neb expressions A scientific update from New England Biolabs Fall 2013

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1 neb expressions scientific update from New England iolabs Fall 0 Restriction Enzymes Mission completed page 8 ddressing hallenges in Microbiome DN nalysis page How Many PR Polymerases Do You Need in Your Freezer? page PR at Special Price Q5 and OneTaq DN Polymerases page

2 Did you receive a copy of the 0 04 NE atalog & Technical Reference? ontents Issue II, 0 feature 6 article new product ddressing hallenges in Microbiome DN nalysis Host genome contamination hinders many microbiome studies NE is working to solve the problem. new product Get more from less! NENext Ultra Kits for Illumina. Recombinant Shrimp lkaline Phosphotase (rsp). TTTGGG TGGTGTT 5 GGG Run-off transcr the top strand 7 8 technical new product RN Synthesis and apping T7 Quick High Yield RN Synthesis Kit and Vaccinia apping System. tips Mission ompleted! NE Restriction Enzymes. 9 technical tips FQ Spotlight NE uffer for Restriction Enzymes. special price How Many DN Polymerases Do You Need in Your Freezer? special price OneTaq and Q5 DN Polymerases enefit from our special prices. To request your personal free copy, visit or contact your local distributor! cover photo Purple pansy (Viola tricolor) on the campus of New England iolabs, photographed by ree Hall. Longmp, NENext, New England iolabs, OneTaq, PRER, Q5, HF and RE-Mix are registered trademarks of New England iolabs, Inc. Phusion DN Polymerase was developed by Finnzymes Oy, now a part of Thermo Fisher Scientific. This product is manufactured by New England iolabs, Inc. under agreement with, and under the performance specifications of Thermo Fisher Scientific. Phusion and Thermo Scientific are registered trademarks and property of Thermo Fisher Scientific. is a trademark of New England iolabs, Inc. ILLUMIN is a registered trademark of Illumina, Inc.

3 While the 6S method is a fast and relatively inexpensive way to survey, at high throughput, the microbial organisms present within a sample, it provides very little information regarding function. dditionally, determining optimal PR primers (for specific sample types and to distinguish between some species) can be challenging. In contrast, sequencing of the total DN of a microbiome sample does not have these limitations and provides a more complex range of information. Through the identification of microbial sequences, genes, variants and polymorphisms, this method enables determination of information on microbiome species diversity and, also, putative functional information. Such sequencing-based studies have enabled the creation of many databases, including the Human Oral Microbiome Database (HOMD) [ (). pproximately 700 prokaryotic species are present in the human oral cavfeature article ddressing hallenges in Microbiome DN nalysis mong the very many -omes now studied and discussed (), microbiomes have received increasing attention in recent months, from both scientists and the general public. Used to describe the communities of microorganisms and their genes in a particular environment, including a body or part of a body, microbiome is becoming an increasingly common term in everyday language. One challenge in microbiome genome analysis is addressing the presence of host DN in samples. s such, improved methods for solving this problem are needed. Fiona Stewart, Ph.D. and Erbay Yigit, Ph.D., New England iolabs, Inc. Introduction wealth of information about the composition of, and interactions between, the constituent microbes of a microbiome can provide insight into both the function and dysfunction of the host organism, as well as the host-microbiome unit as a whole. In particular, the relationships amongst and between resident microbes (bacteria, archea and fungi) and their hosts have recently become the topic of fervent research; the number of microbiome research publications has been steadily increasing since 00 (). Such research has demonstrated that the microbiome communities of individuals are unique, as are the microbiome communities of specific sites within an individual (reviewed in ). In humans, the number of microorganisms present is estimated to exceed the number of human cells by -fold (4). Studies of the human microbiome (including the Human Microbiome Project (HMP) [ (5), and MetaHIT, the metagenomics of the intestinal tract [ (6)) may be the best known, and have led to the understanding that the human microbiome may be critical to health and disease. Until relatively recently, the role of the microbiome was unknown, and an organism s microbial load was considered to be potentially nothing more than cellular hitchhikers, having little impact on the organism s functioning. w, it is understood that an organism s microbiome can influence many processes within the host organism. Discoveries including the role of the microbiome in conditions and disease states, such as obesity, diabetes mellitus and cardiovascular disease (reviewed in 7), have led to the potential for development of microbiome-based diagnostic and therapeutic tools. dditionally, the unique nature of an individual s microbiome has enabled matching of skin-associated bacteria, on objects such as a keyboard, to specific individuals, leading to the potential for use in forensic applications (8). It should be noted that microbiome research is not limited to humans, and research into microbiomes of non-human organisms is also increasing rapidly in environmental and agricultural areas of research (9). lthough it is still not possible to isolate and culture the vast majority of microorganisms (estimated to be over 95%), analysis of total nucleic acid from microbiome samples has enabled significant advances in the field. Furthermore, advances in sequencing technologies have enabled significant progress in microbiome nucleic acid analysis. urrent Methods of nalysis The majority of microbiome DN studies to date have employed 6S analysis (Figure ). This analysis method takes advantage of the 6S rrn gene that is specific to prokaryotes and some of the archaea and is not found in eukaryotes. 6S rrn genes from different species have significant homology, but the gene also includes hypervariable regions that are generally speciesspecific, and are determined by the microbial composition of the community. These characteristics enable the use of universal primer pairs to amplify 6S genes from many organisms in the Figure. Microbiome DN nalysis Methods same PR reaction and then, through subsequent sequencing of the PR products, the individual species represented can be identified. While 6S analysis is fast and inexpensive, it provides little information regarding function. More detailed information can be obtained through microbiome sequencing, particularly once host DN is removed. * For many samples, host DN constitutes a high percentage of sequence reads. Removal of host DN, and enrichment of microbial DN substantially increases the percentage of sequence reads from the microbial sequences of interest.

4 feature article continued ity, and the stated goal of the HOMD database project is to provide taxonomic and genomic information on these species. omparison of microbiome sample sequences to databases, such as HOMD, further enables discovery, including genes, pathways and their relative frequencies in the sample. Overcoming Difficulties with Microbiome Samples Many microbiome samples are overwhelmed with host DN, and the HMP has reported especially high levels of human DN in soft tissue samples, such as mid-vagina and throat samples. Saliva samples also contain high levels of human DN (). In contrast, although human DN is generally all but absent from fecal samples, some infections can substantially increase the level of human DN in such samples, likely due to widespread cell lysis during bacterial infection. The presence of contaminating host genomic DN in a microbiome sample complicates the genetic analysis of these samples. Since a single human cell contains approximately,000 times more DN than a single bacterial cell (approximately 6 billion bp versus 4-5 million bp), even a low level of human cell contamination within a Figure. Microbiome Diversity is Retained fter Enrichment with the NENext Microbiome DN Enrichment Kit DN was purified from pooled human saliva DN (Innovative Research) and enriched using the NENext Microbiome DN Enrichment Kit. Libraries were prepared from unenriched and enriched samples, followed by sequencing on the SOLiD4 platform. The graph shows a comparison between relative abundance of each bacterial species listed in HOMD[] before and after enrichment with the NENext Microbiome DN Enrichment Kit. bundance is inferred from the number of reads mapping to each species as a percentage of all reads mapping to HOMD. High concordance continues even to very low abundance species (inset). We compared M bp SOLiD4 reads in the enriched dataset to 57M bp SOLiD4 reads in the unenriched dataset. Reads were mapped using owtie 0..7[] with typical settings ( mismatches in a 8 bp seed region, etc). * Niesseria flavescens This organism may have unusual methylation density, allowing it to bind the enriching beads at a low level. Other Niesseria species (N. mucosa, N. sicca and N. elognata) are represented, but do not exhibit this anomalous enrichment. Figure. Salivary Microbiome DN Enrichment DN was purified from pooled human saliva DN (Innovative Research) and enriched using the NENext Microbiome DN Enrichment Kit. Libraries were prepared from unenriched and enriched samples and sequenced on the SOLiD 4 platform. The graph shows percentages of 0M-57M SOLiD4 bp reads that mapped to either the Human reference sequence (hg9) or to a microbe listed in the Human Oral Microbiome Database (HOMD) []. (ecause the HOMD collection is not comprehensive, ~80% of reads in the enriched samples do not map to either database.) Reads were mapped using owtie 0..7[] with typical settings ( mismatches in a 8 bp seed region, etc.). microbiome sample can substantially complicate the sample processing and sequencing. s a result, in the case of total microbiome DN sequencing studies, only a small percentage of sequencing reads from such samples pertain to the microbes of interest, and therefore a large percentage of sequencing reads (host) have to be discarded. onsequently, obtaining sufficient sequence coverage of the microbiome DN can become costprohibitive or even technically infeasible. Therefore, methods to enrich microbiome DN are useful, and, in some cases, critical for sequencing of the microbiome. However, until now, options for such enrichment have been limited to selective cell lysis, with the disadvantages of a requirement for live cells, and low bacterial DN recovery. The NENext Solution The NENext Microbiome DN Enrichment Kit addresses this problem by providing a quick and effective way to remove contaminating host DN, thereby enriching for microbiome DN. The kit exploits the different prevalences of pg methylation in the genomes of microbial and eukaryotic organisms. Eukaryotic DN, including human DN, is methylated at pgs, while methylation at pg sites in microbial species is rare. The NENext Microbiome DN Enrichment Kit uses a magnetic bead-based method to selectively bind and remove pg-methylated host DN. The kit contains the MD-Fc protein, which is composed of the methylated pg-specific binding protein MD, fused to the Fc fragment of human IgG. The Fc fragment binds readily to Protein, enabling effective attachment to Protein -bound magnetic beads. The MD domain of this protein binds specifically and tightly to pg methylated DN. pplication of a magnetic field then pulls out the pg-methylated (eukaryotic) DN, leaving the non-pg-methylated (microbial) DN in the supernatant (see workflow figure on Page 5). Microbiome Enrichment of Human Saliva Human saliva samples can be especially challenging, due to high levels of human genomic DN and the poor-quality of the DN itself. Despite these sample challenges, the data shown in Figure demonstrates that substantial enrichment of microbiome DN from saliva was achieved using the NENext Microbiome DN Enrichment Kit. n important consideration when assessing the validity of microbiome enrichment is that the enrichment should not be biased, and the diversity of microbiome organisms in the sample should 4

5 remain intact after enrichment. s shown in Figure, measurement of the relative abundance of species represented in HOMD was equivalent between unenriched and enriched samples. Interestingly, Neisseria flavescens, highlighted with * in Figure, Page 4, was a unique outlier in this comparison and may have unusual methylation density, which enables binding to the MD-Fc beads at a low level. It is notable that other Neisseria species (N. mucosa, N. sicca and N. elognata) are also represented, but do not exhibit this anomalous enrichment. onclusion From forensic microbial fingerprints to diseasecausing pathogens, microbiomes comprise a vast and varied microcosm with a surprising degree of influence over the health and function of the host organism. The potential for significant and exciting discoveries to be achieved with microbiome analysis is enormous, but will require improved tools and methods to make this a reality. s a step towards this goal, the NENext Microbiome DN Enrichment Kit now makes it possible to substantially enrich a variety of sample types for non-host, microbial DN, while retaining microbial diversity, and thereby improving the quality and cost-effectiveness of downstream analyses and data generation. References. lphabetically ordered list of -omes and -omics (0) Omics. org Retrieved on May, 0, from Jones, S. (0) Nature iotechnology,, 77.. Morgan, X.., et al. (0) Trends in Genetics, 9, ackhed, F., et al. (005) Science, 07, Peterson, J., et al. (009) Genome Res. 9, Qin, J., et al. (0) Nature, 464, Pflughoeft, K.J. and Versalovic J. (0) nnu. Rev. of Pathol. 7, Fierer N., et al. (0) Proc. Natl. cad. Sci. US, 7, Jansson, J.K. and Prosser, J. I. (0) Nature, 494, hen, T., et al. (0) The Human Oral Microbiome Database Retrieved on May, 0, from The Human Microbiome Project onsortium (0) Nature, 486, 5.. Langmead,., et al. (009). Genome iol. (), R. Scientific ontribution The scientific contributors to this article include: George R. Feehery, Erbay Yigit, radley W. Langhorst, Fiona J. Stewart, Eileen T. Dimalanta, Sriharsa Pradhan, James MacFarland, hristine Sumner and Theodore. Davis. New product NENext Microbiome DN Enrichment Kit Microbiome DN analysis can be challenging due to the high percentage of host DN present in samples. The NENext Microbiome DN Enrichment Kit facilitates enrichment of microbial genomic DN from samples containing methylated host DN (including human), by selective binding and removal of pg-methylated host DN. Importantly, microbial diversity remains intact after enrichment. Microbiome DN Enrichment Kit Workflow dvantages Effective enrichment of microbial genomic DN from contaminating host DN Fast, simple protocol Enables microbiome whole genome sequencing, even for samples with high levels of host DN ompatible with downstream applications, including next generation sequencing on all platforms, qpr and end point PR Suitable for a wide range of sample types requirement for live cells For more information, visit NENext Reagents FOR NEXT GENERTION SEQUENING The MD-Fc protein binds specifically to pg methylated DN. In the NENext Microbiome DN Enrichment workflow, MD-Fc is attached to Protein magnetic beads, enabling capture of methylated DN, while the microbial DN remains in the supernatant. Ordering Information Product NE # SIZE NENext Microbiome DN Enrichment Kit E6S 6 reactions NENext Reagents rochure request your free copy now! 5

6 New product Get more from less! NENext Ultra DN and RN Kits for Illumina Take the next step! Ordering Information Product NE # SIZE NENext Ultra DN Library Prep Kit for Illumina E770S/L 4/96 rxns NENext Ultra Directional RN Library Prep Kit for Illumina E740S/L 4/96 rxns NENext Ultra RN Library Prep Kit for Illumina E0S/L 4/96 rxns dvantages road range of sample input amounts DN: 5 ng- μg RN: Total RN: ng- µg Purified mrn: ng- ng rrn-depleted RN: ng- ng Fast workflow (< hours for DN), For information with minimal about hands-on other NENext time products, visit NENext.com Robust, reliable performance Ultra high fidelity amplification with minimized G bias Value pricing Shrimp lkaline Phosphatase (rsp) superior choice for PR clean-up or cloning Shrimp lkaline Phosphatase (rsp) is a heat labile alkaline phosphatase of superior quality purified from a recombinant source. rsp nonspecifically catalyzes the dephosphorylation of 5 and ends of DN and RN as well as deoxyribonucleoside triphosphates (rntps and dntps). rsp is useful in many molecular biology applications such as the removal of phosphorylated ends of DN and RN for subsequent use in cloning or end-labeling of probes. The enzyme acts on 5 protruding, 5 recessed and blunt ends. In cloning, dephosphorylation prevents religation of linearized plasmid DN. rsp may also be used to degrade unincorporated dntps in PR reactions to prepare templates for DN sequencing or SNP analysis. rsp is completely and irreversibly inactivated by heating at for 5 minutes, thereby making removal of rsp prior to ligation or end-labeling unnecessary. Remaining ctivity (%) Incubation at, minutes ompletely and irreversibly heat inactivated in just 5 min at 0 dvantages Recombinant quality with significantly improved consistency compared to native enzyme ompletely and irreversibly heat inactivated in just 5 min at ctive in all NE restriction enzyme uffers Does not require supplemental additives such as zinc, as is the case with ntarctic Phosphatase Ordering Information Product NE # SIZE recombinant Shrimp lkaline Phosphatase M07S/L 0/,0 u also available: Exonuclease I (E. coli) - degrades ssdn primers/oligos M09S/L,000/5,000 u 6

7 New product T7 Quick High Yield RN Synthesis Kit The T7 Quick High Yield RN Synthesis Kit offers robust in vitro RN transcription for a variety of applications, incl. capped RN (R, NE #S4) or dye-labeled RN synthesis, anti-sense RN and RNi experiments, generation of RN probes for microarray analysis and microinjection etc., as well as in vitro translation and RN vaccines. Yields of up to 80 µg of RN can be obtained from a standard 0 µl reaction. The kit formulation allows for quick reaction setup, requiring pipetting of two master mix reagents, i.e. the NTP buffer mix, T7 RN Polymerase mix and a suitable template DN. This results in a reduced chance of errors due to fewer pipetting steps during reaction set up. DNase I and Lil are included for DN template removal and quick RN purification. dvantages Streamlined format Flexibility enables incorporation of cap analogs (R), radiolabeled and modified nucleotides High Yield up to 80 µg of RN from a standard 0 µl reaction High Quality Transcripts optimized formulation for increased transcript integrity over extended reaction times Transcription by T7 RN Polymerase 5 T7 Promoter TTGTTTGGG TTTGTGGTGTT Start of Transcription 5 DN template T7 Transcription 5 GGG Run-off transcript has the top strand sequence. RN transcript Ordering Information Product NE # SIZE T7 Quick High Yield RN Synthesis Kit E0S rxns T7 RN Polymerase utilizes a minimal promoter sequence (shown in gold). Upon transcription, the run-off transcript has the top strand sequence. Featured product Vaccinia apping System ased on the Vaccinia Virus apping Enzyme, the Vaccinia apping System provides the necessary components to add 7-methylguanylate cap structures (ap 0) to the 5 end of RN. In eukaryotes, these terminal cap structures are involved in stabilization, transport and translation of mrns. Enzymatic production of capped RN is an easy way to improve the stability and translational competence of RN used for in vitro translation, transfection and microinjection. lternatively, use of labeled GTP in a reaction provides a convenient way to label any RN containing a 5 terminal triphosphate. This single enzyme is composed of two subunits (D and D) and has three enzymatic activities (RN triphosphatase and guanylyltransferase by the D subunit and guanine methyltransferase by the D subunit), all necessary for addition of a complete ap 0 structure, m7gppp(5 )N. apping reaction is efficient and all capped structures are added in the proper orientation, unlike co-transcriptional addition of some cap analogs. pplications apping mrn prior to in vivo or in vitro translation Labeling 5 end of mrn apping reaction is complete in less than one hour Ordering Information Product NE # SIZE Vaccinia apping System also available: mrn ap -O-Methyltransferase M080S M066S 400 u.000 u 7

8 Technical tips ut Smart! The dvantage > 00 restriction enzymes are supplied in a single buffer, uffer Over 00 Restriction Enzymes 76 restriction enzymes are currently sold by NE Only uffer > of NE s restriction enzymes are recombinant % ctivity! 8 unique restriction enzymes are available from NE > 80 restriction enzymes are Time-Saver qualified Mission completed! w over 00 restriction enzymes supplied with one single buffer - NE uffer! NE is continuously looking for ways to enhance the convenience and performance of its products for its customers. With the now finalized update of the reaction buffer system, we are now able to offer >00 restriction enzymes that are % active in a single buffer. This improves ease-of-use, especially when performing double digests. Since the new buffer system includes S, there are fewer tubes and pipetting steps to worry about. dditionally, many DN modifying enzymes (including T4 DN Ligase etc.) are % active in the new uffer, eliminating the need for subsequent purification. High-Fidelity (HF ) Restriction Enzymes 8 Enzyme ompetitor FastDigest EcoRI NE EcoRI-HF 5 M 5 atii cci cc65i cii cli cui fei flii fliii gei gei-hf hdi lei lui lwi lwni pai pali peki poi sci sei sisi vai vaii vrii aei aegi amhi amhi-hf ani anii bsi bvi bvi cci cei cgi civi cli codi fai fui fui gli glii lpi mgi mri mti mti-hf pmi pui puei sai sai-hf sai sai sahi saji sawi saxi seri seyi sgi siei sihki siwi sli smi smi smi smfi soi sp86i spni spdi spei sphi spmi spqi sri sri srdi srfi srgi sshii sski sssi stpi sti steii steii-hf stni stui stxi styi stz7i su6i tgi tgzi tsi tsimuti tsi ac8i lai spi viii viki- viqi DdeI DpnI DpnII DraI DraIII-HF DrdI EaeI EagI EagI-HF EarI EciI Eco5kI EcoNI EcoO9I EcoP5I EcoRI EcoRI-HF EcoRV EcoRV-HF FatI FauI Fnu4HI FokI FseI FspI FspEI Supplied NEuffer Incubation % ctivity in NEuffers Temp.. ( ) * * Inactivation Temp. Methylation ( ) Diluent Sensitivity < < < < < < + SM < <. * + SM * * * < < * + SM <.. < < + SM * U < * < * < < < < < < * * <. + TP U < < < * < <. * * * + SM < * < < * < < + SM. HaeII HaeIII HgaI HhaI HincII HindIII HindIII-HF HinfI HinPI HpaI HpaII HphI Hpy99I Hpy66II Hpy88I Hpy88III HpyV HpyH4III HpyH4IV HpyH4V KasI KpnI KpnI-HF LpnPI MboI MboII MfeI MfeI-HF MluI MluI MlyI MmeI MnlI MscI MseI MslI MspI MspI MspJI MwoI NaeI NarI Nb.bvI Nb.smI Nb.srDI Nb.tsI NciI NcoI NcoI-HF NdeI NgoMIV NheI NheI-HF NlaIII NlaIV NmeIII ti ti-hf NruI NsiI NspI Nt.lwI Nt.bvI Nt.smI Nt.spQI Nt.stNI Nt.viPII PacI PaeR7I PciI PflFI PflMI PhoI PleI PluTI PmeI PmlI PpuMI PshI PsiI PspGI PspOMI PspXI PstI PstI-HF PvuI PvuI-HF PvuII PvuII-HF RsaI RsrII SacI SacI-HF SacII SalI SalI-HF SapI SauI Sau96I SbfI SbfI-HF ScaI-HF ScrFI SexI SfaNI SfcI SfiI SfoI SgrI SmaI SmlI SnaI SpeI SphI SphI-HF SspI SspI-HF StuI StyD4I StyI StyI-HF SwaI TaqI TfiI TseI Tsp45I TspMI TspRI TthI XbaI XcmI XhoI XmaI XmnI ZraI Enzyme < < < te < < + SM. TEHNIL GUIDE S < < < Updates to this chart can be found at NE.com. hart produced March 0 UTSMRT and TiME-SvER are trademarks of New England iolabs, inc. NEw ENGlND iols is a registered trademark of New England ioblabs, inc. Supplied NEuffer... Incubation % ctivity in NEuffers Temp.. ( ) < Inactivation Temp. Methylation ( ) Diluent Sensitivity < < < < < < + SM * < 4 + S < < < < < < < < < + SM < < * < < < < < < <.. < < < < < < * < < < < * < < < <. Requires ND + Requires DTT Requires TP + PEG Requires TP Requires TP Requires TP Requires TP + PEG + + % functional activity New England iolabs, Inc. toll free info@neb.com < full functional activity US Requires a+ + 0 % functional activity * < * Requires Zn+ Requires Zn+ * Required Supplements lkaline Phosphatase (IP) ntarctic Phosphatase st DN Polymerase pg Methyltransferase (M. SssI) DN Polymerase I DN Polymerase I, Large (Klenow) Fragment DN Polymerase Klenow Exo DNase I (RNase free) E. coli DN Ligase Endonuclease III (Nth), recombinant Endonuclease VIII Exonuclease III Gp Methyltransferase (M. vipi) Mcr Micrococcal Nuclease Nuclease L- phi9 DN Polymerase RecJf T DN Ligase T4 DN Ligase T4 DN Polymerase T4 Phage β-glucosyltransferase (T4-GT) T4 Polynucleotide Kinase T4 PNK ( phosphatase minus) T7 DN Ligase T7 DN Polymerase (unmodified) T7 Exonuclease USER Enzyme, recombinant <. < U < ctivity in Enzyme < < ctivity of DN Modifying Enzymes in uffer selection of DN modifying enzymes were assayed in uffer, in lieu of their supplied buffers. Functional activity was compared to the activity in its supplied buffer, plus required supplements. Reactions were set up according to the recommended reaction conditions, with uffer replacing the supplied buffer. NEuffer : mm Nal, mm Tris-Hl, mm Mgl, µg/ml S (ph ). uffer: mm Potassium acetate, 0 mm Tris-acetate, mm Magnesium acetate, µg/ml S (ph ). uffers should be thawed completely and mixed thoroughly before using. mm is Tris Propane-Hl, mm Mgl, µg/ml S (ph ). NEuffer.: mm Nal, mm Tris-Hl, mm Mgl, µg/ml S (ph ). NEuffer.: dcm methylation sensitivity < < RE-Mix Master Mix version of this enzyme is available < NEuffer ompositions (X) pg methylation sensitivity (applies to eukaryotic genomic DN only) Time-Saver qualified Engineered for maximum performance < dam methylation sensitivity Recombinant. Supplied with a separate vial of S-adenosylmethionine (SM). To obtain % activity, SM should be added to the X reaction mix as specified on the product data card. Supplied with its own unique reaction buffer that is different from the four standard NEuffers. ompatibility with the four standard NEuffers is indicated by the chart. SM hart Legend U < 4 + S selection of High Fidelity (HF) enzymes have been engineered for reduced star activity and flexibility in reaction setup < > 00 restriction enzymes are % active in uffer Over 80 Time-Saver qualified enzymes digest DN in 5-5 minutes and additionally can be safely used overnight with no loss of sample More recombinant enzymes than any other supplier ut Smarter with NE Restriction Enzymes The largest selection of enzyme specificities. < < te < anada New England iolabs, Ltd. toll free info.ca@neb.com < < te: The values listed in this table are approximate. They were obtained using each enzyme s specific unit assay substrate DN.. Star activity may result from extended digestion, high enzyme concentration or a glycerol concentration of > 5%.. Star activity may result from extended digestion.. Star activity may result from a glycerol concentration of > 5%. * May exhibit star activity in this buffer hina, People s Republic New England iolabs (eijing), Ltd. telephone /87866 info@neb-china.com France New England iolabs France free call info.fr@neb.com Germany & ustria New England iolabs GmbH free call 0800/46 57 (Germany) free call 00800/ (ustria) info.de@neb.com Japan New England iolabs Japan, Inc. telephone +8 (0) info@neb-japan.com Singapore New England iolabs Pte. Ltd. telephone sales.sg@neb.com United Kingdom New England iolabs (UK), Ltd. call free info.uk@neb.com For a complete list of international offices, please visit Restriction Endonuclease rochure & Performance hart Poster request your free copy now! L. < Restriction Endonucleases Over 80 of our restriction enzymes are able to digest DN in 5-5 minutes, and can safely be used overnight with no loss of sample. For added convenience and flexibility, most of these are supplied with our new uffer. Performance hart for NE Restriction Enzymes NE has developed a line of High-Fidelity (HF) restriction enzymes. These engineered enzymes have the same specificity as the native enzyme, with the added benefit of dramatically reduced star activity, rapid digestion (5-5 minutes), and % activity in uffer. Enjoy the improved performance of NE s engineered enzymes at the same price as the native enzymes! Speed up digestions with Time-Saver Qualified Restriction Enzymes 6 High-Fidelity (HF) Restriction Enzymes are currently available 5 min 5 min hr o/n µl Unwanted leavage HF enzymes out perform the competition. EcoRI-HF (NE #R) exhibits no star activity in overnight digests, even when used at higher concentrations. μl reactions were set up using μg of Lambda DN, the indicated amount of enzyme and the recommended reaction buffer. Reactions were incubated overnight at. Marker M is the kb DN Ladder (NE# N). Optional Time-Saver digest: pxba DN was digested with EcoRV-HF according to the recommended protocol. Lane L is the TriDye -Log DN Ladder (NE #N70). omplete digestion, free of unwanted star activity, is seen whether incubated for 5 5 minutes, hour or overnight.

9 Technical tips FQ Spotlight uffer for Restriction Enzymes Q: How is NE s new buffer system going to help me? : lthough the previous buffer system worked well, NE is continuously looking for ways to enhance the convenience and performance of its products for our customers. Our new buffer system includes S in all reaction buffers, and no longer contains DTT. y adding S to the reaction buffer, we were able to offer even more enzymes that cut in a single buffer (> 00). This improves ease-of-use, especially when performing double digests. In addition, it eliminated the need to add S when setting up restriction enzyme digests. hallenge your friends and colleagues to a game of G Q: If I have an old tube of Restriction Enzyme, what NEuffer should I use? : ll NE Restriction Enzymes have color coded labels for the appropriate NEuffer; this system can either be used with the previously supplied NEuffer or with the newly recommended buffer. G Q: I currently have an old tube of Restriction Enzyme is it still active in the new buffer? : Yes. The new buffers are mostly identical, except that S has been added directly to the buffer and DTT has been removed. S will not harm the reaction and may even enhance it in some cases. Extensive testing has shown that DTT is not required in the reaction. New multi-player format Test your skills at NEcutitout.com by Q: I am still using your previous buffer system. Where can I find performance information for the previous buffers? : Information for the previous buffer system is available on every restriction enzyme product page at www. neb.com, as well as from the Double Digest Finder and ctivity Performance hart, which are both available online. Visit NE.com for the full list of FQs. Learn More with Our Video Tech Tips at Want More? use the video carousel on the restriction enzyme Learn More page to find additional video tech tips from NE scientists. How will NE s uffer help me? Setting up a restriction enzyme digest with RE-Mix Master Mixes Standard protocol for restriction enzyme digests 9

10 Special prices How many DN Polymerases do you need in your freezer? You only need TWO OneTaq and Q5 DN Polymerases, the complete solution for your PR needs. Whether you are performing routine PR, or amplifying more challenging templates, New England iolabs has the solution for you. hoose OneTaq DN Polymerase for routine amplification of standard, T- and G-rich templates, at a price-point that beats the competition. hoose Q5 High- Fidelity DN Polymerase when you need high-fidelity amplification. With a fidelity > X Taq DN Polymerase and X Thermo Scientific Phusion DN Polymerase, Q5 delivers superior performance for complex templates with high-t and -G content. Don t fill your freezer with DN polymerases that can only amplify some of your templates. hoose OneTaq and Q5 and clean out your freezer once and for all! dvantages of OneTaq exceptional performance across a wide range of templates Robust yields with minimal optimization onvenient product formats Hot start version allows room temperature reaction setup and does not require a separate activation step ompatible with standard Taq protocols dvantages of Q5 Highest fidelity amplification available (> X higher than Taq and X higher than Thermo Scientific Phusion) High specificity and robust yields with minimal optimization Superior performance for a broad range of amplicons (from high T to high G) Fast with short extension times mplifications up to 0 kb for simple templates, and kb for complex ones OneTaq Q5 High-Fidelity DN Polymerases DN Polymerases chieve robust amplification for routine, T- and G-rich templates with OneTaq chieve the highest fidelity amplification available with Q5 kb M %G T-rich Standard G-rich High G Standard Reaction uffer G Reaction uffer Plus High G Enhancer Fidelity (x Taq) >x Taq * Q5 >x Taq Phusion 6x Taq 0x Taq 9x Taq 6x Taq 4x Taq ccuprime Pfx PfuUltra II Fusion HS PfuUltra Platinum Taq HiFi KOD Of my 6 reactions each showed higher yield in the OneTaq experiment compared to standard Taq. Graduate Student, Drexel University ollege of Medicine Q5 DN Polymerase gave me wonderful results on the first shot. Research technologist, University of Nebraska Medical enter We switched from KOD to Phusion polymerase which at the time seemed the best. ut now, the new Q5 polymerase is among the best available in the market, even better than Phusion. Highly recommended to others! Ph.D. Student, University of ambridge

11 Order Q5 and OneTaq Polymerases now and benefit from our special prices! Product NE # size Q5 High-Fidelity DN Polymerase M049S/L u/0 u Q5 High-Fidelity X Master Mix M049S/L rxns/0 rxns ( µl) Q5 Hot Start High-Fidelity DN Polymerase M049S/L u/0 u Q5 Hot Start High-Fidelity X Master Mix M0494S/L rxns/0 rxns ( µl) OneTaq DN Polymerase M0480 S/L/X 00/,000/5,000 u OneTaq Quick-Load X Master Mix with Standard uffer OneTaq Quick-Load X Master Mix with G uffer OneTaq Hot Start DN Polymerase OneTaq Hot Start Quick-Load X Master Mix with Standard uffer OneTaq Hot Start Quick-Load X Master Mix with G uffer M0486 S/L M0487 S/L M048 S/L/X M0488 S/L M0489 S/L /0 rxns ( µl) /0 rxns ( µl) 00/,000/5,000 u /0 rxns ( µl) /0 rxns ( µl) For more information, availability of samples as well as your special prices, please contact your local distributor! lso available: the updated NE PR rochure! For technical tips visit and Q5 & OneTaq Special Prices! offer closes 5..0 ontact your local distributor for details! PR Reagents POLYMERSES, NULEOTIDES, & DN LDDERS PR rochure request your free copy now! Learn More with Our Video Tech Tips at Want More? neb scientists have the expertise you need. Use the video carousel on Q5PR.com to find additional video tech tips from NE scientists. How to mplify G-rich DN hoose the Right DN Polymerase for PR Why hoose Q5 High-Fidelity Polymerase?

12 New England iolabs, Inc., 40 ounty Road, Ipswich, M PURExpress In Vitro Protein Synthesis Kit The PURExpress In Vitro Protein Synthesis Kit is a cell-free transcription/translation system reconstituted from purified components necessary for E. coli translation. ll of the activities have been expressed from recombinant sources, enabling PURExpress to be free of contaminating exonucleases, RNases and proteases. This prevents degradation of template DN and results in proteins that are free of modification and degradation. Transcription and translation are carried out in a one-step reaction, and require the mixing of only two tubes. With results available in only a few hours, PURExpress saves valuable laboratory time and is ideal for high throughput technologies. Schematic diagram of protein synthesis and purification using PURExpress. Protein synthesis using PURExpress PURExpress ddition of template DN initiates protein synthesis reaction Incubation at for hours µl reactions containing ng template DN were incubated at for hours..5 µl of each reaction was analyzed by SDS-PGE using a 0% Tris-glycine gel. te that proteins can be purified using affinity chromatography (reagents not supplied). The red dot indicates the protein of interest. Marker M is the Protein Ladder (NE #P770). Ordering Information kda dvantages: Easy-to-use - requires the mixing of two tubes followed by the addition of template DN Fast - reaction is complete in approximately just two hours Efficient - purified components enable more control of translational, ecretory and folding machinery leaner system - lack of endogenous proteases or nucleases eliminates sample degradation Flexible - can be used for circular (plasmids) or linear (PR) template Protein expression using the PURExpress In Vitro Protein Synthesis Kit M no DN DHFR styi mss-blac MP SV9HI T4 ligase NLS-re-E Vent exo b-gal DN loning DN mplification & PR Epigenetics RN nalysis Library Prep for Next Gen Sequencing Protein Expression & nalysis ellular nalysis Your local NE distributor: enelux: IOKÉ Schuttersveld 6 Z Leiden The Netherlands Tel: +(0) Tel: elgium: Fax: +(0)7 568 info@bioke.com ZEH REPULI: IOTEH a.s. Sluzeb Praha Tel: (Toll Free) Fax: biotech@biotech.cz DENMRK: iordika Denmark /S Marielundvej Herlev DENMRK Tel: (9) Fax: (9) info@bionordika.dk FINLND: iordika Oy Kutomotie Helsinki Tel: +58/07/4 70 Fax +58/07/4 77 info@bionordika.fi GREEE: IOLINE SIENTIFI Meg. lexandrou Str. 47 thens Tel: Fax: demagos@hol.gr HUNGRY: Kvalitex Kft Pannónia u udapest Phone: () Fax: () info@kvalitex.hu ISREL: Ornat iochemicals and Laboratory Equipment Ltd. PO 07 Rehovot 760 Tel: Fax: ornatbio@ornat.co.il ITLY: Eurolone S.P.. Via Figino 0/ 006 Pero (Milan) Free call: Tel: (0) 895 Fax: (0) 8465 info@euroclone.it rway: iordika rway S P.O. ox 98 6 Lysaker Tel Fax info@bionordika.no POLND: Lab-JOT Ltd. Sp.z o.o. l. Jerozolimskie 4, Warsaw stacjonarny: (+48 ) komórkowy: fax: (+48 ) labjot@labjot.com SWEDEN: iordika Sweden ox 60 S- Stockholm Tel: (08) 0 60 Fax: (08) info@bionordika.se SPIN/PORTUGL: IZS S.. Plaza de Europa, nº L Hospitalet de Llobregat (arcelona) Tel: Fax: consultasbiotec@izasa.es SWITZERLND: IOONEPT Paradiesrain 4 Postfach 47 H 4 llschwil Tel: (06) Fax: (06) info@bioconcept.ch TURKEY: SEM HYT TEKNOLOJILERI.D.T IS MERKEZI LOK NO:5 Macunkoy/NKR 0670 Tel: Fax: sacem@sacem.com.tr Product NE # SIZE PURExpress in vitro Protein Synthesis Kit E6800S/L / rxns PURExpress Ribosome Kit ES rxns PURExpress (aa, trn) Kit E6840S rxns PURExpress RF Kit E68S rxns Printed in Germany PURExpress Disulfide ond Enhancer E680S rxns