RESTRICTION Technical Guide 2009|10 restriction enzymes FROM NEB

For over 35 years, New England Biolabs has led the industry in Cloning & Mapping innovation. As the first company to commercialize restriction enzymes, we are DNA AMPLIFICATION & PCR committed to providing a wide selection of high quality reagents at exceptional value. Our dedication to research has led to key advances in the restriction enzyme field RNA ANALYSIS that allow us to offer maximum performance and convenience under a broader range Protein Expression & Analysis of reaction conditions. Gene Expression & Cellular Analysis Selection In 1975, New England Biolabs (NEB) became the first company to commercialize Restriction Enzymes – restriction enzymes, thereby providing researchers with a key set of tools that proved to be Inside the Numbers* invaluable for the early development of the biotechnology industry. Since then, NEB 2 31 – restriction enzymes sold by 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 222 – enzyme specificities analysis of sequenced genomes. As a result, we now supply more than 220 specificities, 173 – recombinant enzymes the largest number commercially available. 162 – enzymes exhibiting 100% activity in a single buffer (NEBuffer 4) 15 3 – Time-Saver enzymes that digest substrate DNA in Quality 5 minutes – High Fidelity (HF) enzymes As a result of our active screening and cloning program, NEB supplies the largest 15 engineered for reduced selection of recombinant enzymes available. Expression using a recombinant source star activity improves product yield and removes the uncertainties of potential contaminants. Each 10 – 8-base cutters enzyme is carefully quality controlled using a number of sensitive assays that specifically – nicking enzymes that cleave address nuclease contamination. This results in a higher quality product with proven 10 only one strand of duplex DNA lot-to-lot consistency. 4 – homing endonucleases that r Choose recombinant enzymes for a higher quality product with lot-to-lot consistency. have large asymmetric recognition sites 52 – Type IIS enzymes

* as of 03/26/09 Performance High Fidelity enzymes offer reduced star activity

Cloning and sequencing restriction enzymes has enabled EcoRI-HF our researchers to enhance screening efforts as well as NEB Supplier A Supplier B alter enzyme properties through engineering. This has 10 30 50 100 M 10 30 50 100 M 10 30 50 100 led to the discovery of nicking enzymes, the generation of new specificities, and most recently the introduction of a new line of High Fidelity (HF) enzymes. These engi- neered enzymes have the same specificity as their estab- lished counterparts, and will maximize performance under a wider range of conditions, including reaction volume, incubation time and buffer compatibility.

E High Fidelity enzymes have been engineered for maximum performance. EcoRI from other suppliers produces the correct banding pattern when 10 units are used; however, star activity is observed with larger amounts of enzyme. Star activity is not observed with EcoRI-HF™, even at higher enzyme amounts. Reactions were set up according to recommended reaction conditions of each manufacturer. Reactions contained 1 µg Lambda DNA in a 50 µl volume and were incubated overnight at 37°C. Marker M is the 1 kb DNA Ladder (NEB #N3232). 2 Quality & Innovation

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

www.neb.com 3 maximum performance

New England Biolabs is the only company to engineer restriction enzymes to address the High Fidelity (HF) Enzymes undesirable effects of star activity The latest innovation in restriction enzyme technology As part of our ongoing commitment to the study and improvement of restriction enzymes, FAQs NEB is pleased to introduce a line of High Fidelity (HF) restriction enzymes. These engineered enzymes have the same specificity as their established counterparts with the Q. Why does the HF version of the enzyme benefit of reduced star activity. Star activity, or relaxed specificity, is an intrinsic property have a different recommended buffer than of restriction enzymes. Most restriction enzymes will not exhibit star activity when used as the wild type enzyme? recommended. However, for enzymes that have reported star activity, extra caution must A. In many cases, the mutation introduced be taken to set up reactions according to the recommended conditions to avoid unwanted into the HF enzyme results in significant cleavage. changes in buffer preference. For example, wild type SalI has a strict requirement for Many techniques such as cloning, genotyping, mutational analysis, mapping, probe NEBuffer 3, a high ionic strength buffer. preparation, sequencing and methylation detection employ a wide range of reaction However, SalI-HF™ works well in NEBuffer conditions and require the use of enzymes under suboptimal conditions. These new 4, which is a moderate ionic strength buffer. products with reduced star activity offer increased flexibility to reaction setup and help maximize results under a broader range of conditions. Q. What is the advantage of using NEBuffer 4? Why choose an HF enzyme? A. All HF enzymes work optimally In addition to reduced star activity, all of these engineered enzymes work optimally in in NEBuffer 4, which has the highest level NEBuffer 4, which has the highest level of enzyme compatibility and will simplify double of enzyme compatibility in the NEBuffer digest reactions. They are all Time-Saver qualified and will digest substrate DNA in five system. This can be advantageous when minutes. To distinguish these engineered enzymes, the letters -HF™ have been added to designing double digests. the restriction enzyme name and they are packaged in unique purple-capped tubes.

The following table indicates the number of units of HF-enzyme that can be used compared to the wild type counterparts before significant star activity is detected. The HF Factor refers to the x-fold increase that is achieved by choosing an HF enzyme, and clearly illustrates the flexibility that is offered by using an HF restriction enzyme.

Maximum Maximum Product Product Units with no HF Product Product Units with no HF Name Number Buffer† Star Activity* factor Name Number Buffer† Star Activity* factor BamHI-HF™ #R3136 4 4,000 125 NotI-HF™ #R3189 4 + BSA 64,000 16 BamHI #R0136 3 + BSA 32 NotI #R0189 3 + BSA 4,000 EagI-HF™ #R3505 4 500 2 PvuII-HF™ #R3151 4 500 31 EagI #R0505 3 250 PvuII #R0151 2 16 EcoRI-HF™ #R3101 4 16,000 64 SacI-HF™ #R3156 4 + BSA 4,000 33 EcoRI #R0101 U 250 SacI #R0156 1 + BSA 120 EcoRV-HF™ #R3195 4 64,000 64 SalI-HF™ #R3138 4 2,000 500 EcoRV #R0195 3 + BSA 1,000 SalI #R0138 3 + BSA 4 MfeI-HF™ #R3589 4 500 15 SbfI-HF™ #R3642 4 250 31 MfeI #R0589 4 32 SbfI #R0642 4 8 NcoI-HF™ #R3193 4 16,000 133 ScaI-HF™ #R3122 4 250 62 NcoI #R0193 3 120 ScaI #R0122 3 4 NheI-HF™ #R3131 4 + BSA 32,000 266 SphI-HF™ #R3182 4 2,000 62 NheI #R0131 2 + BSA 120 SphI #R0182 2 32 SspI-HF™ #R3132 4 500 † Wild type enzymes were tested in supplied buffer for comparisons. SspI #R0132 U ND * Wei, H. et al (2008) Nucleic Acids Reseach 36, e50.

4 maximum performance

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

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

www.neb.com 5 additional convenience

Time-Saver Qualified Restriction Enzymes

Whether you are quickly screening large numbers of clones or setting up overnight Only NEB can offer enzymes with power and digests, you will benefit from our high quality enzymes. Typically, a restriction digest purity – the power to digest in 5 minutes and the involves the incubation of 1 µl of enzyme with 1 µg of purified DNA in a final vol- purity to withstand overnight digestions with no ume of 50 µl for 1 hour. However, to speed up the screening process, choose one of loss of sample. NEB’s enzymes that are Time-Saver qualified. These enzymes will digest 1 µg of DNA in 5 minutes using 1 µl of enzyme under recommended reaction conditions. Unlike other suppliers, there is no special formulation, change in concentration or need to buy TIME- UNIT ASSAY PLASMID more expensive new lines of enzymes to achieve digestion in 5 minutes. In fact, 63% of ENZYME SAVER SUBSTRATE SUBSTRATE our enzymes will digest 1 µg of DNA in 5 minutes, while 83% will fully digest in 15 Bme1580I n s minutes (see our website for a comprehensive list). That means >199 of our restriction BmgBI C l l enzymes have the power to get the job done fast. BmrI n s In an effort to provide as much information as possible, NEB has tested all of its enzymes BpmI n s on unit assay substrate as well as plasmid substrate. The rate of cutting for plasmid sub- BsaAI C l l strate can be used as a guide as it is not definitive for all plasmids. Restriction enzymes BsaBI C l s can often show site preference, presumably determined by the sequence flanking the BsaHI n n

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

Information on site preferences and cleavage of supercoiled DNA is found in the BsaXI C l s technical reference section of our catalog and on our website. BseRI C l l Since all of our enzymes are rigorously tested for nuclease contamination, you can also BsgI C l l safely set up digests for long periods of time without sample degradation. BsiEI C l s BsiHKAI C l n BsiWI C l l TIME- UNIT ASSAY PLASMID TIME- UNIT ASSAY PLASMID ENZYME SAVER SUBSTRATE SUBSTRATE ENZYME SAVER SUBSTRATE SUBSTRATE BslI C l n AatII n s AvaI C l s BsmI C l l AccI n s AvaII C l l BsmAI C l s

Acc65I C l s AvrII C l s BsmBI n s

AciI C l l BaeI n l BsmFI C l l

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

AcuI n s BanII C l n Bsp1286I C l l

AflII C l l BbsI n s BspCNI n s

AgeI C l l BbvI C l s BspEI C l s AhdI C l l BbvCI C l s BspHI n l

AluI C l s BccI n s BsrI C l n

AlwI C l l BceAI n n BsrBI C l n

AlwNI C l l BciVI C l n BsrDI C l n ApaI C l l BclI C l NT BsrFI C l s

ApaLI C l l BfaI n s BsrGI n s

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

ApoI C l l BfuCI n s BssKI n s AscI C l l BglI C l l BstBI C l l

AseI C l l BglII C l n BstEII C l l AsiSI C l s BlpI C l l BstNI C l l

6 additional convenience

Legend: l digests in 5 minutes Over 150 of our enzymes are Time-Saver qualified

n digests in 15 minutes and will digest 1µg of substrate DNA in 5 minutes.

s not completely digested in 15 minutes Look for the Time-Saver icon C on our website.

C Time-Saver qualified - will digest substrate DNA in 5 minutes under recommended reaction conditions

NT not tested

TIME- UNIT ASSAY PLASMID TIME- UNIT ASSAY PLASMID TIME- UNIT ASSAY PLASMID ENZYME SAVER SUBSTRATE SUBSTRATE ENZYME SAVER SUBSTRATE SUBSTRATE ENZYME SAVER SUBSTRATE SUBSTRATE BstUI C l l HinfI C l l PmlI C l s BstXI C l l HinP1I C l s PpuMI C l s

BstYI n l HpaI n l PshAI n n

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

Bsu36I n s HphI C l s PvuI C l s

BtgI C l l Hpy188I n s PvuII C l l

BtsCI l n HpyAV C l l RsaI n l BtsI C l l HpyCH4IV C l l SacI C l l Cac8I n s HpyCH4V C l l SacII C l s ClaI C l l KpnI C l l SalI C l n CspCI C l l MboI C l s SapI n s CviAII n l MboII C l l SbfI C l l CviKI-1 n n MfeI C l l ScaI C l l CviQI C l l MluI C l l ScrFI C l s DdeI C l n MlyI C l s SfiI C l s DpnI C l l MmeI C l l SfoI C l l DpnII n s MnlI C l l SmaI C l n

DraI C l l MseI n n SnaBI C l l DraIII C l l MslI C l l SpeI C l l DrdI n l MspI C l l SphI C l l EagI C l s MspA1I C l l SspI C l l

EarI n n MwoI n s StuI n s

EcoNI C l n NciI C l l StyI n s

EcoO109I C l s NcoI C l n StyD4I n s EcoP15I n s NdeI C l l SwaI n s

EcoRI C l l NgoMIV n l TaqI C l l

EcoRV C l l NheI n n TfiI n l

Fnu4HI C l n NlaIII n s TseI n s FokI C l l NotI C l l Tsp509I C l l

FseI C l l NruI C l n TspMI C l n FspI n s NsiI C l l TspRI C l n

HaeII n s NspI C l n Tth111I n n HaeIII C l l PacI C l l XbaI C l l HgaI n s PaeR7I C l s XcmI n l

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

HincII n s PflMI C l s XmaI n s

HindIII C l n PmeI l n XmnI C l l

www.neb.com 7 technical tips

Optimizing Restriction Enzyme Reactions

There are several key factors to consider when setting up a restriction endonuclease Typical Restriction Enzyme Digest

digest. Using the recommended amounts of DNA, enzyme and buffer components in COMPONENT SPECIFICATION the correct reaction volume enables optimal digestion. By definition, 1 unit of restriction Restriction Enzyme 10 units is sufficient

enzyme will completely digest 1 µg of substrate DNA in a 50 µl reaction in 60 minutes. DNA 1 µg

This enzyme : DNA : reaction volume ratio can be used as a guide when designing 10X NEBuffer 5 µl (1X) reactions. However, most researchers follow the “typical” reaction conditions listed, Add to a final concentration of BSA where a 5-10-fold overdigestion is recommended to overcome variability in DNA source, 100 µg/ml (1X) if necessary

quantity and purity. The following tips will help achieve maximum success in your Total Reaction Volume 50 µl

restriction endonuclease reactions. Incubation Time 1 hour*

Incubation Temperature Enzyme Dependent Enzyme Incubation Time * This can be decreased by using a Time-Saver qualified enzyme. • Keep on ice when not in the freezer • Can often be decreased by using an excess of enzyme, or by using one of our Should be the last component added to • Time-Saver qualified enzymes (see page 6) Reactions in Smaller Volumes reaction • With many enzymes, it is possible to use Many techniques such as cloning, Mix components after addition of enzyme • fewer units and digest for up to 16 hours. genotyping, mutational analysis, mapping, by pipetting the reaction mixture up and For more information, visit www.neb.com. probe preparation, sequencing and down, or by “flicking” the reaction tube. methylation detection require the use Follow with a quick (“touch”) spin-down Stopping a Reaction of enzymes under suboptimal conditions. in a microcentrifuge. Do not vortex the Additives in the restriction enzyme reaction. If no further manipulation of DNA is storage buffer (e.g., glycerol, salt) as well required: as contaminants found in the substrate DNA • Terminate with a stop solution [50% solution (e.g., salt, EDTA, or alcohol) can • Should be free of contaminants such glycerol, 50 mM EDTA (pH 8.0), and be problematic in smaller reaction volumes. as phenol, chloroform, alcohol, EDTA, 0.05% bromophenol blue]. se 10 µl NEB has introduced a line of High Fidelity detergents, nucleases or excessive salts per 50 µl reaction. (HF) enzymes that provide added flexibility to reaction setup. For more information on • Methylation of DNA can inhibit digestion When further manipulation of DNA is required: HF enzymes, see page 4. The following with certain enzymes guidelines can be used for techniques that Heat inactivation can be used (buffer • require smaller reaction volumes. Buffer chart indicates if the enzyme can be heat inactivated) • Use at a 1X concentration Restriction digests in • If required, add BSA to a final concentra- • Remove enzyme by using a spin column smaller reaction volumes tion of 100 µg/ml (1:100 dilution) or phenol/chloroform extraction 10 µl † 25 µl 50 µl • Restriction enzymes that do not require Control Reactions REACTION REACTION REACTION Restriction BSA for optimal activity are not adversely 1 unit 5 units 10 units For difficulty cleaving DNA substrate, Enzyme* affected if BSA is present in the reaction we recommend the following controls: DNA 0.2 µg 0.5 µg 1 µg • Experimental DNA without restriction Reaction Volume 10X 1 µl 2.5 µl 5 µl enzyme to check for contamination in the NEBuffer • A 50 µl reaction volume is recommended DNA preparation or reaction buffer for digestion of 1 µg of substrate 100X BSA** 0.1 µl 0.25 µl 0.5 µl • Control DNA (DNA with multiple known • Keep glycerol concentration at less than sites for the enzyme) with restriction 5% of total reaction volume to prevent star * Restriction enzyme can be diluted using the recommended diluent enzyme to test enzyme viability buffer when smaller amounts are needed. activity † 10 µl reactions should not be incubated for longer than • If the control DNA is cleaved and the 1 hour to avoid evaporation. • The restriction enzyme (supplied in 50% experimental DNA resists cleavage, the ** BSA can be diluted in 1X buffer glycerol) should not exceed 10% of the two can be mixed to determine if total reaction volume 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. 8 troubleshooting

Troubleshooting Guide

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

www.neb.com 9 technical reference

For help choosing double digest conditions, Double Digestion try the Double Digest Finder (page 13).

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

Setting up a Double Digest • If two different incubation temperatures Additional double digest information with are necessary, choose the optimal reac- unique buffers can be found on our website, • Choose an NEBuffer that results in the tion buffer and set up reaction accordingly. www.neb.com. most activity for both enzymes, provided Add the first enzyme and incubate at the that star activity is not a concern. desired temperature. Heat inactivate the first Setting up a Sequential Digest • If BSA is required for either enzyme, enzyme, add the second enzyme and • Set up a reaction using the restriction add it to the double digest reaction (BSA incubate at the recommended temperature. endonuclease that has the lowest salt does not inhibit restriction enzymes). concentration in its recommended buffer • Depending on an enzyme’s activity rating • Set up reaction according to recom- and incubate to completion. in a non-optimal NEBuffer, the number of mended conditions (see p. 8). The final units or incubation time may be adjusted • Adjust the salt concentration of the concentration of glycerol in any reaction to compensate for slower rate of cleavage. reaction (using a small volume of a should be less than 5% to minimize the concentrated salt solution) to approxi- possibility of star activity (see p. 5). Setting up a Double Digest mate the reaction conditions of the For example, in a 50 µl reaction, the second restriction endonuclease. total amount of enzyme added should with a unique buffer • Add the second enzyme and incubate not exceed 5 µl. • Our buffer system has been streamlined, to complete the second reaction. • Incubate at recommended temperature. leaving three enzymes that have unique • Alternatively, a spin column can be used NEBuffers: EcoRI (included in activity Overnight digests should be avoided due to isolate the DNA prior to the second chart), SspI (same buffer composition as to the possibility of star activity. reaction. EcoRI) and DpnII. In most cases, DpnII requires a sequential digest. Double Digest Chart

ENZYME AatII AvrII BamHI BglII BsgI EagI EcoRI EcoRV HindIII KpnI MseI NcoI NdeI NheI NotI PstI PvuI SacI SacII SalI SmaI SpeI SphI XbaI XhoI NEBuffer 4 4 3 3 4 3 U 3 2 1 4 3 4 2 3 3 3 1 4 3 4 4 2 4 4 AvrII 4 4 BamHI 3 seq 3 BglII 3 seq 2 3 BsgI 4 4 4 3 3 Enzymes in purple are also available in EagI 3 seq 3 3 3 seq a High Fidelity (HF) format. HF enzymes EcoRI U seq EcoRI EcoRI EcoRI seq EcoRI are 100% active in NEBuffer 4 and will EcoRV 3 4 2 3 3 4 3 EcoRI simplify double digest reactions. HindIII 2 4 2 seq 2 2 seq seq 2 KpnI 1 4 1 seq 2 seq seq 1 2 2 MseI 4 4 4 3 2 4 3 EcoRI 2 2 1 NcoI 3 4 4 3 3 4 3 EcoRI 3 2 1 4 NdeI 4 4 4 3 3 4 3 EcoRI 2 2 1 4 4 NheI 2 4 2 seq 2 4 seq 1 2 2 1 2 2 4 NotI 3 seq 3 3 3 3 3 EcoRI 3 2 2 2 3 3 2 PstI 3 4 3 3 3 3 3 EcoRI 3 2 1 3 3 3 2 3 PvuI 3 seq 2 3 3 3 3 EcoRI 3 2 2 3 3 3 2 3 3 SacI 1 4 1 seq 2 4 seq 1 2 2 1 4 1 4 1 2 1 2 SacII 4 4 4 seq seq 4 seq EcoRI 2 2 4 4 4 4 4 2 2 2 4 SalI 3 seq 3 3 3 3 3 EcoRI 3 seq seq 3 3 3 seq 3 3 3 seq seq SmaI 4 4 4 seq seq 4 seq seq 4 4 seq 4 4 4 4 seq 4 seq 4 4 seq SpeI 4 4 4 seq 2 4 seq EcoRI 2 2 1 4 4 4 2 2 2 2 1 4 seq 4 SphI 2 4 2 3 2 4 3 EcoRI 2 2 1 2 2 2 2 2 2 2 1 4 3 4 2 XbaI 4 4 4 3 2 4 3 seq 2 2 2 4 4 4 2 3 3 3 4 4 3 4 4 2 XhoI 4 4 4 3 3 4 3 EcoRI 3 2 1 4 4 4 2 3 3 3 4 4 3 4 4 2 4 XmaI 4 4 4 seq seq 4 seq seq 4 seq 4 4 4 4 4 2 4 seq 4 4 seq 4 4 4 4 4 10 technical reference

DNA Methylation & Restriction Digests Two common types of methylation that can block cutting at a restriction site are Dam and Dam/Dcm Sensitive Dcm methylation. Both arise from replicating DNA in a strain of E. coli that has functional Dam and Dcm methylation systems. Restriction Enzymes available from NEB Site Blocked by Dam/Dcm Methylation The following enzymes are blocked or Restriction by some enzymes can be inhibited due to methylation caused by the common impaired by Dam or Dcm methylation. E. coli methyltransferases. Dam methyltransferase causes methylation of the adenine in In order to achieve cleavage with these the sequence GATC while Dcm methyltransferase causes methylation of the first cytosine enzymes, the DNA should be passed in the sequence C(A/T)GG. Any restriction enzyme whose site contains either of these through a dam–/dcm– strain. sequences may be affected by the relevant methylation. For example, the site for AlwI Acc651 BspHI PflMI (GGATC4/5) contains the recognition site for Dam methyltransferase, GATC. If the DNA AlwI BssKI PhoI is produced in a methylating E. coli strain, the adenine is methylated and cleavage by AlwI is blocked. In the case of AlwI or any other restriction enzyme blocked by Dam methy- AlwNI BstXI PpuMI lation, blocked cleavage can be avoided by cloning the DNA into a dam– strain such as ApaI ClaI PspGI dam–/dcm– Competent E. coli (NEB #C2925). AvaII DpnII PspOMI BanI EaeI Sau96I On the other hand BamHI is not Dam/Dcm sensitive; the BamHI site contains GATC BcgI EcoO1091 ScrFI but cleavage by this enzyme is not blocked even when the adenine is methylated. Bc1I HphI SexAI The same principles apply for Dcm methylation but the enzyme sites affected would BsaI Hpy188I SfiI contain the sequence CC(A/T)GG. BsaBI Hpy1881II SfoI dam/dcm Overlapping Sites BsaHI Mbol StuI Bs1I MboII StyD41 Restriction sites can also be blocked if an overlapping site is present. In this case, BsmFI MscI TaqIa part of the dam or dcm sequence is generated by the restriction enzyme sequence followed BspDI NlaIV XbaI by the flanking sequence. For example, the site for ClaI (AT/CGAT) contains GAT. BspEI Nrul If it is followed by a C, the A can be methylated, and cleavage will be blocked. Methylation sensitivity information can More information regarding methylation sensitivity can be found in the technical also be found on REBASE (Restriction Enzyme reference section of the NEB Catalog & Technical Reference, or www.neb.com. Database), a comprehensive database of Key Points to Consider: information about restriction enzymes and related proteins. For more information, see • Genomic DNA directly isolated from a mammalian source is not Dcm or Dam methylated, and is therefore not an issue when digesting mammalian DNA. page 13. • Mammalian and plant DNA that has been cloned into a methylating E. coli strain will be Dam/Dcm methylated. ost commonly used laboratory E. coli strains methylate DNA. • Directly isolated mammalian and plant genomic DNA are CpG methylated. Some enzymes are inhibited by CpG methylation. (See www.neb.com for more information). • Most bacterial DNA (including E. coli DNA) is not CpG methylated. Inhibition of enzyme activity by CpG methylation is not an issue for DNA prepared from E. coli strains. • DNA amplified by PCR does not contain any methylated bases. • To avoid Dam/Dcm methylation when subcloning in bacteria, NEB offers the methyltransferase deficient cloning strain dam–/dcm– Competent E. coli (NEB #C2925) for propagation.

www.neb.com 11 web tools

Online Tools

As the leader in enzyme technology, NEB’s technical support is an invaluable resource. Our scientists provide assistance via [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.

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

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

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

Zoom in to view cleavage overhangs

Identify enzymes affected by methylation Calculate GC Content

Introduce silent mutation sites into ORF sequence with a single mouse click Expand region of interest to obtain sequence information

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

Choose linear or circular maps Summarize ORF sequences 12 Web tools

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

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

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

Lists available suppliers

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

View a complete list of enzymes in the database including recognition sequences, isoschizomers Contains detailed methylation and commercial suppliers sensitivity testing data

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

Additional web-based tools can be found on our website, www.neb.com.

www.neb.com 13 technical reference

Cleavage Close to the Ends of DNA Fragments

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

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

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

14 selection

Cleavage Close to the Ends of DNA Fragments Activity Chart for Restriction Enzymes

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

This buffer is not recommended for NR HEAT use with this enzyme. SUPPLIED % ACTIVITY in NEBuffers INACTIVATION INCUBATION ENZYME NEBuffer 1 2 3 4 EcoRI (TEMP) TEMPERATURE r Produced from a recombinant source. r AatII 4 0 50 50 100 25 65° 37° r AccI 4 50 50 10 100 5 80° 37° Time-Saver qualified (digests 1 µg r C Acc65I 3 + BSA 10 75 100 25 50 65° 37° C of substrate DNA in 5 minutes under recommended reaction conditions). C AciI 3 25 50 100 50 100 65° 37° r C AclI 4 + BSA 10 10 0 100 5 No 37° E Engineered enzyme for maximum r AcuI 4 + SAM 50 100 50 100 100 65° 37° performance. r AfeI 4 25 50 25 100 100 65° 37° r C AflII 4 + BSA 50 100 25 100 0 65° 37° NEBuffer Compositions (1X) r AflIII 3 + BSA 25 75 100 50 100 80° 37° 10 mM Bis Tris Propane-HCl, r C AgeI 1 100 50 10 75 50 65° 37° NEBuffer 1 10 mM MgCl , 1 mM DTT (yellow) 2 r C AhdI 4 + BSA 25 75 0 100 10 65° 37° (pH 7.0 @ 25°C). r AleI 4 10 25 10 100 0 65° 37° 10 mM Tris-HCl, 10 mM r C AluI 4 100 100 75 100 100 65° 37° NEBuffer 2 MgCl , 50 mM NaCl, (blue) 2 r C AlwI 4 50 100 10 100 5 65° 37° 1 mM DTT (pH 7.9 @ 25°C). C AlwNI 4 50 100 50 100 10 65° 37° 50 mM Tris-HCl, 10 mM NEBuffer 3 r C ApaI 4 + BSA 25 50 0 100 5 65° 25° MgCl , 100 mM NaCl, (red) 2 r C ApaLI 4 + BSA 100 100 10 100 5 No 37° 1 mM DTT (pH 7.9 @ 25°C). r C ApeKI 3 25 75 100 50 100 No 75° 20 mM Tris-acetate, 10 mM r C ApoI 3 + BSA 10 75 100 75 100 80° 50° NEBuffer 4 magnesium acetate, 50 mM (green) potassium acetate, 1 mM DTT r C AscI 4 0 10 10 100 5 65° 37° (pH 7.9 @ 25°C). r C AseI 3 NR 75 dd100 NR 50 65° 37° 50 mM NaCI, 100 mM r C AsiSI 4 + BSA 50 100 100 100 0 80° 37° NEBuffer Tris-HCI, 10 mM MgCI2, r C AvaI 4 10 75 10 100 50 80° 37° EcoRI 0.025% Triton X-100 (pH 7.5 @ 25°C). r C AvaII 4 50 75 10 100 50 65° 37° r C AvrII 4 100 100 50 100 50 80° 37° Buffers should be thawed completely and BaeI 4 + BSA + SAM 50 100 50 100 100 65° 25° mixed thoroughly before using. C BaeGI 1 100 75 10 50 100 65° 37° r C BamHI 3 + BSA 75 100 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.

www.neb.com 15 selection

HEAT SUPPLIED % ACTIVITY in NEBuffers INACTIVATION INCUBATION ENZYME NEBuffer 1 2 3 4 EcoRI (TEMP) TEMPERATURE BbsI 2 100 100 25 75 50 65° 37° r C BbvCI 4 50 100 10 100 100 80° 37° r C BbvI 2 100 100 25 75 100 65° 37° r BccI 1 + BSA 100 50 10 50 15 65° 37° BceAI 3 + BSA 100 100 100 100 100 65° 37° r BcgI 3 + SAM NR NR 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° New England Biolabs offers C BsrI 3 0 50 100 10 100 80° 65° over 220 enzyme specificities. C BsrBI 4 50 100 100 100 100 80° 37° C BsrDI 2 + BSA 50 100 50 75 50 80° 65° r C BsrFI 4 10 100 100 100 100 No 37° BsrGI 2 + BSA 25 100 10 100 50 80° 37° r C BssHII 3 100 100 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°

16 selection

HEAT SUPPLIED % ACTIVITY in NEBuffers INACTIVATION INCUBATION ENZYME NEBuffer 1 2 3 4 EcoRI (TEMP) TEMPERATURE r C BstBI 4 75 50 25 100 50 No 65° r C BstEII 3 + BSA 50 75 100 75 100 No 60° r C BstNI 2 + BSA 10 100 100 75 50 No 60° C BstUI 4 100 100 50 100 100 No 60° Chart Legend r C BstXI 3 0 50 100 25 100 65° 37° BstYI 2 Supplied with a unique reaction buffer r 50 100 75 100 50 80° 60° that is different from the four standard C BstZ17I 4 NR NR 100 100 100 No 37° U NEBuffers. The compatibility with the Bsu36I 3 + BSA four standard NEBuffers is indicated r 0 25 100 0 15 80° 37° in the chart. r C BtgI 3 + BSA 25 50 100 100 100 80° 37° Supplied with a separate vial of bovine BtgZI 4 + BSA 10 25 0 100 100 80° 60° serum albumin (10 mg/ml). To obtain r C BtsI 4 + BSA 100 50 50 100 25 80° 55° BSA 100% activity, BSA should be added r C BtsCI 4 to the 1X reaction mix to a final 50 100 50 100 25 No 50° concentration of 100 µg/ml. Cac8I 4 50 75 100 100 50 65° 37° r C ClaI 4 + BSA 10 50 50 100 50 65° 37° Supplied with a separate vial of CspCI 4 + SAM S-adenosylmethionine (SAM). r C 10 100 10 100 40 65° 37° SAM To obtain 100% activity, SAM should r CviAII 4 75 25 10 100 100 65° 25° be added to the 1X reaction mix as specified on the product data card. r CviKI-1 4 10 50 25 100 100 80° 37° r C CviQI 3 + BSA 75 100 100 75 0 No 25° This NEBuffer is recommended for r C DdeI 3 75 100 100 75 100 65° 37° dd a double digest because it minimizes star activity. r C DpnI 4 100 100 75 100 100 80° 37° r DpnII U NR NR dd100 NR 100 65° 37° This buffer is not recommended for NR r C DraI 4 75 75 50 100 100 65° 37° use with this enzyme. r C DraIII 3 + BSA 100 75 100 25 50 65° 37° r Produced from a recombinant source. DrdI 4 25 50 10 100 100 65° 37° r EaeI 4 100 100 50 100 100 80° 37° Time-Saver qualified (digests 1 µg EagI 3 r C 10 25 100 10 100 65° 37° C of substrate DNA in 5 minutes under r C E EagI-HF 4 25 50 10 100 100 65° 37° recommended reaction conditions). r EarI 4 100 100 50 100 100 65° 37° EciI 2 + BSA Engineered enzyme for maximum 50 100 100 50 100 65° 37° E performance. C EcoNI 4 100 100 75 100 100 65° 37° r C EcoO109I 4 + BSA 100 100 75 100 15 65° 37° r EcoP15I 3 + BSA + ATP 75 100 100 100 50 65° 37° NEBuffer Compositions (1X) r C EcoRI Udd 100 100 100 100 100 65° 37° 10 mM Bis Tris Propane-HCl, NEBuffer 1 r C E EcoRI-HF 4 10 mM MgCl , 1 mM DTT 10 100 0 100 5 65° 37° (yellow) 2 (pH 7.0 @ 25°C). 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° 10 mM Tris-HCl, 10 mM NEBuffer 2 MgCl , 50 mM NaCl, r FatI 2 10 100 50 50 100 65° 55° (blue) 2 1 mM DTT (pH 7.9 @ 25°C). r FauI 4 100 50 10 100 50 65° 55° r C Fnu4HI 4 10 25 25 100 10 65° 37° 50 mM Tris-HCl, 10 mM NEBuffer 3 MgCl , 100 mM NaCl, r C FokI 4 100 100 75 100 25 65° 37° (red) 2 1 mM DTT (pH 7.9 @ 25°C). r C FseI 4 + BSA 100 75 0 100 50 65° 37° r FspI 4 10 75 10 100 50 65° 37° 20 mM Tris-acetate, 10 mM NEBuffer 4 magnesium acetate, 50 mM r HaeII 4 + BSA 75 100 50 100 25 80° 37° (green) potassium acetate, 1 mM DTT r C HaeIII 4 50 100 25 100 100 80° 37° (pH 7.9 @ 25°C). r HgaI 1 100 75 50 100 100 65° 37° 50 mM NaCI, 100 mM NEBuffer Tris-HCI, 10 mM MgCI , r C HhaI 4 + BSA 75 100 100 100 100 65° 37° 2 EcoRI 0.025% Triton X-100 r HincII 3 + BSA 75 100 100 100 100 65° 37° (pH 7.5 @ 25°C). r C HindIII 2 50 100 10 50 25 65° 37° r C HinfI 4 75 100 75 100 100 80° 37° Buffers should be thawed completely and r C HinP1I 4 100 100 100 100 100 65° 37° mixed thoroughly before using. r HpaI 4 25 50 10 100 100 No 37° r C HpaII 1 100 50 10 100 15 65° 37° r C HphI 4 NR 75 0 100 10 65° 37° r Hpy99I 4 + BSA 10 10 0 100 25 65° 37° r C Hpy166II 4 100 100 50 100 40 65° 37° r Hpy188I 4 50 25 10 100 50 65° 37°

www.neb.com 17 selection

HEAT SUPPLIED % ACTIVITY in NEBuffers INACTIVATION INCUBATION ENZYME NEBuffer 1 2 3 4 EcoRI (TEMP) TEMPERATURE r Hpy188III 4 + BSA 100 100 10 100 5 65° 37° r C HpyAV 4 + BSA 100 100 25 100 25 65° 37° r HpyCH4III 4 100 50 25 100 20 80° 37° r C HpyCH4IV 1 100 25 10 25 100 65° 37° r C HpyCH4V 4 50 50 25 100 50 65° 37° r KasI 4 + BSA 25 100 0 100 100 65° 37° r C KpnI 1 + BSA 100 75 0 50 25 No 37° r C MboI 4 75 100 100 100 100 65° 37° r C MboII 4 100 100 50 100 100 65° 37° r C MfeI 4 75 50 10 100 5 65° 37° r C E MfeI-HF 4 75 50 10 100 0 65° 37° r C MluI 3 25 75 100 50 100 65° 37° r C MlyI 4 + BSA 50 50 25 100 25 65° 37° r C MmeI 4 + SAM NR NR NR 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° New England Biolabs offers r Nt.CviPII 4 25 100 50 100 N/A 65° 37° over 170 recombinant enzymes. r C PacI 1 + BSA 100 75 10 100 5 65° 37° r C PaeR7I 4 25 100 10 100 50 No 37° r PciI 3 50 75 100 50 0 80° 37° C PflFI 4 + BSA 0 25 25 100 5 65° 37° r C PflMI 3 + BSA 0 100 100 50 100 65° 37° r PhoI 3 50 50 100 75 100 No 75° r PleI 4 100 100 75 100 50 65° 37° r C PmeI 4 + BSA 0 50 10 100 5 65° 37° C PmlI 1 + BSA 100 75 0 75 15 65° 37° r C PpuMI 4 0 25 0 100 10 No 37° 18 selection

HEAT SUPPLIED % ACTIVITY in NEBuffers INACTIVATION INCUBATION ENZYME NEBuffer 1 2 3 4 EcoRI (TEMP) TEMPERATURE r PshAI 4 + BSA 50 75 10 100 5 65° 37° r PsiI 4 10 100 10 100 10 65° 37° r PspGI 4 75 100 50 100 100 No 75° r PspOMI 4 25 25 10 100 15 65° 37° Chart Legend r PspXI 4 0 100 10 100 100 80° 37° Supplied with a unique reaction buffer r C PstI 3 + BSA 75 75 100 50 50 80° 37° that is different from the four standard C PvuI 3 + BSA 10 75 100 10 100 80° 37° U NEBuffers. The compatibility with the four standard NEBuffers is indicated r C PvuII 2 100 100 100 100 100 No 37° in the chart. r C E PvuII-HF 4 0 25 0 100 0 80° 37° r C Supplied with a separate vial of bovine RsaI 4 100 100 50 100 15 65° 37° serum albumin (10 mg/ml). To obtain r RsrII 4 25 75 10 100 25 65° 37° BSA 100% activity, BSA should be added r C to the 1X reaction mix to a final SacI 1 + BSA 100 50 10 100 15 65° 37° concentration of 100 µg/ml. r C E SacI-HF 4 + BSA 100 50 10 100 15 65° 37° r C SacII 4 25 75 10 100 100 65° 37° Supplied with a separate vial of S-adenosylmethionine (SAM). r C SalI 3 + BSA 0 0 100 0 50 65° 37° SAM To obtain 100% activity, SAM should r C E SalI-HF 4 10 100 100 100 100 65° 37° be added to the 1X reaction mix as specified on the product data card. r SapI 4 75 50 0 100 50 65° 37° r Sau3AI 1 + BSA 100 50 10 100 100 65° 37° This NEBuffer is recommended for r Sau96I 4 50 100 100 100 50 80° 37° dd a double digest because it minimizes star activity. r C SbfI 4 75 50 0 100 25 65° 37° r C E SbfI-HF 4 50 25 0 100 0 65° 37° This buffer is not recommended for NR r C ScaI 3 NR NR dd100 NR 50 80° 37° use with this enzyme. r C E ScaI-HF 4 100 100 10 100 15 65° 37° r Produced from a recombinant source. C ScrFI 4 100 100 100 100 100 65° 37° r SexAI 4 + BSA 100 75 50 100 50 65° 37° Time-Saver qualified (digests 1 µg r SfaNI 3 0 75 100 25 50 65° 37° C of substrate DNA in 5 minutes under r SfcI 4 + BSA 75 50 10 100 25 65° 37° recommended reaction conditions). r C SfiI 4 + BSA 0 100 10 100 15 No 50° Engineered enzyme for maximum r C SfoI 4 25 100 50 100 100 No 37° E performance. r SgrAI 4 100 50 10 100 50 65° 37° r C SmaI 4 0 0 0 100 0 65° 25° r SmlI 4 + BSA 25 75 25 100 50 No 55° NEBuffer Compositions (1X)

dd r C SnaBI 4 + BSA 25 50 25 100 5 80° 37° 10 mM Bis Tris Propane-HCl, NEBuffer 1 10 mM MgCl , 1 mM DTT r C SpeI 4 + BSA 75 100 25 100 50 80° 37° (yellow) 2 (pH 7.0 @ 25°C). r C SphI 2 100 100 50 100 50 65° 37° r C E SphI-HF 4 50 25 10 100 0 65° 37° 10 mM Tris-HCl, 10 mM NEBuffer 2 MgCl , 50 mM NaCl, r C SspI U 50 100 50 50 100 65° 37° (blue) 2 1 mM DTT (pH 7.9 @ 25°C). r C E SspI-HF 4 25 100 0 100 25 65° 37° r C 50 mM Tris-HCl, 10 mM StuI 4 100 100 50 100 50 65° 37° NEBuffer 3 MgCl , 100 mM NaCl, r (red) 2 StyD4I 2 10 100 100 100 20 65° 37° 1 mM DTT (pH 7.9 @ 25°C). r StyI 3 + BSA 25 75 100 10 15 65° 37° r SwaI 3 + BSA 10 10 100 10 15 65° 25° 20 mM Tris-acetate, 10 mM NEBuffer 4 magnesium acetate, 50 mM r C TaqI 4 + BSA 50 75 100 100 100 80° 65° (green) potassium acetate, 1 mM DTT (pH 7.9 @ 25°C). r TfiI 4 100 100 dd100 100 100 No 65° r TliI 3 + BSA 25 25 100 10 50 No 75° 50 mM NaCI, 100 mM NEBuffer Tris-HCI, 10 mM MgCI , TseI 4 75 100 100 100 100 No 65° 2 EcoRI 0.025% Triton X-100 Tsp45I 1 + BSA 100 25 0 75 50 No 65° (pH 7.5 @ 25°C). r C Tsp509I 1 100 100 100 NR 100 No 65° C TspMI 4 50 75 50 100 20 No 75° Buffers should be thawed completely and r C TspRI 4 + BSA 25 50 25 100 5 No 65° mixed thoroughly before using. r Tth111I 4 50 25 25 100 100 No 65° r C XbaI 4 + BSA 0 100 75 100 15 65° 37° r XcmI 2 10 100 50 50 20 65° 37° r C XhoI 4 + BSA 75 100 100 100 100 65° 37° r XmaI 4 + BSA 25 50 0 100 100 65° 37° r C XmnI 4 + BSA 100 100 50 100 5 65° 37° r ZraI 4 100 25 10 100 10 No 37° www.neb.com 19 Canada New England Biolabs, Ltd. PRSRT STD MAIL Toll Free: 1-800-387-1095 US POSTAGE PAID [email protected] BOSTON, MA PERMIT NO. 54302 China, People’s Republic New England Biolabs, Inc. New England Biolabs (Beijing), Ltd. 240 County Road Telephone: 010-82378265/82378266 Ipswich, MA 01938-2723 [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

Version 1.0