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restriction enDonucleAsesTechnical Guide 2009|10

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For over 35 years, New England Biolabs has led the industry in restriction enzyme innovation. As the first company to commercialize restriction enzymes, we are committed to providing a wide selection of high quality reagents at exceptional value. 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 of reaction conditions.

Cloning & Mapping

DNA AMPLIFICATION & PCR

RNA ANALYSIS

PROTEIN ExPRESSION & ANALYSIS

GENE ExPRESSION & CELLuLAR ANALYSIS

restriction enzymes From neB

selection in 1975, new england Biolabs (neB) became the first company to commercialize restriction enzymes, thereby providing researchers with a key set of tools that proved to be invaluable for the early development of the biotechnology industry. since then, neB has continued an aggressive program of cloning and overexpressing restriction enzymes. this is done biochemically, by the analysis of cell extracts, and computationally, by the analysis of sequenced genomes. As a result, we now supply more than 220 specificities, the largest number commercially available.

QualityAs a result of our active screening and cloning program, neB supplies the largest selection of recombinant enzymes available. expression using a recombinant source improves product yield and removes the uncertainties of potential contaminants. each 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 lot-to-lot consistency.

r Choose recombinant enzymes for a higher quality product with lot-to-lot consistency.

Performancecloning and sequencing restriction enzymes has enabled our researchers to enhance screening efforts as well as alter enzyme properties through engineering. this has 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.

Restriction Enzymes – Inside the Numbers*

231 – restriction enzymes sold by NEB

222 – enzyme specificities

173 – recombinant enzymes

162 – enzymes exhibiting 100% activity in a single buffer (NEBuffer 4)

153 – Time-Saver enzymes that digest substrate DNA in 5 minutes

15 – High Fidelity (HF) enzymes engineered for reduced star activity

10 – 8-base cutters

10 – nicking enzymes that cleave only one strand of duplex DNA

4 – homing endonucleases that have large asymmetric recognition sites

52 – Type IIS enzymes

* as of 03/26/09

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).

High Fidelity enzymes offer reduced star activity

10 30 50 100 M

EcoRI-HFNEB Supplier A Supplier B

10 30 50 100 M 10 30 50 100

www.neb.com 3

convenienceour dedication to restriction enzyme research has led to improvements that simplify reaction setup. neB utilizes a 4 buffer system, with over 160 enzymes recommended for use in a single buffer (neBuffer 4). this improves efficiency and ease-of-use, especially when performing double digests. over 150 of our enzymes are time-saver qualified, and will digest substrate DnA in five minutes under recommended reaction conditions. time-saver qualified enzymes are easy-to-use as there is no special formulation or change in concentration. these same enzymes can be used in overnight digests without sample loss, providing additional flexibility to reaction setup.

4 Over 160 restriction enzymes are recommended for use in NEBuffer 4.

C Over 150 enzymes are Time-Saver qualified and will digest substrate DNA in 5 minutes.

Valuethe development of recombinant enzymes allows neB to offer products at a lower cost per unit, enabling substantial savings, improved purity and consistency of product. there is no added cost for the convenience of time-saver qualified enzymes. Additionally, the scientific expertise of our research staff and extensive technical information available through our catalog and website are invaluable resources for questions regarding restriction enzymes and for support in experimental design.

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

Restriction enzymes usually occur in •combination with one or two modifying enzymes (DNA methyltransferases) that protect the cell’s own DNA from cleavage

Type I enzymes are multisubunit proteins •that cut DNA randomly at a distance far from their recognition sequence

Type II enzymes cut DNA at defined •positions close to or within their recogni-tion 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 technolo-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 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

QuAlity & innoVAtion

Power and purity of time-saver qualified restriction enzymesHindIII is powerful enough to digest 1 µg of DNA in 5 minutes, but can also be used in overnight digests with no indication of nuclease contamination. Marker (M) is the 1 kb DNA Ladder (NEB #N3232).

Incubation time (minutes) 0 5 15 30 60 0/N M

High Fidelity (HF) enzymesThe latest innovation in restriction enzyme technologyAs part of our ongoing commitment to the study and improvement of restriction enzymes, 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 benefit of reduced star activity. star activity, or relaxed specificity, is an intrinsic property of restriction enzymes. most restriction enzymes will not exhibit star activity when used as recommended. 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.

many techniques such as cloning, genotyping, mutational analysis, mapping, probe preparation, sequencing and methylation detection employ a wide range of reaction conditions and require the use of enzymes under suboptimal conditions. these new products with reduced star activity offer increased flexibility to reaction setup and help maximize results under a broader range of conditions.

Why choose an HF enzyme? in addition to reduced star activity, all of these engineered enzymes work optimally in neBuffer 4, which has the highest level of enzyme compatibility and will simplify double digest reactions. they are all time-saver qualified and will digest substrate DnA in five minutes. to distinguish these engineered enzymes, the letters -HF™ have been added to the restriction enzyme name and they are packaged in unique purple-capped tubes.

mAximum PerFormAnce

PRODuCT NAME

PRODuCT NuMBER BuFFER†

MAxIMuM uNITS WITH NO STAR ACTIVITY*

HF FACTOR

BamHi-HF™ #r3136 4 4,000 125

BamHi #r0136 3 + BsA 32

eagi-HF™ #r3505 4 500 2

eagi #r0505 3 250

ecori-HF™ #r3101 4 16,000 64

ecori #r0101 u 250

ecorV-HF™ #r3195 4 64,000 64

ecorV #r0195 3 + BsA 1,000

mfei-HF™ #r3589 4 500 15

mfei #r0589 4 32

ncoi-HF™ #r3193 4 16,000 133

ncoi #r0193 3 120

nhei-HF™ #r3131 4 + BsA 32,000 266

nhei #r0131 2 + BsA 120

PRODuCT NAME

PRODuCT NuMBER BuFFER†

MAxIMuM uNITS WITH NO STAR ACTIVITY*

HF FACTOR

noti-HF™ #r3189 4 + BsA 64,000 16

noti #r0189 3 + BsA 4,000

Pvuii-HF™ #r3151 4 500 31

Pvuii #r0151 2 16

saci-HF™ #r3156 4 + BsA 4,000 33

saci #r0156 1 + BsA 120

sali-HF™ #r3138 4 2,000 500

sali #r0138 3 + BsA 4

sbfi-HF™ #r3642 4 250 31

sbfi #r0642 4 8

scai-HF™ #r3122 4 250 62

scai #r0122 3 4

sphi-HF™ #r3182 4 2,000 62

sphi #r0182 2 32

sspi-HF™ #r3132 4 500

sspi #r0132 u nD† Wild type enzymes were tested in supplied buffer for comparisons.

* Wei, H. et al (2008) Nucleic Acids Reseach 36, e50.

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.

FAQsQ. Why does the HF version of the enzyme have a different recommended buffer than the wild type enzyme?

A. in many cases, the mutation introduced into the HF enzyme results in significant changes in buffer preference. For example, wild type sali has a strict requirement for neBuffer 3, a high ionic strength buffer. However, sali-HF™ works well in neBuffer 4, which is a moderate ionic strength buffer.

Q. What is the advantage of using NEBuffer 4?

A. All HF enzymes work optimally in neBuffer 4, which has the highest level of enzyme compatibility in the neBuffer system. this can be advantageous when designing double digests.

New England Biolabs is the only company

to engineer restriction enzymes to address the

undesirable effects of star activity

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www.neb.com

mAximum PerFormAnce

Tips for preventing unwanted cleavage in restriction enzyme digestsunder non-standard reaction conditions, some restriction enzymes are capable of cleav-ing sequences that 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 description found in the catalog or 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, so a good rule of thumb is to limit the total amount of enzyme added to 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 strength and pH may contribute to star activity.

Prolonged reaction timeuse the minimum reaction time required for complete digestion. Prolonged incubation may result in increased star activity.

Presence of organic solvents [Dmso, ethanol (2), ethylene glycol, dimethylacetamide, dimethylformamide, sulphalane (3)]

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 perform-ing restriction enzyme digests in smaller reaction volumes, extra caution must be taken to avoid star activity. alternatively, using our new line of HF restriction enzymes allows greater flexibility in reaction setup. Please visit our website to learn about new additions to the HF restriction enzyme product line.

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.

FAQsQ. I would like to digest DNA with EcoRI and Xbal at the same time. The Double Digest Finder recommends a sequential digest, using each enzyme in its supplied NEBuffer. However, both of these enzymes show 100% activity in NEBuffer 2. Why is a double digest not recommended?

A. Although ecori shows 100% activity in neBuffer 2, it also exhibits significant star activity in this buffer. this is observed when using neBuffer 4 as well. For this reason, a sequential digest is recommended. Alternatively, ecori-HF™ could be used, which has 100% activity in neBuffer 4. since xbai is also 100% active in neBuffer 4, a double digest could easily be set up.

Q. Why is BamHI now supplied with NEBuffer 3 rather than a unique buffer? Why is BamHI not recommended for use in NEBuffer 2 or 4, even though it is listed as being 100% active in these buffers?

A. in an effort to simplify our buffer system, BamHi is now supplied with neBuffer 3. BamHi has been carefully purified and characterized so there is no loss of activity in this buffer. BamHi works in most of our buffers but, like ecori, has significant star activity, particularly in buffers with low salt concentrations. therefore, BamHi is not recommended in neBuffers 1, 2 and 4. As an alternative, neB offers BamHi-HF™ which has the same activity as BamHi, but is 100% active in neBuffer 4, which will simplify double digest reactions.

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time-saver Qualified restriction enzymesWhether you are quickly screening large numbers of clones or setting up overnight digests, you will benefit from our high quality enzymes. typically, a restriction digest involves the incubation of 1 µl of enzyme with 1 µg of purified DnA in a final vol-ume of 50 µl for 1 hour. However, to speed up the screening process, choose one of 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 more expensive new lines of enzymes to achieve digestion in 5 minutes. in fact, 63% of our enzymes will digest 1 µg of DnA in 5 minutes, while 83% will fully digest in 15 minutes (see our website for a comprehensive list). that means >199 of our restriction enzymes have the power to get the job done fast.

in an effort to provide as much information as possible, neB has tested all of its enzymes on unit assay substrate as well as plasmid substrate. the rate of cutting for plasmid sub-strate can 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. information on site preferences and cleavage of supercoiled DnA is found in the technical reference section of our catalog and on our website.

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.

ADDitionAl conVenience

ENZYMETIME-SAVER

uNIT ASSAY SuBSTRATE

PLASMID SuBSTRATE

Aatii n s

Acci n s

Acc65i C l s

Acii C l l

Acli C l n

Acui n s

Aflii C l l

Agei C l l

Ahdi C l l

Alui C l s

Alwi C l l

Alwni C l l

Apai C l l

Apali C l l

ApeKi C l n

Apoi C l l

Asci C l l

Asei C l l

Asisi C l s

ENZYMETIME-SAVER


PLASMID SuBSTRATE

Avai C l s

Avaii C l l

Avrii C l s

Baei n l

BamHi C l l

Banii C l n

Bbsi n s

Bbvi C l s

Bbvci C l s

Bcci n s

BceAi n n

BciVi C l n

Bcli C l nt

Bfai n s

BfuAi C l l

Bfuci n s

Bgli C l l

Bglii C l n

Blpi C l l

ENZYMETIME-SAVER


PLASMID SuBSTRATE

Bme1580i n s

BmgBi C l l

Bmri n s

Bpmi n s

BsaAi C l l

BsaBi C l s

BsaHi n n

BsaWi n s

Bsaxi C l s

Bseri C l l

Bsgi C l l

Bsiei C l s

BsiHKAi C l n

BsiWi C l l

Bsli C l n

Bsmi C l l

BsmAi C l s

BsmBi n s

BsmFi C l l

BsoBi C l n

Bsp1286i C l l

Bspcni n s

Bspei C l s

BspHi n l

Bsri C l n

BsrBi C l n

BsrDi C l n

BsrFi C l s

BsrGi n s

BssHii C l s

BssKi n s

BstBi C l l

Bsteii C l l

Bstni C l l

Only NEB can offer enzymes with power and purity – the power to digest in 5 minutes and the purity to withstand overnight digestions with no loss of sample.

www.neb.com 7

ADDitionAl conVenience

ENZYMETIME-SAVER


PLASMID SuBSTRATE

Bstui C l l

Bstxi C l l

Bstyi n l

Bstz17i C l s

Bsu36i n s

Btgi C l l

Btsci l n

Btsi C l l

cac8i n s

clai C l l

cspci C l l

cviAii n l

cviKi-1 n n

cviQi C l l

Ddei C l n

Dpni C l l

Dpnii n s

Drai C l l

Draiii C l l

Drdi n l

eagi C l s

eari n n

econi C l n

ecoo109i C l s

ecoP15i n s

ecori C l l

ecorV C l l

Fnu4Hi C l n

Foki C l l

Fsei C l l

Fspi n s

Haeii n s

Haeiii C l l

Hgai n s

Hhai C l n

Hincii n s

Hindiii C l n

ENZYMETIME-SAVER


PLASMID SuBSTRATE

Hinfi C l l

HinP1i C l s

Hpai n l

Hpaii C l l

Hphi C l s

Hpy188i n s

HpyAV C l l

HpycH4iV C l l

HpycH4V C l l

Kpni C l l

mboi C l s

mboii C l l

mfei C l l

mlui C l l

mlyi C l s

mmei C l l

mnli C l l

msei n n

msli C l l

mspi C l l

mspA1i C l l

mwoi n s

ncii C l l

ncoi C l n

ndei C l l

ngomiV n l

nhei n n

nlaiii n s

noti C l l

nrui C l n

nsii C l l

nspi C l n

Paci C l l

Paer7i C l s

Pflfi C l n

Pflmi C l s

Pmei l n

ENZYMETIME-SAVER


PLASMID SuBSTRATE

Pmli C l s

Ppumi C l s

PshAi n n

Psti C l l

Pvui C l s

Pvuii C l l

rsai n l

saci C l l

sacii C l s

sali C l n

sapi n s

sbfi C l l

scai C l l

scrFi C l s

sfii C l s

sfoi C l l

smai C l n

snaBi C l l

spei C l l

sphi C l l

sspi C l l

stui n s

styi n s

styD4i n s

swai n s

taqi C l l

tfii n l

tsei n s

tsp509i C l l

tspmi C l n

tspri C l n

tth111i n n

xbai C l l

xcmi n l

xhoi C l l

xmai n s

xmni C l l

legend:

l digests in 5 minutes

n digests in 15 minutes

s not completely digested in 15 minutes

C time-saver qualified - will digest substrate DnA in 5 minutes under recommended reaction conditions

nt not tested

Over 150 of our enzymes are Time-Saver qualified

and will digest 1µg of substrate DNA in 5 minutes.

Look for the Time-Saver icon C on our website.

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tecHnicAl tiPs

optimizing restriction enzyme reactions

there are several key factors to consider when setting up a restriction endonuclease digest. using the recommended amounts of DnA, enzyme and buffer components in the correct reaction volume enables 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 following tips will help achieve maximum success in your restriction endonuclease reactions.

EnzymeKeep 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.

DNAshould be free of contaminants such •as phenol, chloroform, alcohol, eDtA, detergents, nucleases or excessive salts

methylation of DnA can inhibit digestion •with certain enzymes

Bufferuse 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 •BsA for optimal activity are not adversely affected if BsA is present in the reaction

Reaction VolumeA 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

Incubation Timecan 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 Reactionif 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 ReactionsFor difficulty cleaving DnA substrate, we recommend the following controls:

experimental DnA without restriction • enzyme to check for contamination in the DnA preparation or reaction buffer

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.

typical restriction enzyme Digest

COMPONENT SPECIFICATION

Restriction Enzyme 10 units is sufficient

DNA 1 µg

10x NEBuffer 5 µl (1x)

BSA Add to a final concentration of 100 µg/ml (1x) if necessary

Total Reaction Volume 50 µl

Incubation Time 1 hour*

Incubation Temperature enzyme Dependent

Reactions in Smaller Volumes

* 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 1 hour to avoid evaporation.

** BSA can be diluted in 1X buffer

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 † REACTION

25 µl REACTION

50 µl REACTION

Restriction Enzyme*

1 unit 5 units 10 units

DNA 0.2 µg 0.5 µg 1 µg

10x NEBuffer 1 µl 2.5 µl 5 µl

100x BSA** 0.1 µl 0.25 µl 0.5 µl

* This can be decreased by using a Time-Saver qualified enzyme.

www.neb.com

FAQsQ. Do restriction enzymes cleave single-stranded 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 unconstrained 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 incuba-tion. incubation time may be shortened if additional units of restriction enzyme are added to the reaction. conversely, longer incubation times are often used to allow a reaction to proceed to completion with fewer units of enzyme. 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.

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trouBlesHootinG

troubleshooting Guide

references

Blakesley, r.W., Wells, r.D. (1975) 1. Nature 257, 421-422.

Blakesley, r.W., et al. (1977) 2. J. Biol. Chem. 252, 7300-7306.

yoo, o.J., Agarwal, K.l, (1980) 3. J. Biol. Chem. 255, 10559-10562.

PROBLEM POSSIBLE CAuSE SOLuTION

INCOMPLETE OR NO DIGESTION

Enzyme is inactivetest enzyme on control DnA with known multiple sites •enzyme should be stored at –20°c. enzymes stored at –70°c will •freeze, and repeated thaw/freeze cycles reduce enzyme activity.

Reaction conditions are not optimal

use recommended buffer supplied with restriction enzyme•Follow double digest recommendations, or try a sequential digest•repeat with fresh buffer. Additives present in buffer •(e.g., Dtt, sAm) may degrade over time.

Enzyme concentration is too low

some plasmids or genomic DnAs may require up to 10-20 units/µg

Additive is missing repeat reaction setup, being sure that enzyme and/or additives (e.g., BsA) are added

DNA concentration is not optimal

neB recommends 1 µg of DnA in a 50 µl reaction. excess DnA may result in incomplete cleavage.

Incubation time is too short

some enzymes can exhibit slower cleavage towards specific sites. in most cases, 1-2 hours are sufficient.

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 is present. miniprep DnA is particularly susceptible to contaminants. clean DnA with a spin column, resin or drop dialysis, •or increase volume to dilute contaminant.

Recognition site is not present

confirm DnA sequence

Cleavage is blocked by methylation

DnA isolated from a bacterial source may be blocked by Dam and Dcm •methylation. DnA should be passed through a dam-/dcm- strain (neB #c2925). eukaryotic genomic DnA may be blocked by cpG methylation. •this can be overcome by cloning into a bacterial host.Pcr products are not methylated.•

DNA may be supercoiled

restriction enzymes cleave supercoiled DnA with varying efficiency. Additional enzyme may be required.

Recognition site may be too close to the end of the DNA fragment

As a general rule, add 6 base pairs on either side of the recognition site for efficient cleavage

Site preference enzyme requires two recognition sites for efficient cleavage

uNExPECTED CLEAVAGE PATTERN

Determine nature of pattern

run uncut substrate DnA alongside the digest. A partial digest will show bands found in the uncut, whereas star activity will show bands of unexpected size.

DNA sample is contaminated

Prepare a new DnA sample

Additional recognition sites are present in DNA

confirm DnA sequence

Star Activitysee tips for avoiding star activity (see page 5) and/or use a High Fidelity restriction enzyme (see page 4)

SMEARING OF DNA ON GEL

Enzyme has a high binding affinity to DNA and will not dissociate well

Add sDs to the gel loading dye/stop solution to a final concentration of 0.1–0.5% to help dissociate the enzyme from the DnA, or treat with protease before loading

Nuclease contamination

care should be taken to avoid cross contamination when setting up reactions

Agarose running conditions use fresh running buffer and appropriate voltage to avoid overheating

10

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.

Double Digestion

tecHnicAl reFerence

Setting up a Double Digestchoose an neBuffer that results in the •most activity for both enzymes, provided that star activity is not a concern.

if BsA is required for either enzyme, •add it to the double digest reaction (BsA does not inhibit restriction enzymes).

set up reaction according to recom-•mended conditions (see p. 8). the final concentration of glycerol in any reaction should be less than 5% to minimize the possibility of star activity (see p. 5). For example, in a 50 µl reaction, the total amount of enzyme added should not exceed 5 µl.

incubate at recommended temperature. •overnight digests should be avoided due to the possibility of star activity.

For help choosing double digest conditions,

try the Double Digest Finder (page 13).

if two different incubation temperatures •are necessary, choose the optimal reac-tion buffer and set up reaction accordingly. Add the first enzyme and incubate at the desired temperature. 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 slower rate of cleavage.

Setting up a Double Digest with a unique buffer

our buffer system has been streamlined, •leaving three enzymes that have unique neBuffers: ecori (included in activity chart), sspi (same buffer composition as ecori) and Dpnii. in most cases, Dpnii requires a sequential digest.

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

AvrII

BamHI

BglII

BsgI

EagI

EcoRI

EcoRV

HindIII

KpnI

MseI

NcoI

NdeI

NheI

NotI

PstI

PvuI

SacI

SacII

SalI

SmaI

SpeI

SphI

XbaI

XhoI

XmaI

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

4 4

3 seq 3

3 seq 2 3

4 4 4 3 3

3 seq 3 3 3 seq

U seq EcoRI EcoRI EcoRI seq EcoRI

3 4 2 3 3 4 3 EcoRI

2 4 2 seq 2 2 seq seq 2

1 4 1 seq 2 seq seq 1 2 2

4 4 4 3 2 4 3 EcoRI 2 2 1

3 4 4 3 3 4 3 EcoRI 3 2 1 4

4 4 4 3 3 4 3 EcoRI 2 2 1 4 4

2 4 2 seq 2 4 seq 1 2 2 1 2 2 4

3 seq 3 3 3 3 3 EcoRI 3 2 2 2 3 3 2

3 4 3 3 3 3 3 EcoRI 3 2 1 3 3 3 2 3

3 seq 2 3 3 3 3 EcoRI 3 2 2 3 3 3 2 3 3

1 4 1 seq 2 4 seq 1 2 2 1 4 1 4 1 2 1 2

4 4 4 seq seq 4 seq EcoRI 2 2 4 4 4 4 4 2 2 2 4

3 seq 3 3 3 3 3 EcoRI 3 seq seq 3 3 3 seq 3 3 3 seq seq

4 4 4 seq seq 4 seq seq 4 4 seq 4 4 4 4 seq 4 seq 4 4 seq

4 4 4 seq 2 4 seq EcoRI 2 2 1 4 4 4 2 2 2 2 1 4 seq 4

2 4 2 3 2 4 3 EcoRI 2 2 1 2 2 2 2 2 2 2 1 4 3 4 2

4 4 4 3 2 4 3 seq 2 2 2 4 4 4 2 3 3 3 4 4 3 4 4 2

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

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

Double Digest chart

enzymes in purple are also available in a High Fidelity (HF) format. HF enzymes are 100% active in neBuffer 4 and will simplify double digest reactions.

Additional double digest information with unique buffers can be found on our website, www.neb.com.

Setting up a Sequential Digestset 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 approxi-mate 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.

www.neb.com 11

tecHnicAl reFerence

DnA methylation & restriction Digeststwo common types of methylation that can block cutting at a restriction site are Dam and Dcm methylation. Both arise from replicating DnA in a strain of E. coli that has functional Dam and Dcm methylation systems.

Site Blocked by Dam/Dcm Methylationrestriction by some enzymes can be inhibited due to methylation caused by the common E. coli methyltransferases. Dam methyltransferase causes methylation of the adenine in the sequence GAtc while Dcm methyltransferase causes methylation of the first cytosine in the sequence cc(A/t)GG. Any restriction enzyme whose site contains either of these sequences may be affected by the relevant methylation. For example, the site for Alwi (GGAtc4/5) contains the recognition site for Dam methyltransferase, GAtc. if the DnA is produced in a methylating E. coli strain, the adenine is methylated and cleavage by Alwi 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 dam–/dcm– competent E. coli (neB #c2925).

on the other hand BamHi is not Dam/Dcm sensitive; the BamHi site contains GAtc but cleavage by this enzyme is not blocked even when the adenine is methylated. the same principles apply for Dcm methylation but the enzyme sites affected would contain the sequence cc(A/t)GG.

dam/dcm Overlapping Sitesrestriction 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. For example, the site for clai (At/cGAt) contains GAt. if it is followed by a c, the A can be methylated, and cleavage will be blocked.

more information regarding methylation sensitivity can be found in the technical reference section of the neB catalog & technical reference, or www.neb.com.

Key Points to Consider: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.

Dam/Dcm Sensitive Restriction Enzymes available from NEBthe following enzymes are blocked or impaired by Dam or Dcm methylation. in order to achieve cleavage with these enzymes, the DnA should be passed through a dam–/dcm– strain.

Methylation sensitivity information can

also be found on REBASE (Restriction Enzyme

Database), a comprehensive database of

information about restriction enzymes and

related proteins. For more information, see

page 13.

Acc651 BspHi Pflmi

Alwi BssKi Phoi

Alwni Bstxi Ppumi

Apai clai PspGi

Avaii Dpnii Pspomi

Bani eaei sau96i

Bcgi ecoo1091 scrFi

Bc1i Hphi sexAi

Bsai Hpy188i sfii

BsaBi Hpy1881ii sfoi

BsaHi mbol stui

Bs1i mboii styD41

BsmFi msci taqia

BspDi nlaiV xbai

Bspei nrul

WeB tools

online tools

NEBcutterneBcutter 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

Calculate GC Content

Zoom in to view cleavage overhangs

Identify enzymes affected by methylation

Introduce silent mutation sites into ORF sequence with a single mouse click

Generate a custom digest and create a virtual gel by choosing appropriate markers and gel types

Summarize ORF sequencesChoose linear or circular maps

Expand region of interest to obtain sequence information

List enzymes by number of sites produced, alphabetically or by cut frequency

Submit your sequence to find largest ORF, identify restriction sites and generate custom digests

Enzyme Finderenzyme 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

As the leader in enzyme technology, neB’s technical support is an invaluable resource. our scientists provide assistance via [email protected] 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.

12

www.neb.com

WeB tools

Double Digest Finderselect optimal conditions for double digest including buffer, incubation, temperature and supplement requirements.

www.neb.com/nebecomm/DoubleDigestCalculator.asp

REBASEreBAse (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/

Summary of enzyme source, status and properties

Lists available suppliers

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

Access available tools (theoretical digests, BLAST, NEBcutter, REBpredictor)

Click here for specialized infor-mation (star activity, methylation, cleavage type, etc.), REBASE genomes and sequence collections

View a complete list of enzymes in the database including recognition sequences, isoschizomers and commercial suppliers

Contains detailed methylation sensitivity testing data

Lists all references containing this enzyme, with links to reference details

13

Additional web-based tools can be found on

our website, www.neb.com.

14

tecHnicAl reFerence

cleavage close to the ends of DnA Fragmentsto 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.

ENZYMEBASE 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

ENZYMEBASE PAIRS FROM END

% CLEAVAGE EFFICIENCY VECTOR

INITIAL CuT

mlui 2 99 litmus 39 eagi

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.

The technical reference section of our website

contains additional charts, protocols and

technical tips related to restriction enzymes.

www.neb.com

cleavage close to the ends of DnA Fragments Activity chart for restriction enzymesnew england Biolabs utilizes a simple 4 buffer system. A color-coded 10x neBuffer is supplied with every restriction endonuclease, ensuring 100% activity. to help select the best conditions for double digests, this chart shows the optimal (supplied) neBuffer and approximate activity in the four standard neBuffers and neBuffer ecori for each enzyme. 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.

Buffers should be thawed completely and mixed thoroughly before using.

neBuffer compositions (1x)

NEBuffer 1 (yellow)

10 mm Bis tris Propane-Hcl, 10 mm mgcl

2, 1 mm Dtt

(pH 7.0 @ 25°c).

NEBuffer 2 (blue)

10 mm tris-Hcl, 10 mm mgcl

2, 50 mm nacl,

1 mm Dtt (pH 7.9 @ 25°c).

NEBuffer 3 (red)


2, 100 mm nacl,

1 mm Dtt (pH 7.9 @ 25°c).

NEBuffer 4 (green)

20 mm tris-acetate, 10 mm magnesium acetate, 50 mm potassium acetate, 1 mm Dtt (pH 7.9 @ 25°c).

NEBufferEcoRI

50 mm naci, 100 mm tris-Hci, 10 mm mgci

2,

0.025% triton x-100 (pH 7.5 @ 25°c).

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.

BSA

supplied with a separate vial of bovine serum albumin (10 mg/ml). to obtain 100% activity, BsA should be added to the 1x reaction mix to a final concentration of 100 µg/ml.

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.

ddthis neBuffer is recommended for a double digest because it minimizes star activity.

NR this buffer is not recommended for use with this enzyme.

r Produced from a recombinant source.

C

time-saver qualified (digests 1 µg of substrate DnA in 5 minutes under recommended reaction conditions).

Eengineered enzyme for maximum performance.

enzymesuPPlieD neBuffer

% ActiVity in neBuffers 1 2 3 4 ecori

HeAt inActiVAtion(temP)

incuBAtion temPerAture

r Aatii 4 0 50 50 100 25 65° 37°

r Acci 4 50 50 10 100 5 80° 37°

r C Acc65i 3 + BsA 10 75 100 25 50 65° 37°

C Acii 3 25 50 100 50 100 65° 37°

r C Acli 4 + BsA 10 10 0 100 5 no 37°

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°

r Afliii 3 + BsA 25 75 100 50 100 80° 37°

r C Agei 1 100 50 10 75 50 65° 37°

r C Ahdi 4 + BsA 25 75 0 100 10 65° 37°

r Alei 4 10 25 10 100 0 65° 37°

r C Alui 4 100 100 75 100 100 65° 37°

r C Alwi 4 50 100 10 100 5 65° 37°

C Alwni 4 50 100 50 100 10 65° 37°

r C Apai 4 + BsA 25 50 0 100 5 65° 25°

r C Apali 4 + BsA 100 100 10 100 5 no 37°

r C ApeKi 3 25 75 100 50 100 no 75°

r C Apoi 3 + BsA 10 75 100 75 100 80° 50°

r C Asci 4 0 10 10 100 5 65° 37°

r C Asei 3 nr 75 dd100 nr 50 65° 37°

r C Asisi 4 + BsA 50 100 100 100 0 80° 37°

r C Avai 4 10 75 10 100 50 80° 37°

r C Avaii 4 50 75 10 100 50 65° 37°

r C Avrii 4 100 100 50 100 50 80° 37°

Baei 4 + BsA + sAm 50 100 50 100 100 65° 25°

C BaeGi 1 100 75 10 50 100 65° 37°

r C BamHi 3 + BsA 75 100 dd100 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.

Activity Chart HighlightsOver 160 restriction enzymes exhibit 100% activity in a single buffer (NEBuffer 4)•

Over 150 enzymes are Time-Saver qualified and will digest substrate DNA in 5 minutes•

A selection of High Fidelity (HF) enzymes are available that have been engineered for •reduced star activity

selection

15

16

selection

New England Biolabs offers

over 220 enzyme specificities.





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 dd100 nr 50 65° 37°

r C BciVi 4 100 50 0 100 25 65° 37°

r C Bcli 3 50 100 dd100 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°

C Bsri 3 0 50 100 10 100 80° 65°

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 dd100 100 100 80° 50°

r BssKi 3 + BsA 0 50 100 50 100 80° 60°

r Bsssi 3 0 50 dd100 10 100 80° 37°

r BstAPi 4 25 100 100 100 100 80° 60°

www.neb.com 17

selection



NEBuffer 1 (yellow)


2, 1 mm Dtt

(pH 7.0 @ 25°c).

NEBuffer 2 (blue)


2, 50 mm nacl,

1 mm Dtt (pH 7.9 @ 25°c).

NEBuffer 3 (red)


2, 100 mm nacl,

1 mm Dtt (pH 7.9 @ 25°c).

NEBuffer 4 (green)


NEBufferEcoRI


2,

0.025% triton x-100 (pH 7.5 @ 25°c).

chart legend

u


BSA


SAM





C







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°

C Bstui 4 100 100 50 100 100 no 60°

r C Bstxi 3 0 50 100 25 100 65° 37°

r Bstyi 2 50 100 75 100 50 80° 60°

C Bstz17i 4 nr nr 100 100 100 no 37°

r Bsu36i 3 + BsA 0 25 100 0 15 80° 37°

r C Btgi 3 + BsA 25 50 100 100 100 80° 37°Btgzi 4 + BsA 10 25 0 100 100 80° 60°

r C Btsi 4 + BsA 100 50 50 100 25 80° 55°

r C Btsci 4 50 100 50 100 25 no 50°cac8i 4 50 75 100 100 50 65° 37°

r C clai 4 + BsA 10 50 50 100 50 65° 37°

r C cspci 4 + sAm 10 100 10 100 40 65° 37°

r cviAii 4 75 25 10 100 100 65° 25°

r cviKi-1 4 10 50 25 100 100 80° 37°

r C cviQi 3 + BsA 75 100 100 75 0 no 25°

r C Ddei 3 75 100 100 75 100 65° 37°

r C Dpni 4 100 100 75 100 100 80° 37°

r Dpnii u nr nr dd100 nr 100 65° 37°

r C Drai 4 75 75 50 100 100 65° 37°

r C Draiii 3 + BsA 100 75 100 25 50 65° 37°Drdi 4 25 50 10 100 100 65° 37°

r eaei 4 100 100 50 100 100 80° 37°

r C eagi 3 10 25 100 10 100 65° 37°

r C E Eagi-HF 4 25 50 10 100 100 65° 37°

r eari 4 100 100 50 100 100 65° 37°ecii 2 + BsA 50 100 100 50 100 65° 37°

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°

r C ecori udd100 100 100 100 100 65° 37°

r C E EcoRi-HF 4 10 100 0 100 5 65° 37°

r C ecorV 3 + BsA 50 75 100 50 50 80° 37°

r C E EcoRV-HF 4 25 100 100 100 100 65° 37°

r Fati 2 10 100 50 50 100 65° 55°

r Faui 4 100 50 10 100 50 65° 55°

r C Fnu4Hi 4 10 25 25 100 10 65° 37°

r C Foki 4 100 100 75 100 25 65° 37°

r C Fsei 4 + BsA 100 75 0 100 50 65° 37°

r Fspi 4 10 75 10 100 50 65° 37°

r Haeii 4 + BsA 75 100 50 100 25 80° 37°

r C Haeiii 4 50 100 25 100 100 80° 37°

r Hgai 1 100 75 50 100 100 65° 37°

r C Hhai 4 + BsA 75 100 100 100 100 65° 37°

r Hincii 3 + BsA 75 100 100 100 100 65° 37°

r C Hindiii 2 50 100 10 50 25 65° 37°

r C Hinfi 4 75 100 75 100 100 80° 37°

r C HinP1i 4 100 100 100 100 100 65° 37°

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°

18

selection

New England Biolabs offers

over 170 recombinant enzymes.





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 dd100 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°

r nt.cviPii 4 25 100 50 100 n/A 65° 37°

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°

www.neb.com 19

selection



NEBuffer 1 (yellow)


2, 1 mm Dtt

(pH 7.0 @ 25°c).

NEBuffer 2 (blue)


2, 50 mm nacl,

1 mm Dtt (pH 7.9 @ 25°c).

NEBuffer 3 (red)


2, 100 mm nacl,

1 mm Dtt (pH 7.9 @ 25°c).

NEBuffer 4 (green)


NEBufferEcoRI


2,

0.025% triton x-100 (pH 7.5 @ 25°c).

chart legend

u


BSA


SAM





C







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°

r Pspxi 4 0 100 10 100 100 80° 37°

r C Psti 3 + BsA 75 75 100 50 50 80° 37°

C Pvui 3 + BsA 10 75 100 10 100 80° 37°

r C Pvuii 2 100 100 100 100 100 no 37°

r C E pvuii-HF 4 0 25 0 100 0 80° 37°

r C rsai 4 100 100 50 100 15 65° 37°

r rsrii 4 25 75 10 100 25 65° 37°

r C saci 1 + BsA 100 50 10 100 15 65° 37°

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°

r C sali 3 + BsA 0 0 100 0 50 65° 37°

r C E Sali-HF 4 10 100 100 100 100 65° 37°

r sapi 4 75 50 0 100 50 65° 37°

r sau3Ai 1 + BsA 100 50 10 100 100 65° 37°

r sau96i 4 50 100 100 100 50 80° 37°

r C sbfi 4 75 50 0 100 25 65° 37°

r C E Sbfi-HF 4 50 25 0 100 0 65° 37°

r C scai 3 nr nr dd100 nr 50 80° 37°

r C E Scai-HF 4 100 100 10 100 15 65° 37°

C scrFi 4 100 100 100 100 100 65° 37°

r sexAi 4 + BsA 100 75 50 100 50 65° 37°

r sfani 3 0 75 100 25 50 65° 37°

r sfci 4 + BsA 75 50 10 100 25 65° 37°

r C sfii 4 + BsA 0 100 10 100 15 no 50°

r C sfoi 4 25 100 50 100 100 no 37°

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°

r C snaBi 4 + BsA 25 50 25 dd100 5 80° 37°

r C spei 4 + BsA 75 100 25 100 50 80° 37°

r C sphi 2 100 100 50 100 50 65° 37°

r C E Sphi-HF 4 50 25 10 100 0 65° 37°

r C sspi u 50 100 50 50 100 65° 37°

r C E Sspi-HF 4 25 100 0 100 25 65° 37°

r C stui 4 100 100 50 100 50 65° 37°

r styD4i 2 10 100 100 100 20 65° 37°

r styi 3 + BsA 25 75 100 10 15 65° 37°

r swai 3 + BsA 10 10 100 10 15 65° 25°

r C taqi 4 + BsA 50 75 100 100 100 80° 65°

r tfii 4 100 100 dd100 100 100 no 65°

r tlii 3 + BsA 25 25 100 10 50 no 75°

tsei 4 75 100 100 100 100 no 65°

tsp45i 1 + BsA 100 25 0 75 50 no 65°

r C tsp509i 1 100 100 100 nr 100 no 65°

C tspmi 4 50 75 50 100 20 no 75°

r C tspri 4 + BsA 25 50 25 100 5 no 65°

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°

Canada

new england Biolabs, ltd.

toll Free: 1-800-387-1095

[email protected]

China, people’s Republic

new england Biolabs (Beijing), ltd.

telephone: 010-82378265/82378266

[email protected]

germany

new england Biolabs GmbH

Free call: 0800/246 5227

[email protected]

United Kingdom

new england Biolabs (uK), ltd.

call Free 0800 318486

[email protected]

Japan

new england Biolabs Japan, inc.

telephone: +81 (0)3 5669 6191

[email protected]

www.neb.com

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BOSTON, MA

PERMIT NO. 54302

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240 county road

ipswich, mA 01938-2723

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