7.9 @ 25°C).
CutSmart Buffer 20 mM Tris-acetate, 10 mM magnesium acetate, 50 mM potassium acetate, 100 µg/ml BSA (pH 7.9 @ 25°C).
Diluent A 50 mM KCI, 10 mM Tris-HCl, 0.1 mM EDTA, 1 mM dithiothreitol, 200 µg/ml BSA (pH 7.4 @ 25°C).
Diluent B 300 mM NaCI, 10 mM Tris-HCl, 0.1 mM EDTA, 1 mM dithiothreitol, 500 µg/ml BSA, 50% glycerol (pH 7.4 @ 25°C).
Diluent C 50 mM KCI, 10 mM Tris-HCl, 0.1 mM EDTA, 1 mM dithiothreitol, 0.15% Triton X-100, 200 µg/ml BSA 50% glycerol (pH 7.4 @ 25°C).
FOR STAR ACTIVITY
1.	Star activity may result from extended digestion,
high enzyme concentration or a glycerol
concentration of > 5%.
2.	Star activity may result from extended digestion.
3.	Star activity may result from a glycerol
concentration of > 5%.
* May exhibit star activity in this buffer.
FOR LIGATION AND RECUTTING
a. Ligation is less than 10%
b. Ligation is 25% – 75%
c. Recutting after ligation is < 5%
d. Recutting after ligation is 50% – 75%
e. Ligation and recutting after ligation is not applicable since
the enzyme is either a nicking enzyme, is affected by
methylation, or the recognition sequence contains variable
sequences.
Activity Notes (see last column)
AatII CutSmart < 10 50* 50 100 37° 80° B Lambda
AbaSI 4 25 50 50 100 25° 65° C T4 wt Phage e
AccI CutSmart 50 50 10 100 37° 80° A Lambda
Acc65I 3.1 10 75* 100 25 37° 65° A pBC4
AciI CutSmart < 10 25 100 100 37° 65° A Lambda
AclI CutSmart < 10 < 10 < 10 100 37° No B Lambda
AcuI CutSmart + SAM 50 100 50 100 37° 65° B Lambda 3, b, d
AfeI CutSmart 25 100 25 100 37° 65° B pXba
AflII CutSmart 50 100 10 100 37° 65° A phiX174
AflIII 3.1 10 50 100 50 37° 80° B Lambda
AgeI 1.1 100 75 25 75 37° 65° C Lambda 2
AgeI-HF CutSmart 100 50 10 100 37° 65° A Lambda
AhdI CutSmart 25 25 10 100 37° 65° A Lambda a
AleI CutSmart < 10 < 10 < 10 100 37° 80° B Lambda
AluI CutSmart 25 100 50 100 37° 80° B Lambda b
AlwI CutSmart 50 50 10 100 37° No A Lambda dam- 1, b, d
AlwNI CutSmart 10 100 50 100 37° 80° A Lambda
ApaI CutSmart 25 25 < 10 100 25° 65° A pXba
ApaLI CutSmart 100 100 10 100 37° No A Lambda HindIII
ApeKI 3.1 25 50 100 10 75° No B Lambda
Chart Legend
U
Supplied with a unique reaction buffer that is
different from the four standard NEBuffers. The
compatibility with the four standard NEBuffers is
indicated in the chart.
SAM
Supplied with a separate vial of
S-adenosylmethionine (SAM). To obtain
100% activity, SAM should be added to the 1X
reaction mix as specified on the product data card.
Recombinant dcm methylation sensitivity
Time-Saver qualified CpG methylation sensitivity
Engineered enzyme for maximum performance RE-Mix Master Mix version available
dam methylation sensitivity
PERFORMANCE CHART
19
	 	 INCUB.	INACTIV.
	 SUPPLIED	 % ACTIVITY IN NEBUFFERS	 TEMP.	 TEMP.	 METHYLATION
ENZYME	 NEBUFFER	 1.1	2.1	3.1	CUTSMART	 (°C)	(°C)	DIL.	SUBSTRATE	SENSITIVITY	NOTE(S)
ApoI 3.1 10 75 100 75 50° 80° A Lambda
AscI CutSmart < 10 10 10 100 37° 80° A Lambda
AseI 3.1 < 10 50* 100 10 37° 65° B Lambda 3
AsiSI CutSmart 50 100 100 100 37° 80° B pXba (Xho digested) 2, b
AvaI CutSmart < 10 100 25 100 37° 80° A Lambda
AvaII CutSmart 50 75 10 100 37° 80° A Lambda
AvrII CutSmart 100 50 50 100 37° No B Lambda HindIII
BaeI CutSmart + SAM 50 100 50 100 25° 65° A Lambda e
BaeGI 3.1 75 75 100 25 37° 80° A Lambda
BamHI 3.1 75* 100* 100 100* 37° No A Lambda 3
BamHI-HF CutSmart 100 50 10 100 37° No A Lambda
BanI CutSmart 10 25 < 10 100 37° 65° A Lambda 1
BanII CutSmart 100 100 50 100 37° 80° A Lambda 2
BbsI 2.1 100 100 25 75 37° 65° B Lambda
BbvI CutSmart 100 100 25 100 37° 65° B pBR322 3
BbvCI CutSmart 10 100 50 100 37° No B Lambda
1, a
BccI CutSmart 100 50 10 100 37° 65° A pXba 3, b
BceAI 3.1 100* 100* 100 100* 37° 65° A pBR322 1
BcgI 3.1 + SAM 10 75* 100 50* 37° 65° A Lambda e
BciVI CutSmart 100 25 < 10 100 37° 80° C Lambda b
BclI 3.1 50 100 100 75 50° No A Lambda damBcoDI
CutSmart 50 75 75 100 37° No B Lambda
BfaI CutSmart < 10 10 < 10 100 37° 80° B Lambda 2, b
BfuAI 3.1 < 10 25 100 10 50° 65° B Lambda 3
BfuCI CutSmart 100 50 25 100 37° 80° B Lambda
BglI 3.1 10 25 100 10 37° 65° B Lambda
BglII 3.1 10 10 100 < 10 37° No A Lambda
BlpI CutSmart 50 100 10 100 37° No A Lambda d
BmgBI 3.1 < 10 10 100 10 37° 65° B Lambda 3, b, d
BmrI 2.1 75 100 75 100* 37° 65° B Lambda HindIII b
BmtI 3.1 100 100 100 100 37° 65° B pXba 2
BmtI-HF CutSmart 50 100 10 100 37° 65° B pXba
BpmI 3.1 75 100 100 100 37° 65° B Lambda 2
Bpu10I 3.1 10 25 100 25 37° 80° B Lambda 3, b, d
BpuEI CutSmart + SAM 50* 100 50* 100 37° 65° B Lambda d
BsaI CutSmart 75* 75 100 100 37° 65° B pXba 3
BsaI-HF CutSmart 50 100 25 100 37° 65° B pXba
BsaAI CutSmart 100 100 100 100 37° No C Lambda
BsaBI CutSmart 50 100 75 100 60° 80° B Lambda dam- 2
BsaHI CutSmart 50 100 100 100 37° 80° A Lambda
BsaJI CutSmart 50 100 100 100 60° 80° A Lambda
BsaWI CutSmart 10 100 50 100 60° 80° A Lambda
BsaXI CutSmart 50* 100* 10 100 37° No B Lambda e
BseRI CutSmart 100* 100 75 100 37° 80° A Lambda d
BseYI 3.1 10 50 100 50 37° 80° B Lambda d
BsgI CutSmart + SAM 25 50 25 100 37° 65° B Lambda d
BsiEI CutSmart 25 50 < 10 100 60° No A Lambda
BsiHKAI CutSmart 25 100 100 100 65° No B Lambda
BsiWI 3.1 25 50* 100 25 55° 65° B phiX174
BslI CutSmart 50 75 100 100 55° No A Lambda b
BsmI CutSmart 25 100 < 10 100 65° 80° A pBR322
BsmAI CutSmart 50 100 100 100 55° No B Lambda
1.	 Star activity may result from extended digestion, high enzyme
concentration or a glycerol concentration of > 5%.
2.	 Star activity may result from extended digestion.
3.	 Star activity may result from a glycerol concentration of > 5%.
* May exhibit star activity in this buffer.
PERFORMANCE CHART
20
	 	 INCUB.	INACTIV.
	 SUPPLIED	 % ACTIVITY IN NEBUFFERS	 TEMP.	 TEMP.	 METHYLATION
ENZYME	 NEBUFFER	 1.1	2.1	3.1	CUTSMART	 (°C)	(°C)	DIL.	SUBSTRATE	SENSITIVITY	NOTE(S)
BsmBI 3.1 10 50* 100 25 55° 80° B Lambda
BsmFI CutSmart 25 50 50 100 65° 80° A pBR322 1
BsoBI CutSmart 25 100 100 100 37° 80° A Lambda
Bsp1286I CutSmart 25 25 25 100 37° 65° A Lambda 3
BspCNI CutSmart + SAM 100 75 10 100 25° 80° A Lambda b
BspDI CutSmart 25 75 50 100 37° 80° A Lambda
BspEI 3.1 < 10 10 100 < 10 37° 80° B Lambda damBspHI
CutSmart < 10 50 25 100 37° 80° A Lambda
BspMI 3.1 10 50* 100 10 37° 65° B Lambda
BspQI 3.1 100 100 100 100 50° 80° B Lambda 3
BsrI 3.1 < 10 50 100 10 65° 80° B phiX174 b
BsrBI CutSmart 50 100 100 100 37° 80° A Lambda d
BsrDI 2.1 10 100 75 25 65° 80° A Lambda 3, d
BsrFI CutSmart 10 100* 100* 100 37° No C pBR322 1
BsrGI 2.1 25 100 100 25 37° 80° A Lambda
BsrGI-HF CutSmart 10 100 100 100 37° 80° A Lambda
BssHII CutSmart 100 100 100 100 50° 65° B Lambda
BssKI CutSmart 50 100 100 100 60° 80° A Lambda b
BssSa
I CutSmart 10 25 < 10 100 37° No B Lambda
BstAPI CutSmart 50 100 25 100 60° 80° A Lambda b
BstBI CutSmart 75 100 10 100 65° No A Lambda
BstEII 3.1 10 75* 100 75* 60° No A Lambda 3
BstEII-HF CutSmart < 10 10 < 10 100 37° No A Lambda
BstNI 3.1 10 100 100 75 60° No A Lambda a
BstUI CutSmart 50 100 25 100 60° No A Lambda b
BstXI 3.1 < 10 50 100 25 37° 80° B Lambda 3
BstYI 2.1 25 100 75 100 60° No A Lambda
BstZ17I CutSmart 75 100 100 100 37° No B Lambda 3, b
Bsu36I CutSmart 25 100 100 100 37° 80° A Lambda HindIII b
BtgI CutSmart 50 100 100 100 37° 80° B pBR322
BtgZI CutSmart 10 25 < 10 100 60° 80° A Lambda 3, b, d
Btsa
l CutSmart 100 100 25 100 55° No A Lambda
BtsIMutI CutSmart 100 50 10 100 55° 80° A pUC19 b
BtsCI CutSmart 10 100 25 100 50° 80° B Lambda
Cac8I CutSmart 50 75 100 100 37° 65° B Lambda b
ClaI CutSmart 10 50 50 100 37° 65° A Lambda damCspCI
CutSmart + SAM 10 100 10 100 37° 65° A Lambda e
CviAII CutSmart 50 50 10 100 25° 65° C pUC19
CviKI-1 CutSmart 25 100 100 100 37° No A pBR322 1, b
CviQI 3.1 75 100* 100 75* 25° No C Lambda b
DdeI CutSmart 75 100 100 100 37° 65° B Lambda
DpnI CutSmart 100 100 75 100 37° 80° B pBR322 b
DpnII U 25 25 100* 25 37° 65° B Lambda damDraI
CutSmart 75 75 50 100 37° 65° A Lambda
DraIII-HF CutSmart < 10 50 10 100 37° No B Lambda b
DrdI CutSmart 25 50 10 100 37° 65° A pUC19 3, b
EaeI CutSmart 10 50 < 10 100 37° 65° A Lambda b
EagI 3.1 10 25 100 10 37° 65° C pXba
EagI-HF CutSmart 25 100 100 100 37° 65° B pXba
EarI CutSmart 50 10 < 10 100 37° 65° B Lambda b, d
EciI CutSmart 100 50 50 100 37° 65° A Lambda 2
Eco53kI CutSmart 100 100 < 10 100 37° 65° A pXba 3, b
a. Ligation is less than 10%
b. Ligation is 25% – 75%
c. Recutting after ligation is <5%
d. Recutting after ligation is 50% – 75%
e. Ligation and recutting after ligation is not applicable since the enzyme
is either a nicking enzyme, is affected by methylation, or the recognition
sequence contains variable sequences.
PERFORMANCE CHART
21
EcoNI CutSmart 50 100 75 100 37° 65° A Lambda b
EcoO109I CutSmart 50 100 50 100 37° 65° A Lambda HindIII 3
EcoP15I 3.1 + ATP 75 100 100 100 37° 65° A pUC19 e
EcoRI U 25 100* 50 50* 37° 65° C Lambda
EcoRI-HF CutSmart 10 100 < 10 100 37° 65° C Lambda
EcoRV 3.1 10 50 100 10 37° 80° A Lambda
EcoRV-HF CutSmart 25 100 100 100 37° 65° B Lambda
FatI 2.1 10 100 50 50 55° 80° A pUC19
FauI CutSmart 100 50 10 100 55° 65° A Lambda 3, b, d
Fnu4HI CutSmart < 10 < 10 < 10 100 37° No A Lambda a
FokI CutSmart 100 100 75 100 37° 65° A Lambda 3, b, d
FseI CutSmart 100 75 < 10 100 37° 65° B Adenovirus-2
FspI CutSmart 10 100 10 100 37° No C Lambda b
FspEI 4 + BSA < 10 < 10 < 10 100 37° 80° B pBC4 2, e
HaeII CutSmart 25 100 10 100 37° 80° A Lambda
HaeIII CutSmart 50 100 25 100 37° 80° A Lambda
HgaI 1.1 100 100 25 100 37° 65° A phiX174 1
HhaI CutSmart 25 100 100 100 37° 65° A Lambda
HincII 3.1 25 100 100 100 37° 65° B Lambda
HindIII 2.1 25 100 50 50 37° 80° B Lambda 2
HindIII-HF CutSmart 10 100 10 100 37° 80° B Lambda
HinfI CutSmart 50 100 100 100 37° 80° A Lambda
HinP1I CutSmart 100 100 100 100 37° 65° A Lambda
HpaI CutSmart < 10 75* 25 100 37° No A Lambda 1
HpaII CutSmart 100 50 < 10 100 37° 80° A Lambda
HphI CutSmart 50 50 < 10 100 37° 65° B Lambda b, d
Hpy99I CutSmart 50 10 < 10 100 37° 65° A Lambda
Hpy166II CutSmart 100 100 50 100 37° 65° C pBR322
Hpy188I CutSmart 25 100 50 100 37° 65° A pBR322 1, b
Hpy188III CutSmart 100 100 10 100 37° 65° B pUC19 3, b
HpyAV CutSmart 100 100 25 100 37° 65° B Lambda 3, b, d
HpyCH4III CutSmart 100 25 < 10 100 37° 65° A Lambda b
HpyCH4IV CutSmart 100 50 25 100 37° 65° A pUC19
HpyCH4V CutSmart 50 50 25 100 37° 65° A Lambda
KasI CutSmart 50 100 50 100 37° 65° B pBR322 3
KpnI 1.1 100 75 < 10 50* 37° No A pXba 1
KpnI-HF CutSmart 100 25 < 10 100 37° No A pXba
LpnPI 4 + BSA < 10 < 10 < 10 50 37° 65° B pBR322 2, e
MboI CutSmart 75 100 100 100 37° 65° A Lambda damMboII
CutSmart 100* 100 50 100 37° 65° C Lambda dam- b
MfeI CutSmart 75 50 10 100 37° No A Lambda 2
MfeI-HF CutSmart 75 25 < 10 100 37° No A Lambda
MluI 3.1 10 50 100 25 37° 80° A Lambda
MluI-HF CutSmart 25 100 100 100 37° No A Lambda
MluCI CutSmart 100 10 10 100 37° No A Lambda
MlyI CutSmart 50 50 10 100 37° 65° A Lambda b, d
MmeI CutSmart + SAM 50 100 50 100 37° 65° B phiX174 b, c
MnlI CutSmart 75 100 50 100 37° 65° B Lambda b
MscI CutSmart 25 100 100 100 37° 80° B Lambda
MseI CutSmart 75 100 75 100 37° 65° A Lambda
MslI CutSmart 50 50 < 10 100 37° 80° A Lambda
MspI CutSmart 75 100 50 100 37° No A Lambda
	 	 INCUB.	INACTIV.
	 SUPPLIED	 % ACTIVITY IN NEBUFFERS	 TEMP.	 TEMP.	 METHYLATION
ENZYME	 NEBUFFER	 1.1	2.1	3.1	CUTSMART	 (°C)	(°C)	DIL.	SUBSTRATE	SENSITIVITY	NOTE(S)
PERFORMANCE CHART
22
	 	 INCUB.	INACTIV.
	 SUPPLIED	 % ACTIVITY IN NEBUFFERS	 TEMP.	 TEMP.	 METHYLATION
ENZYME	 NEBUFFER	 1.1	2.1	3.1	CUTSMART	 (°C)	(°C)	DIL.	SUBSTRATE	SENSITIVITY	NOTE(S)
MspA1I CutSmart 10 50 10 100 37° 65° B Lambda
MspJI 4 + BSA < 10 < 10 < 10 50 37° 65° B pBR322 2, e
MwoI CutSmart < 10 100 100 100 60° No B Lambda
NaeI CutSmart 25 25 < 10 100 37° No A pXba b
NarI CutSmart 100 100 10 100 37° 65° A pXba
Nb.BbvCI CutSmart 25 100 100 100 37° 80° A pUB e
Nb.BsmI 3.1 < 10 50 100 10 65° 80° A pBR322 e
Nb.BsrDI CutSmart 25 100 100 100 65° 80° A pUC19 e
Nb.BtsI CutSmart 75 100 75 100 37° 80° A phiX174 e
NciI CutSmart 100 25 10 100 37° No A Lambda b
NcoI 3.1 100 100 100 100 37° 80° A Lambda
NcoI-HF CutSmart 50 100 10 100 37° 80° B Lambda
NdeI CutSmart 75 100 100 100 37° 65° A Lambda
NgoMIV CutSmart 100 50 10 100 37° No A pXba 1
NheI 2.1 100 100 10 100 37° 65° C Lambda HindIII
NheI-HF CutSmart 100 25 < 10 100 37° 80° C Lambda HindIII
NlaIII CutSmart < 10 < 10 < 10 100 37° 65° B phiX174
NlaIV CutSmart 10 10 10 100 37° 65° B pBR322
NmeAIII CutSmart + SAM 10 10 < 10 100 37° 65° B phiX174 c
NotI 3.1 < 10 50 100 25 37° 65° C pBC4
NotI-HF CutSmart 25 100 25 100 37° 65° A pBC4
NruI 3.1 < 10 10 100 10 37° No A Lambda b
NruI-HF CutSmart 0 25 50 100 37° No A Lambda
NsiI 3.1 10 75 100 25 37° 65° B Lambda
NsiI-HF CutSmart < 10 20 < 10 100 37° 80° B Lambda
NspI CutSmart 100 100 < 10 100 37° 65° A Lambda
Nt.AlwI CutSmart 10 100 100 100 37° 80° A pUC101 dam-dcm- e
Nt.BbvCI CutSmart 50 100 10 100 37° 80° A pUB e
Nt.BsmAI CutSmart 100 50 10 100 37 65° A pBR322 e
Nt.BspQI 3.1 < 10 25 100 10 50° 80° B pUC19 e
Nt.BstNBI 3.1 0 10 100 10 55° 80° A T7
Nt.CviPII CutSmart < 10 100 25 100 37° 65° A pUC19 e
PacI CutSmart 100 75 10 100 37° 65° A pNEB193
PaeR7I CutSmart 25 100 10 100 37° No A Lambda HindIII
PciI 3.1 50 75 100 50* 37° 80° B pXba
PflFI CutSmart 25 100 25 100 37° 65° A pBC4 b
PflMI 3.1 0 100 100 50 37° 65° A Lambda 3, b, d
PleI CutSmart 25 50 25 100 37° 65° A Lambda b
PluTI CutSmart 100 25 < 10 100 37° 65° A pXba
PmeI CutSmart < 10 50 10 100 37° 65° A Lambda
PmlI CutSmart 100 50 < 10 100 37° 65° A Lambda HindIII
PpuMI CutSmart < 10 < 10 < 10 100 37° No B Lambda HindIII
PshAI CutSmart 25 50 10 100 37° 65° A Lambda
PsiI CutSmart 10 100 10 100 37° 65° B Lambda 3
PspGI CutSmart 25 100 50 100 75° No A T7 3
PspOMI CutSmart 10 10 < 10 100 37° 65° B pXba
PspXI CutSmart < 10 100 25 100 37° No B Lambda HindIII
PstI 3.1 75 75 100 50* 37° 80° C Lambda
PstI-HF CutSmart 10 75 50 100 37° No C Lambda
PvuI 3.1 < 10 25 100 < 10 37° 80° B pXba
PvuI-HF CutSmart 25 100 100 100 37° No B pXba
1.	 Star activity may result from extended digestion, high enzyme
concentration or a glycerol concentration of > 5%.
2.	 Star activity may result from extended digestion.
3.	 Star activity may result from a glycerol concentration of > 5%.
* May exhibit star activity in this buffer.
PERFORMANCE CHART
23
	 	 INCUB.	INACTIV.
	 SUPPLIED	 % ACTIVITY IN NEBUFFERS	 TEMP.	 TEMP.	 METHYLATION
ENZYME	 NEBUFFER	 1.1	2.1	3.1	CUTSMART	 (°C)	(°C)	DIL.	SUBSTRATE	SENSITIVITY	NOTE(S)
PvuII 3.1 50 100 100 100* 37° No B Lambda
PvuII-HF CutSmart < 10 < 10 < 10 100 37° 80° B Lambda
RsaI CutSmart 25 50 < 10 100 37° No A Lambda
RsrII CutSmart 25 75 10 100 37° 65° C Lambda
SacI 1.1 100 50 10 100 37° 65° A Lambda HindIII
SacI-HF CutSmart 10 50 < 10 100 37° 65° A Lambda HindIII
SacII CutSmart 10 100 10 100 37° 65° A pXba
SalI 3.1 < 10 < 10 100 < 10 37° 65° A Lambda HindIII
SalI-HF CutSmart 10 100 100 100 37° 65° A Lambda HindIII
SapI CutSmart 75 50 < 10 100 37° 65° B Lambda
Sau3AI 1.1 100 50 10 100 37° 65° A Lambda b
Sau96I CutSmart 50 100 100 100 37° 65° A Lambda
SbfI CutSmart 50 25 < 10 100 37° 80° A Lambda 3
SbfI-HF CutSmart 50 25 < 10 100 37° 80° B Lambda
ScaI-HF CutSmart 100 100 10 100 37° 80° B Lambda
ScrFI CutSmart 100 100 100 100 37° 65° C Lambda 2, a
SexAI CutSmart 100 75 50 100 37° 65° A pBC4 dcm- 3, b, d
SfaNI 3.1 < 10 75 100 25 37° 65° B phiX174 3, b
SfcI CutSmart 75 50 25 100 37° 65° B Lambda 3
SfiI CutSmart 25 100 50 100 50° No C pXba
SfoI CutSmart 50 100 100 100 37° No B Lambda HindIII
SgrAI CutSmart 100 100 10 100 37° 65° A Lambda 1
SmaI CutSmart < 10 < 10 < 10 100 25° 65° B Lambda HindIII b
SmlI CutSmart 25 75 25 100 55° No A Lambda b
SnaBI CutSmart 50 50 10 100 37° 80° A T7 1
SpeI CutSmart 75 100 25 100 37° 80° C pXba-XbaI digested
SpeI-HF CutSmart 25 50 10 100 37° 80° C pXba-XbaI digested
SphI 2.1 100 100 50 100 37° 65° B Lambda 2
SphI-HF CutSmart 50 25 10 100 37° 65° B Lambda
SspI U 50 100 50 50 37° 65° C Lambda
SspI-HF CutSmart 25 100 < 10 100 37° No B Lambda
StuI CutSmart 50 100 50 100 37° No A Lambda
StyD4I CutSmart 10 100 100 100 37° 65° B Lambda
StyI 3.1 10 25 100 10 37° 65° A Lambda b
StyI-HF CutSmart 25 100 25 100 37° 65° A Lambda
SwaI 3.1 10 10 100 10 25° 65° B pUPS b, d
Taqa
I CutSmart 50 75 100 100 65° 80° B Lambda
TfiI CutSmart 50 100 100 100 65° No C Lambda
TseI CutSmart 75 100 100 100 65° No B Lambda 3
Tsp45I CutSmart 100 50 < 10 100 65° No A Lambda
TspMI CutSmart 50* 75* 50* 100 75° No B pUCAdeno d
TspRI CutSmart 25 50 25 100 65° No B Lambda
Tth111I CutSmart 25 100 25 100 65° No B pBC4 b
XbaI CutSmart < 10 100 75 100 37° 65° A Lambda HindIII damXcmI
2.1 10 100 25 100 37° 65° C Lambda 2
XhoI CutSmart 75 100 100 100 37° 65° A Lambda HindIII b
XmaI CutSmart 25 50 < 10 100 37° 65° A pXba 3
XmnI CutSmart 50 75 < 10 100 37° 65° A Lambda b
ZraI CutSmart 100 25 10 100 37° 80° B Lambda
a. Ligation is less than 10%
b. Ligation is 25% – 75%
c. Recutting after ligation is <5%
d. Recutting after ligation is 50% – 75%
e. Ligation and recutting after ligation is not applicable since the enzyme
is either a nicking enzyme, is affected by methylation, or the recognition
sequence contains variable sequences.
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