A Scientific Update

Fall/Winter 2017 expressions a scientific update

® ! No witchcraft required in this issue – Clone with Confidence 2 Overcoming the challenges of applying target enrichment for translational research 5 Now available: the NEBNext Direct® BRCA1/BRCA2 Panel 6 NEBNext® Ultra™ II RNA Library Prep kits – Streamlined workflows for lower input amounts 8 No witchcraft required: Special Prices on all Restriction Enzymes and more! 14 Get free tube opener with NEB competent cells 16 New to Molecular Biology? Get your free NEB Starter Pack be INSPIRED drive DISCOVERY stay GENUINE FEATURE ARTICLE

Overcoming the challenges of applying target enrichment for translational research by Andrew Barry, M.S., New England Biolabs, Inc.

Target enrichment is used to describe a variety of strategies to selectively isolate specific genomic regions of interest for sequencing analysis. The wide array of approaches presents challenges in selecting the appropriate technology for the growing number of research and clinical applications to which the sequencing data will ultimately be applied.

INTRODUCTION strates the practical need for continued use of Examples of these hybrid approaches include In recent years, several techniques have emerged target enrichment strategies across the gamut of multiplex extension ligation (3), molecular to enrich for specific genes of interest. When translational research activities. inversion probes (MIPS)/padlock probes (4), determining the appropriate target enrichment nested patch PCR (5), and selector probes (6). technology to use, one must first consider the TARGET ENRICHMENT These technologies can be broadly characterized primary goal of the study. For example, different APPROACHES as having more complex workflows, requiring approaches will be used if the aim is to identify splitting of samples into separate reactions, and known variants already shown to have clinical There are a number of different target enrich- creating challenges in target coverage uniformity. implications, versus discovering novel nucleic ment approaches that can be grouped into three generalized categories: in-solution hybridization, acid variants that may be associated with a given NEBNext Direct® for target enrichment phenotype. Variant identification lends itself multiplex PCR, and “alternative approaches”, which span a wide variety of techniques. NEBNext Direct is a novel target enrichment to more focused enrichment strategies, while method that addresses several drawbacks that variant discovery is driven by trade-offs between In-solution hybridization-based approaches, exist in alternative enrichment technologies sequencing costs and target territory, as well as originally developed for whole exome sequenc- (Table 1). Enrichment is achieved through available sample cohort sizes for a given study. ing, use biotinylated oligonucleotides to capture direct hybridization of biotinylated DNA baits As translational research seeks to bridge funda- genomic regions of interest (1). Commercial- to denatured, fragmented molecules, which mental laboratory research and clinical treatment ly-available kits use DNA or RNA baits ranging are subsequently captured using magnetic regimens for patients, there is an emerging from 50-150 nucleotides. Researchers have streptavidin beads (Figure 1, page 3). Unlike need to balance discovery of novel nucleic acid adapted this technique for more focused panels, alternative in-solution hybridization protocols, variants, identification of known variants, and ranging down to tens of kilobases in target the NEBNext Direct protocol does not require studies aimed at revealing associations with clin- territory, with limited success in maintaining library preparation prior to hybridization of ical phenotypes. Recent advances in sequencing specificity for target regions. oligonucleotide probes. This feature reduc- technologies have revolutionized the field of ge- Multiplex PCR-based enrichment is most often es the overall amount of amplification that is nomic research, making tractable the application employed for highly focused panels targeting a required throughout the protocol and enables of whole genome and whole exome sequenc- smaller territory than in-solution hybridization, single-stranded DNA to be captured along with ing for broad discovery of germline genomic and is typically limited to 150-200 amplicons denatured, double-stranded DNA. variants. However, despite these advances, the (2). Using a pool of primers, enrichment is Conversion of captured fragments to sequence- oncology field is fraught with the complexity of accomplished through PCR amplification of the ready libraries is achieved by the ligation of detangling the underpinnings of tumorigenesis, targeted regions, which is followed by adaptor a loop adaptor to the proximal 3´ end of the progression, and resistance mechanisms driven ligation or a second round of PCR using tailed captured molecule. During these steps, the bait / by somatic variants present at extremely low primers to include sequencing adaptors. Scaling target molecules remain bound to the magnetic abundance in mixtures of malignant and stromal this technology has presented a challenge in streptavidin beads and are processed in a single cells. These complexities necessitate increases in maintaining target coverage uniformity. reaction tube. This eliminates sample loss and the depth of sequencing coverage to confident- A number of alternative approaches have been improves overall conversion efficiency. ly call somatic variants, making broader scale developed in an attempt to bridge the gap be- approaches infeasible from an economic and tween hybridization and PCR-based approaches. practical standpoint. To overcome these challenges, focused gene panels are being applied to patient samples. The size TABLE 1: of the panel is highly variable, trending toward Enrichment Challenges and Advantages of NEBNext Direct decreased genomic content as assays progress from pure research and discovery applications to Challenge NEBNext Direct Advantage clinical diagnostic assays. Furthermore, clinical Specificity across panel sizes Enzymatic removal of off-target sequence applications raise the question of incidental findings and how to report them, introduc- Uniformity of coverage Individual synthesis of baits & empirical balancing ing challenges for diagnostic assays based on Sensitivity to detect variants Unique Molecule Indexes for PCR duplicate marking & consensus variant calling sequencing entire genomes. This trend demon- Degraded or low quality samples Short baits that extend across molecules, targeting both DNA strands 2 Following ligation of the 3´ adaptor, the bait FIGURE 1: is extended across the entirety of the captured NEBNext Direct target enrichment workflow molecule, resulting in double stranded DNA that is ready for ligation of the 5´ unique molecular 5´ 3´ Genomic identifier (UMI) adaptor. This adaptor contains 3´ 5´ DNA a 12 bp random sequence that is incorporated Target region discretely into each molecule, indexing each Fragmentation molecule prior to amplification. This index can 5´ 3´ be used to identify duplicate molecules, thereby 3´ 5´ reducing artifacts that can lead to false positive variant calls. Denaturation 5´ 3´ probe hybridization Biotin probe targets both 3´ 5´ Once the 5´ adaptor is ligated, the 3´ loop strands (shown for 1 strand) adaptor is cleaved, and the target molecule is PCR Biotin probe amplified off of the bait complex. It is important to note that the bait strand is not perpetuated 3 blunting of DNA 5´ 3´ Enzymatic removal through the PCR amplification and is not present 3´ 5´ of off-target sequence in the final, sequencer-ready library. Streptavidin bead The coverage plots of NEBNext Direct libraries are unique for a hybridization-based approach dA-tailing 5´ A 3´ in that reads have a defined 3´ end, resulting in 3´ 5´ coverage plots that resemble PCR-based libraries, yet the approach allows for flexibility in tiling across longer targets. Disambiguation of PCR duplicates is accomplished by two features of the Ligation of 3 adaptor 5´ A NEBNext Direct library: A variable 5´ end and a 3´ T 3´ adaptor 12 bp randomized UMI that is incorporated into the 5´ adaptor.

5 blunting Probes are extended to 5´ A CHALLENGES OF TARGET of DNA the 5 end of the randomly 3´ T sheared fragment, creating ENRICHMENT FOR a variable 5 end of the TRANSLATIONAL RESEARCH target read Specificity of target enrichment Ligation of 5 Unique Molecular 5´ A For any study that necessitates enrichment of spe- 5 UMI adaptor Identifier (UMI) addition 3´ T cific targets over more comprehensive sequencing 5´ UMI improves sensitivity approaches, specificity becomes more important as adaptor it directly translates to the amount of sequencing required to achieve the minimum coverage thresh- Adaptor cleaving 5´ A 3´ old to reliably detect variants of a given frequen- 3´ T 5´ cy. Specificity is typically measured by looking at the percentage of sequencing data that is derived from the targeted regions relative to the data that PCR amplification 5´ A 3´ is aligned to other parts of the reference genome. 3´ T 5´ Enrichment of genomic regions is typically Sequencing-ready fragment Incorporated sample index achieved by either amplifying the desired regions through PCR to generate enough copies of the targeted regions over the untargeted regions, in which randomly fragmented molecules are Uniformity of coverage across targets or through hybridization of complementary captured overnight, and without any removal One of the drawbacks to many available target biotinylated oligonucleotide probes to fragmented of upstream off-target sequences, read coverage enrichment methods is the inability to enrich DNA molecules, where specificity is driven resembles a normal distribution. different targets with equivalent efficiency. The through careful control of melting temperatures While specificity for targeted regions using tradi- result requires an increase in the overall coverage and buffer composition to promote hybridization. tional hybridization approaches is typically quite for all targets to achieve the minimum depth of Specificity for target regions is enhanced using high for larger panels up to whole exome, speci- coverage required to reliably call variants. One of NEBNext Direct through both the hybridization ficity typically decreases as the size of the target- the main factors influencing coverage uneven- of specific baits, as well as through enzymatic ed region decreases. Thus, smaller panels typically ness is the sequence composition of the targeted removal of off-target sequence. The enzymatic result in an increased proportion of sequencing regions themselves, with different efficiencies for treatment removes both off-target sequence of lost to off-target regions. In contrast, the NEB- sequences comprised of GC or AT rich regions. molecules unbound to baits, as well as the regions Next Direct approach maintains high specificity Depending on the approach, the target enrich- of molecules upstream of where the baits are across a broad range of target territory, from ment strategy being employed may be more bound. This additional means of driving speci- single genes or exons to hundreds of kilobases, or less susceptible to the need for balancing ficity enables the bait hybridization to be shorter, eliminating the need to use different technologies melting temperatures across any complementary lasting only 90 minutes in duration. This differs for different panels (Table 2, page 4). oligonucleotide baits or PCR primers that are from a typical hybridization-based approach, employed in the enrichment process. Challenges continued on page 4... 3 FEATURE ARTICLE continued...

in uniformity can also arise from any down- detect nucleic acid variants that are present at TABLE 2: stream PCR that is used to generate sufficient a given frequency, referred to as variant allele Specificity and uniformity of NEBNext material for the sequencing process, as various frequency (VAF) or mutation allele frequency Direct panels DNA polymerases demonstrate biases toward (MAF). Biologically, in the context of solid Panel Size Specificity Uniformity targets that may include secondary structure. tumors, this is a function of the mixture (kb) (% Reads on Target) (% bp >20% MTC*) of stromal and tumor cells, as well as the Using multiplex PCR-based workflows, primer 15.2 99.4 99.3 design is limiting as melting temperatures must heterogeneity of tumor cells, and the existence 15.9 96.1 100 match within each panel and primer-primer in- of subclonal variants that are associated with teractions and primer cross-talk must be consid- tumorigenesis. Utilization of sequence data for 20.4 99 99.5 ered. These constraints can lead to variations in the approximation of allele frequency is achieved 36.8 92.5 98.7 through counting of sequence reads that coverage uniformity between targets. Partitioning 76.4 91 98.5 individual amplification reactions into emulsion possess a given variant. Quantitative assessment droplets can alleviate some of these constraints of sequence reads is challenged through the 93 95.9 99.35 and improve target uniformity (12), but this presence of duplicate molecules, or molecules 217 90 99.23 that are identified through sequencing as having approach requires investment in instrumentation * bp – base pairs MTC – Mean Target Coverage as well as additional workflow steps. the same genomic coordinates. Depending on the target enrichment method that was Oligonucleotides utilized during NEBNext employed to prepare the samples for sequencing, Direct enrichment are individually synthe- disambiguation of molecules that have arisen The most widely used technique for the storage sized, which enables bait pools to be carefully from discrete copies of genomic DNA versus and preservation of tissue derived from patient optimized based on empirical testing. Individual those resulting from PCR amplification can be samples involves fixing the tissue in formalin, and baits are balanced, allowing fine tuning of target difficult or impossible to ascertain. embedding the fixed sample in paraffin. DNA coverage. Additionally, the bait design algorithm derived from formalin-fixed, paraffin embedded optimizes new bait design based on outcomes Disambiguation of PCR duplicates is accomplished by two features of the NEBNext (FFPE) samples has been shown to contain varying from prior results. Further, because the specificity degrees of degradation, accumulation of base- is not solely driven through melting tempera- Direct library: A variable 5´ end and a 12 bp randomized UMI that is incorporated into the specific errors, DNA breaks with damaged ends, tures alone, NEBNext Direct allows increased and are often present in extremely low quantities flexibility in bait design. 5´ adaptor. The amount of coverage one can expect from a given panel should be measured (7-9). The recent application of target enrichment The result is coverage across targets that can once duplicate molecules are removed in order to circulating cell-free DNA molecules offers a less be optimized, demonstrating high degrees of to determine if the coverage is deep enough to invasive means of monitoring cancer progression. uniformity and diminishing the overall amount reliably call a variant as a true-positive variant Cell-free DNA derived from solid tumors is of sequencing required to identify nucleic acid (Figure 3, page 5). biologically present in relatively short fragments of variants (Figure 2). 150-160 bp, which can present challenges using traditional enrichment approaches as both cell-free Sensitivity to detect nucleic acid variants Difficult sample types and FFPE tissue-derived nucleic acids contain high amounts of ssDNA (10, 11). Perhaps the most critical aspect is the sensitivity Whether for research or clinical applications, of an approach to detect nucleic acid variants, translational genomics often examines samples Using in-solution hybridization based enrichment as this is often the primary goal of studies in that are derived from patients. Patient tissue can presents challenges, as an upfront library must be humans where target enrichment is employed. be compromised by processes used to collect, prepared prior to hybridization to long (>100 bp) This is measured as the ability of an assay to preserve, store, extract nucleic acids from, and baits, and can result in sample loss. Moreover, deg- ultimately prepare for sequencing-based assays. radation of FFPE derived nucleic acids can create shorter library inserts not optimal for hybridization FIGURE 2: to longer baits. Finally, the initial library generation NEBNext direct delivers higher coverage uniformity than step requires dsDNA; thus, the approach disregards alternative approaches. ssDNA that may be present in the original sample Plot shows the uniformity across targets for each panel, measured as the percentage of bases above 25% of the mean due to DNA damage. target coverage. Samples were processed in duplicate according to the manufacturer’s suggested protocol using the recommended amount of DNA input. DNA used was a blend of 24 HapMap samples. Samples were sequenced on an Multiplex PCR also presents challenges in Illumina® MiSeq® per the manufacturer recommendation. Representative data across 2 replicates are shown. targeting degraded samples, as the ability to successfully anneal both primers on a given rse se ere molecule is difficult as DNA input molecule length 100 is decreased due to degradation. 95 The short (~45-55 nucleotide) baits used in 90 NEBNext Direct enrichment provide an increased 85 probability of binding to shorter fragments, and 80 the independent targeting of both strands of DNA 75 offers improved opportunity to capture degraded 70 fragments. The approach also contains an optional 65 phosphorylation step to ensure the ends of target 60 DNA are prepared for ligation of adaptors. 55 Bases 25% Mean Coverage (%) 50 ex re 37 kb 37 kb 23 kb 51.1 kb 20 kb Cancer 35 kb 37 kb Cancer Competitor Competitor Oncology Solid Tumor Research Panel Cancer Panel HotSpot Panel Custom Custom Panel Panel (Selector (Extension Panel A Panel B Probe) Ligation) 4 FIGURE 3: CONCLUSION NEBNext Direct is able to achieve high depths of sequence coverage across NEBNext Direct target enrichment overcomes a broad range of inputs. several challenges translational researchers face Mean depth of coverage relative to sequencing depth is shown across a range of DNA inputs. A blend of 24 HapMap samples were in selectively enriching for certain genomic enriched using the 37 kb NEBNext Direct Cancer HotSpot Panel and sequenced on an Illumina MiSeq using 2 x 75 base pair sequencing. Coverage is shown after the removal of PCR duplicates using the information from the unique molecular identifier (UMI). targets for clinical research. Providing the flexibility to use a single approach across a 2,000 500 ng wide range of target content, NEBNext Direct

1,800 200 ng allows enrichment of a single gene, up to 100 ng panels comprised of hundreds of genes, without 1,600 50 ng compromising performance as targets change. NEBNext Direct provides the specificity and 1,400 25 ng 10 ng coverage uniformity to maximize sequencing 1,200 efficiency, in order to realize the benefits of target enrichment. Furthermore, intrinsic 1,000 properties of the approach lend themselves

800 to improved sensitivity, and have proven amenable to challenging sample types, typical 600 of translational workflows. Combining the best aspects of hybridization-based enrichment and 400 multiplex PCR enrichment, without the trade-

Mean Deduplicated Target Coverage Depth 200 offs, NEBNext Direct is a single-day, easy-to- use protocol that can be applied to advance 0 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 translational research. Millions of Reads

References 1. Gnirke, A., et al. (2009) Nature Biotechnology 27, 182–189. 6. Johansson, H., et al. (2011) Nucleic Acids Research 39:e8. Learn more about NEBNext Direct 2. Mertes, F., et al. (2011) Briefings in Functional Genomics 10, 7. Srinivasan, M., et al. (2002) The American Journal of Pathology 374–386. 161, 1961–1971. at NEBNextDirect.com 3. Shen, M.-J. R., et al. (2011) Multiplex nucleic acid reactions. US 8. Costello, M, et al. (2013) Nucleic Acids Research 41(6):e67. Patent 7955794 B2. 9. Chen, L., et al. (2017) Science 355, 752-756. 4. Porreca, G. J., et al. (2007) Nature Methods 4, 931–936. 10. Stiller, M., et al. (2016) Oncotarget 37(7), 59115-59128. 5. Varley, K. E., and Mitra, R. D. (2008) Genome Research 18, 11. Burnham, P., et al. (2016) Scientific Reports 6, 27859. 1844–1850. 12. Tewhey, R., et al. (2009) Nature Biotechnology 27, 1025–1031.

New target enrichment Product: Benefits

NEBNext Direct BRCA1/BRCA2 Panel • Generate full (100%) coverage of all protein coding regions in BRCA1 and The NEBNext Direct BRCA1/BRCA2 Panel (NEB # E6627S/L/X) enriches the complete exon BRCA2 genes content of BRCA1 and BRCA2 genes for NGS analysis. NEBNext Direct employs a unique enrichment • Obtain highly uniform sequencing workflow that hybridizes baits directly to genomic DNA, without the need for upfront library prepa- across exon target – 100% of base ration. The BRCA1/BRCA2 panel demonstrates high specificity and unmatched coverage uniformity pairs in panel have coverage greater across a wide range of DNA inputs, allowing highly sensitive calling of germline and somatic variants, than 20X of the mean target coverage while maximizing sequencing efficiency. • Save time with a 1-day workflow IGV plot showing complete and even coverage across exon 1 of the BRCA1 gene that combines enrichment with library preparation Integrative Genomics Viewer (IGV) Plot of exon 1 from BRCA1 gene showing read level coverage obtained using 50 ng DNA enriched using the NEBNext Direct BRCA1/BRCA2 Panel. Sequencing was performed on an Illumina MiSeq using 2 x 75 paired-end sequencing. • Distinguish molecular duplicates, reducing false-positive variants and improving assay sensitivity • Produce high depths of target coverage across a wide range of DNA input amounts for germline and somatic variants

PRODUCT NEB # SIZE

NEBNext Direct® E6627S/L/X 8 / 24 / 96 rxns BRCA1 / BRCA2 Panel NEW

ALSO AVAILABLE: NEBNext Direct® E7000S/L/X 8/24/96 rxns HotSpot Panel See NEBNextDirect.com 5 Even more from less

Do you need increased sensitivity and specificity from your RNA-seq experiments? Do you have ever-decreasing amounts of input RNA? To address these challenges, our next generation of RNA library prep kits have been Advantages reformulated at each step, resulting in several fold higher yields of high quality libraries, enabling use of lower input amounts and fewer PCR cycles. The kits • Generate high yield, high-quality have streamlined, automatable workflows and are available for directional libraries even with limited amounts (strand-specific, using the “dUTP method”(1,2)) and non-directional library prep, of RNA: with the option of SPRISelect® beads for size selection and clean-up steps. ° 10 ng – 1 µg total RNA (poly(A) mRNA workflow)

° 5 ng – 1 µg total RNA NEBNext Ultra II Directional RNA produces the highest yields, from a range of (rRNA depletion workflow) input amounts Poly(A)-containing mRNA was isolated from Universal Human Reference RNA (Agilent® #740000) and libraries were made using the NEBNext • Minimize bias, with fewer PCR Ultra II Directional RNA Kit (plus the NEBNext poly(A) mRNA Magnetic Isolation Kit), Kapa Stranded mRNA-Seq Kit, Kapa mRNA HyperPrep Kit cycles required and Illumina TruSeq Stranded mRNA Kit. The input RNA amount and number of PCR cycles are indicated. Library yields from an average of three replicates are shown. • Increase library complexity and transcript coverage 2,000 NEBNext® Ultra II Directional RNA • Increase flexibility by ordering 1,800 Kapa Stranded Kapa HyperPrep reagents specific to your 1,600 Illumina® TruSeq® Stranded workflow needs

1,400 ° Directional and non-directional

1,200 kits available

ield (ng) 1,000 ° rRNA depletion and poly(A) mRNA isolation reagents

otal 800 T supplied separately 600 ° Adaptors and primers 400 (12-, 96-, and dual index) 200 supplied separately

0 • Enjoy the reliability of the gold PCR Cycles 9 13 16 standard SPRISelect size selection Total RNA Input 1 µg 100 ng 10 ng and clean-up beads, supplied in just the amounts you need • Save time with streamlined The Ultra II RNA kit has allowed us to reduce the input for directional workflows, reduced hands-on time, poly(A)+ RNA-seq libraries by a factor of 10 or more. We can now make a and automation compatibility “ library with only 10 ng high quality total RNA and get the same gene expression • Rely on robust performance, even profile as for 1 µg input. We've even pushed the input as low as 1 ng for very with low quality RNA, including FFPE high quality total RNA. The new Ultra II RNA kit makes RNA-seq achievable for low yield samples, we actually need more RNA for quality control than for library prep! Furthermore, the library prep protocol is streamlined compared to the previous Ultra RNA kits, including a reduction in AMPure bead clean-ups and PCR cycles, resulting in better libraries for less time and resources.

– Jen Grenier, Ph.D., Director of RNA Sequencing Core (RSC), Center for Reproductive Genomics, Department of Biomedical Sciences, ” For more information and to request College of Veterinary Medicine, Cornell University a sample, visit UltraIIRNA.com

1. Parkhomchuck, D., et al. (2009) Nucleic Acids Res. 37. e123. 2. Levin, J. Z., et al. (2010) Nature Methods 7, 709–715. 6 NEW PRODUCTS NEBNext® Ultra™ II RNA Library Prep Kits

NEBNext Ultra II Directional RNA libraries provide uniform GC content distribution, for a broad range of input amounts Poly(A)-containing mRNA was isolated from Universal Human Reference RNA (Agilent #740000), and libraries were made using the NEBNext Available kits: Ultra II Directional RNA Kit (plus the NEBNext Poly(A) mRNA Magnetic Isolation Module), Illumina TruSeq Stranded mRNA Kit, Kapa Stranded mRNA-Seq Kit and Kapa mRNA HyperPrep Kit. Libraries were sequenced on an Illumina NextSeq® 500 using paired-end mode (2x76 bp). Reads were mapped to the hg19 reference genome. GC content distribution for each library was calculated using mapped reads. Ultra II Directional RNA • NEBNext Ultra II Directional RNA libraries had uniform GC content distribution across a range of input amounts, as indicated by the curve overlap. For other kits the GC content distribution changed with different input amounts, indicating the introduction of input-dependent sequence bias. Library Prep Kit for Illumina ex r re Includes optimized mixes for 0.05 exre r 1 µg 0.05 re re 1 µg 100 ng 1100 µg ng 1 µg directional RNA library preparation 0.05 re 0.05 re 10 ng 10010 ng ng 100 ng (fragmentation, cDNA synthesis, end 10 ng 10 ng 0.04 0.04 repair/dA-tailing, adaptor ligation 0.04 0.04 and PCR enrichment steps) for sequencing on the Illumina platform. 0.03 0.03 0.03 0.03 NEB # E7760S/L 24/96 rxns

0.02 0.02 Fraction of Reads 0.02 Fraction of Reads 0.02 • NEBNext Ultra II Directional Fraction of Reads Fraction of Reads RNA Library Prep with Sample 0.01 0.01 Purification Beads 0.01 0.01 Includes optimized mixes for directional RNA library preparation 0 0 20 30 40 50 60 70 80 90 20 30 40 50 60 70 80 90 0 0 (fragmentation, cDNA synthesis, end 20 30 40GC Content50 60 (%) 70 80 90 20 30 40 GC 50Content60 (%)70 80 90 repair/dA-tailing, adaptor ligation GC Content (%) GC Content (%) and PCR enrichment steps) plus p re p per SPRISelect® beads for size selection 0.05 p re 1 µg 0.05 p per 1 µg 100 ng and clean-up. 1100 µg ng 1 µg 0.05 0.05 10 ng 10010 ng ng 100 ng 10 ng 10 ng NEB # E7765S/L 24/96 rxns 0.04 0.04 0.04 0.04 • NEBNext Ultra II RNA Library Prep 0.03 0.03 Kit for Illumina 0.03 0.03 Includes optimized mixes for non- directional RNA library preparation 0.02 0.02

Fraction of Reads 0.02 Fraction of Reads 0.02 (fragmentation, cDNA synthesis, end

Fraction of Reads Fraction of Reads repair/dA-tailing, adaptor ligation 0.01 0.01 and PCR enrichment steps) for 0.01 0.01 sequencing on the Illumina platform.

0 0 NEB # E7770S/L 24/96 rxns 20 30 40 50 60 70 80 90 20 30 40 50 60 70 80 90 0 0 20 30 40GC Content50 60 (%) 70 80 90 20 30 40GC 50Content60 (%)70 80 90 GC Content (%) GC Content (%) • NEBNext Ultra II RNA Library Prep PRODUCT NEB # SIZE with Sample Purification Beads NEBNext Ultra II Directional RNA Library Prep Kit for Illumina E7760S/L 24/96 rxns Includes optimized mixes for non- NEBNext Ultra II Directional RNA Library Prep with Sample Purification Beads E7765S/L 24/96 rxns directional RNA library preparation (fragmentation, cDNA synthesis, end NEBNext Ultra II RNA Library Prep Kit for Illumina E7770S/L 24/96 rxns repair/dA-tailing, adaptor ligation NEBNext Ultra II RNA Library Prep with Sample Purification Beads E7775S/L 24/96 rxns and PCR enrichment steps) plus OTHER PRODUCTS YOU MIGHT BE INTERESTED IN: SPRISelect beads for size selection NEBNext rRNA Depletion Kit (Human/Mouse/Rat) E6310S/L/X 6/24/96 rxns and clean-up. NEBNext rRNA Depletion Kit (Human/Mouse/Rat) E6350S/L/X 6/24/96 rxns with RNA Sample Purification Beads NEB # E7775S/L 24/96 rxns NEBNext Poly(A) mRNA Magnetic Isolation Module E7490S/L 24/96 rxns NEBNext Ultra II RNA First Strand Synthesis Module E7771S/L 24/96 rxns NEBNext Ultra II Directional RNA Second Strand Synthesis Module E7550S/L 24/96 rxns see all details in new catalog: NEBNext Ultra II Non-Directional RNA Second Strand Synthesis Module E6111S/L 20/100 rxns NEBNext Multiplex Oligos for Illumina (Index Primers Set 1, 2, 3, 4) E7335, E7500, E7710, E7730S/L 24/96 rxns p. 150 -169 NEBNext Multiplex Oligos for Illumina (96 Index Primers) E6609S/L 96/384 rxns NEBNext Multiplex Oligos for Illumina (Dual Index Primers Set 1) E7600S 96 rxns 7 NEB: No Witchcraft required

SPECIAL PRICE ON RESTRICTION ENZYMES & CLONING PRODUCTS* until 31.01.2018

*No Witchcraft! Get special prices on selected NEB Cloning Products incl. all Restriction Enzymes (Art.# Rnnnn). All other eligible discounted products are marked with the "Special Price" icon within this newsletter (pages 8-13). Campaign ends Jan. 31st, 2018. Please ask your local distributor for details.

For some, Molecular Biology seems like witchcraft. And even professional researchers may have encountered situations during their career, where experiments failed or sometimes even worked and nobody had an explanation, why. Witchcraft?

Molecular Biology does not need no witchcraft. Instead, it needs high quality reagents with superior and reliable performance – even under suboptimal reaction conditions. Especially when it comes to cloning, a work flow of numerous, sometimes cumbersome steps: from DNA preparation, digestion and modification to transformation and analysis – you just don't want to risk or spoil your precious samples and working time. Benefit from NEB's unrivaled reagent quality, reliability, convenience and performance – Clone with Confidence®!

Cut Smart with High-Fidelity & Time-Saver! SPECIAL NEB offers the largest selection of restriction enzymes commercially available: 285 enzymes to choose from including traditional restriction PRICE* enzymes, nicking endonucleases, homing endonucleases and methylation-sensitive enzymes for epigenetics studies. until 31.01.2018

® • High-Fidelity (HF ) Restriction Enzymes – engineered for performance! E R NEB EcoRI-HF Competitor EcoRI 1 3 5 10 M 1 3 5 10 µl Engineered by NEB's R&D Team for superior performance, our unique High-Fidelity (HF) restriction enzymes offer dramatically reduced star activity (up to 62.500-fold less). This means that you encounter virtually "no" off-target cleavage and degradation of end-product leading to higher cloning efficiency, reliability and greater confidence in the accuracy of your results. Unwanted High-Fidelity enzymes have the same specificity as the native counterparts. All HF enzymes Cleavage are also Time-Saver™ qualified for rapid 5-15 minutes digests and offer 100% activity in NEB's regular CutSmart Buffer for easy reaction set-up and double digests. Like all other top selling NEB restriction enzymes, HF enzymes are supplied with free purple gel loading HF enzymes outperform the competition. EcoRI-HF (NEB #R3101) exhibits no star activity in overnight buffer as added convenience and extra bonus. digests, even when used at higher concentrations. 50 μl reactions were set up using 1 μg of Lambda DNA, the indicated amount of enzyme and the recommended reaction buffer. Reactions were incubated overnight at 37°C. Marker M is the 1 kb DNA Ladder (NEB# N3232). For illustration purposes, the part of the gel below approx. 1.2 kb is not shown. 8 Clone with Confidence®!

• Simplify Reaction Setup and Double Digestion with CutSmart® Buffer Advantages With the CutSmart reaction buffer system, we offer >210 restriction enzymes (incl. all HF enzymes) that are 100% active in a single buffer. This improves ease-of-use, especially when performing double digests. Additionally, most commonly used cloning enzymes (including T4 DNA Ligase etc.) are 100% • 285 restriction enzymes to choose from active in CutSmart Buffer, eliminating the need to change buffers in subsequent cloning workflows. • Unique engineered High-Fidelity SPECIAL (HF) restriction enzymes for superior • Speed up digestions with Time-Saver™ Qualified Restriction Enzymes C performance (32 specificities) 193 of our restriction enzymes (incl. all HF enzymes) are able to digest DNA in 5-15 minutes, and can • Over 210 restriction enzymes are safely be used overnight with no loss or degradation of product (see figure below). 100% active in a single buffer – PRICE CutSmart Buffer ON RESTRICTION ENZYMES • 193 restriction enzymes are • Improve your analysis with our Purple Gel Loading Dye Time-Saver qualified (5-15 min digest) * & CLONING PRODUCTS Our Gel Loading Dye, Purple (6X), is supplied as a free bonus with all top selling restriction enzymes (incl. • All Top 30 modifying/cloning enzymes all HF enzymes). It eliminates the UV shadow often seen with other dyes. It also sharpens the bands and are 100% active in CutSmart Buffer prevents from smear since this solution contains SDS, as some restriction enzymes are known to remain • Purple loading dye is included bound to DNA following cleavage. (product dependent)

L 5 min 15 min 1 hr o/n Activity of DNA Modifying Enzymes conventional dye NEB's purple dye in CutSmart Buffer: Clone smarter!

Activity Required Supple- Enzyme in CutSmart ments Phosphatases: Alkaline Phosphatase (CIP) + + + Antarctic Phosphatase + + + Requires Zn2+ Quick CIP + + + Shrimp Alkaline Phosphatase (rSAP) + + + Ligases: No UV-shadow and sharp bands: Optional Time-Saver digest: T4 DNA Ligase + + + Requires ATP Lane 1: UV shadow typical for conventional loading dyes pXba DNA was digested with EcoRV-HF according to the recom- E. coli DNA Ligase + + + Requires NAD (e.g. Bromophenol-Blue); Lane 2: NEB's purple loading T3 DNA Ligase + + + Requires ATP + PEG mended protocol. Lane L is the 2-Log DNA Ladder (NEB #N3200). buffer as supplied with all top selling restriction enzymes T7 DNA Ligase + + + Requires ATP + PEG Complete digestion, free of unwanted star activity, is seen whether incubated for 5–15 minutes, 1 hour or overnight. For illustration Polymerases: purposes, the part of the gel below approx. 0.9 kb is not shown. T4 DNA Polymerase + + + DNA Polymerase I, Large (Klenow) Frag. + + + DNA Polymerase I + + + DNA Polymerase Klenow Exo– + + + NEB Restriction Enzyme Quality outperforms any competitor. Bst DNA Polymerase + + + phi29 DNA Polymerase + + + Example: Nuclease Contamination. T7 DNA Polymerase (unmodified) + + + Transferases/Kinases: Single-strandedSingle-strandedSingle-strandedDouble-strandedDouble-strandedDouble-stranded blunt top blunt blunt top top 3´ OverhangSingle-stranded3´ 3 topOverhang´ Single-strandedOverhang Single-strandedSingle-stranded top top 5´ OverhangDouble-strandedDouble-stranded5´5 Double-stranded tOverhangópDouble-stranded Overhang blunt top top top blunt blunt Overhangblunt top top top OverhangOverhang3´ Overhang3´ 3Overhang3´ Óverhang topOverhang Single-stranded Single-strandedtop top top 5´ Overhang5´5 Overhang5´ Óverhang tOverhangopDouble-strandedDouble-stranded top t opt op Overhang blunt blunt topOverhang topOverhangOverhang 3´3 Óverhang Overhang top top 55´ Óverhang Overhang t optop OverhangOverhang Double-strandedDouble-strandedDouble-stranded blunt bottom blunt blunt bottom bottom3´ Overhang3´ 3 bOverhangó Overhangttom b obtotomttom 5 ´ OverhangDouble-strandedDouble-stranded5´5Double-stranded bOverhang´Double-strandedo Overhangttom blunt b obto tbottomom tblunttom blunt blunt bottom bottom bottom 3´ Overhang3´3 Overhang3´ Óverhang bOverhangottom b o bt btoomotttomtom 5´ Overhang5´5 Overhang5´ Óverhang bOverhangDouble-strandedoDouble-strandedttom b o bt btoomotttomtom blunt blunt bottom bottom 3´3 Óverhang Overhang b bootttomtom T4 Polynucleotide55´ Óverhang Overhang b boot ttomKinasetom + + + Requires ATP + DTT T4 PNK (3´ phosphatase minus) + + + Requires ATP + DTT 16 1210201616 16120 161161216201021020 120 1210161216200 112200 120 121020 120 120 CpG Methyltransferase (M. SssI) + + + 14 1414 14 141414 1414 GpC Methyltransferase (M. CviPI) + Requires DTT 100 100100 100 100100100 100 100100100100 100 100100100 100100 T4 Phage β-glucosyltransferase + + + 12 1212 12 121212 1212 Nucleases, other: 80 8080 80 808080 80 80808080 80 808080 8080 1010 1010 10 1010 10 10 DNase I (RNase free) + + + Requires Ca2+

60 6060 608 6060608 8 860 86086086060 60 60608608 6060 Endonuclease III (Nth), recombinant + + + adation (%) adation (%) adation (%) adation (%) adation (%) adation (%) adation (%) adation (%) adation (%) adation (%) adation (%) adation (%) adation (%) adation (%) adation (%) adation (%) adation (%) adation (%) adation (%) adation (%) adation (%) adation (%) adation (%) adation (%) adation (%) adation (%) adation (%) Endonuclease VIII + + + 6 6 6 66 66

6 Deg r Deg r 6 Deg r Deg r Deg r Deg r Deg r Deg r Deg r Deg r Deg r Deg r Deg r Deg r Deg r Deg r Deg r Deg r Deg r Deg r Deg r Deg r 40 Deg r 4040 Deg r 40 4040Deg r 40 Deg r 40 40Deg r 404040 40 404040 4040 Exonuclease III + + + 4 4 4 4 4 44 44 Lambda Exonuclease + + 20 2020 20 202200 20 22002020 20 202200 2200 McrBC + + + 2 2 2 2 2 22 22 Micrococcal Nuclease + + Requires Ca2+ 0 0 0 00 0 000 0 0 0 0 0000 0 0 0 00000 00 RecJ + + + EcoRI EcoRI-HFEcoRIEcoRI EcoRI-HFEcoRI-HFA ABA BCB CDC DD EcoRINotI EcoRIEcoRI-HFEcoRIEcoRINotI-HFNotINotIEcoRI-HFEcoRI-HFEcoRI-HFNotI-HFNotI-HFAA A AABABA B BBCBCB C CCDCDC D DDDD NotIBamHINotIEcoRINotI-HFNotIBamHI-HFEcoRINotIBamHIBamHINotI-HFEcoRI-HFNotI-HFEcoRI-HFNotI-HFBamHI-HFBamHI-HFAA A AAABABA B BBBCBCB C CCCDCDC D DDDD BamHIBamHIBamHIBamHI-HFNotIBamHINotI BamHI-HFBamHI-HFNotI-HFBamHI-HFNotI-HFA AAAAAB BBBBBC CCCCCD DDDD BamHIBamHI BamHI-HFBamHI-HFf AA BB CC DD NEB EcoRINEBNEB EcoRI EcoRI CompetitorCompetitorCompetitor EcoRI EcoRI EcoRI NEBNEB EcoRINEB NotINEBNEB NEBEcoRI NEBEcoRI EcoRI NotI NotI CompetitorCompetitorCompetitorCompetitorCompetitorCompetitor CompetitorEcoRI NotI EcoRI EcoRI EcoRI NotI NotI NEB NotIBamHINEBNEBNEBNEBNEB NEBNotI EcoRI NotI EcoRINotIBamHI BamHI CompetitorCompetitorCompetitorCompetitorCompetitorCompetitorCompetitor NotIBamHI NotI EcoRI NotI EcoRINotIBamHI BamHI NEB BamHINEBNEBNEBNEB BamHI BamHI NotIBamHI NotI CompetitorCompetitorCompetitorCompetitorCompetitor BamHI BamHI BamHI NotIBamHI NotI NEBNEB BamHI BamHIT5 ExonucleaseCompetitorCompetitor BamHI BamHI + + + T7 Exonuclease + + + ™ EcoRI and BamHI from multiple suppliers were tested in reactions containing a fluorescent USER Enzyme, recombinant + + + labeled single stranded, double stranded blunt, 3’ overhang or 5’ overhang containing + + + full functional activity + + 50–100% functional activity + 0–50% functional activity oligonucleotides. The percent degradation is determined by capillary electrophoresis and peak A selection of DNA modifying enzymes were assayed in CutSmart analysis. The resolution is at the single nucleotide level. Buffer and functional activity was compared to the activity in their supplied buffers. Reactions were set up according to the recommended reaction conditions, with CutSmart Buffer (plus REQUEST YOUR FREE COPY required supplement) replacing the supplied buffer. Tech Tip: When supplements are required, one can see all details in new catalog: OF THE UPDATED PERFORMANCE CHART simply add the supplied buffer of the respective modifying p. 16 - 71 & appendix enzyme at 1× concentration to the CutSmart Buffer to POSTER 2017 achieve appropriate activity for most applications – no FROM YOUR LOCAL change of buffers needed. DISTRIBUTOR. 9 NEB: No Witchcraft required

Designed for sustainability & performace: Monarch Nucleic Acid Purification Advantages Kits from NEB • Improved recovery of concentrated, It’s time to transform your DNA purification experience. NEB’s Monarch Nucleic pure DNA Acid Purification Kits are optimized for maximum performance and minimal • Low volume elutions, resulting in environmental impact. Our unique thin-walled column design uses less plastic, highly-concentrated DNA prevents buffer retention, eliminates the risk of carryover contamination, and • Fast, user-friendly protocols enables elution in smaller volumes. The result: high performing DNA purification • Significantly less plastic in every kit for your downstream applications. • Responsibly sourced and recyclable packaging Labeling tab and frosted surface provide • Reusable kit boxes convenient writing space Unique, tapered design • Buffers and columns available eliminates buffer carryover and allows for separately elution in as little as 30 μl Made with less plastic for reduced environmental impact Column tip is compatible with Binding capacity vacuum manifolds up to 20 μg

Monarch Plasmid Miniprep Kits consistently produce more concentrated plasmid DNA with equivalent yield, purity and functionality as compared to the leading supplier. 2.7 kb 5.2 kb 10 kb Designed for performance N Q N Q N Q NEB Qiagen®

10.0 Buffer 5.2 retention

2.7 N – NEB Q – Qiagen

Conc. (ng/µl) 334.9 202.2 384.2 216.4 607.6 351.0 Total yield (µg) 10.0 10.1 11. 5 10.8 18.2 1 7. 6 1. 9 1. 9 1. 9 1. 9 1. 9 1. 9 A260/280 2.4 2.3 2.4 2.4 2.3 2.4 A260/230

SPECIAL Request a free sample**! PRICE* Visit NEBMonarch.eu until 31.01.2018 Free Sample

ORDERING INFORMATION: PRODUCT NEB # SIZE Monarch DNA Gel Extraction Kit T1020S/L 50/250 preps Monarch PCR & DNA Cleanup Kit (5 µg) T1030S/L 50/250 preps Monarch Plasmid Miniprep Kit T1010S/L 50/250 preps

see all details in new catalog: p. 140-145, 367

*No Witchcraft! Special prices on selected NEB Cloning Products incl. all Restriction Enzymes (Art.# Rnnnn). All other eligible discounted products are marked with the "Special Price" icon within this newsletter pages 8-13. Campaign ends Jan. 31st, 2018. Please ask your local distributor for details. ** Limited supply. As long as stocks last. 10 Clone with Confidence®!

Choose NEB's "Quick" DNA Ligases for performance & convenience Advantages

Fast & versatile: Robust Ligation with No Incubation: • Industry standard for purity NEB's Quick Ligation Kit Instant Sticky-end Ligase Master Mix • Highest reliability Sticky-ends ligations were set up using the • Fast ligations Instant Sticky-end Ligase Master Mix. Without any • Robust ligation efficiency additional incubation time, 2 µl were immediately • Convenient master mix formats withdrawn and used to transform a 50 µl aliquot of The Quick Ligation Kit enables NEB 10-beta Competent E. ligation of cohesive end or coli (NEB #C3019). blunt end DNA fragments in 5 minutes at room temperature (25°C ) SPECIAL Outperforms the competition: Select the Ligase product PRICE* Blunt/TA Ligase Master Mix that works best for you until 31.01.2018 ORDERING INFORMATION: INSTANT 100 T/A ligation QUICK BLUNT/TA STICKY-END PRODUCT NEB # SIZE Blunt ligation LIGATION™ LIGASE LIGASE

75 KIT MASTER MIX MASTER MIX 30 rxn (20 µl vol)/ Quick Ligation Kit M2200S/L DNA APPLICATIONS 150 rxn (20 µl vol) ficiency (% )

50 LIGATION OF STICKY ENDS ««« «« ««« Blunt/TA Ligase 50 rxn (10 µl vol)/ M0367S/L Master Mix 250 rxn (10 µl vol)

ansformation ef 25 Tr LIGATION OF BLUNT ENDS ««« ««« « Instant Sticky-end Ligase 50 rxn (10 µl vol)/ (as compared to Blunt/TA Ligase Master Mix) 0 M0370S/L Company Company Company NEB Master Mix 250 rxn (10 µl vol) A B C Blunt/TA Ligase Master Mix T/A CLONING «« ««« «

Duplicate ligation reactions of blunt or T/A vector/insert pairs were set up according to the master mix vendors’ suggestions. Equal ««« Optimal, recommended ligase for selected application amounts of ligated DNA were used to transform NEB 10-beta «« Works well for selected application see all details in new catalog: « Competent E. coli (NEB #C3019). Will perform selected application, but is not recommended p. 107-113 & 362-366

TECH-TIP: Ligation of Vector and Insert Ligation with the Quick Ligation Kit Vector DNA (3 kb) 50 ng Insert DNA (1 kb) To 50 ng • Use a molar ratio of 1:3 vector to insert 2X Quick Ligation Buffer 10 μl • If using T4 DNA Ligase (NEB #M0202) or the Quick Ligation Kit (NEB #M2200), thaw Quick T4 DNA Ligase 1 μl and resuspend the Ligase Buffer at room temp. If using Ligase Master Mixes, no thaw- Nuclease-free Water 20 μl (mix well) ing is necessary. Incubation Room temperature for 5 minutes

• The Quick Ligation Kit (NEB #M2200) is optimized for ligation of both sticky and blunt Ligation with Instant Sticky-end Ligase Master Mix ends Vector DNA (3 kb) 50 ng • Instant Sticky-end Ligase Master Mix (NEB #M0370) is optimized for instant ligation of Insert DNA (1 kb) 50 ng sticky/cohesive ends Master Mix 5 μl • Blunt/TA Ligase Master Mix (NEB #M0367) is optimized for ligation of blunt or single Nuclease Free Water To 10 μl Incubation None base overhangs, which are the more challenging type of ends for T4 DNA Ligase • Following ligation, chill on ice and transform Ligation with Blunt/TA Ligase Master Mix • DO NOT heat inactivate when using the Quick Ligation Buffer or Ligase Master Mixes, Vector DNA (3 kb) 50 ng as this will inhibit transformation Insert DNA (1 kb) 50 ng • Electroligase (NEB #M0369) is optimized for ligation of both sticky and blunt ends and Master Mix 5 μl is compatible with electroporation (i.e., no cleanup step required) Nuclease Free Water To 10 μl Incubation Room temperature for 15 min

Q5 High-Fidelity DNA Polymerase Advantages – Ultra fidelity for cloning & beyond • Fidelity – the highest fidelity 300 1 amplification available (> 280x Q5 High-Fidelity DNA Polymerase is a high-fidel- 275 ity, thermostable DNA polymerase with 3´-> 5´ higher than Taq and >4x higher 250 than Phusion) exonuclease activity, fused to a processivity-en- 225

hancing Sso7d domain to support robust DNA 200 ) • Robustness – high specificity amplification. With a fidelity >280-fold higher 175 compared to Taq DNA Polymerase, Q5 High-Fi- and yield with minimal 150 delity DNA Polymerase is ideal for cloning and optimization 125 can also be used for long or difficult amplicons. (x TaFidelity q 100 Q5 High-Fidelity DNA Polymerase is supplied with • Coverage – superior performance 75 an optimized buffer system that allows robust 2 for a broad range of amplicons 50 2 amplification regardless of GC content. 2 2 (from high AT to high GC) 25 2 3 0 ® ® ® II • Speed – short extension times Q5 Pfx KOD PfuUltra Phusion Platinum SPECIAL Taq HiFi PfuUltra Fusion HS • Amplicon length – robust * AccuPrime PRICE amplifications up to 20 kb for until 31.01.2018 Highest fidelity DNA amplification available. Fidelity of various PCR Polymerases as compared to Taq DNA Polymerase. simple templates, and 10 kb for ORDERING INFORMATION complex PRODUCT NEB # SIZE Q5 High-Fidelity DNA Polymerase M0491S/L 100/500 units Q5 Hot Start High-Fidelity DNA Polymerase M0493S/L 100/500 units see all details in new catalog: p. 76-77

NEB PCR Cloning Kit: Advantages With or without competent cells

BtgZI 100 This PCR Cloning Kit (available with or without SapI 2526 BsaXI 2541 AfeI 144 • Works with both blunt and TA ends PciI - AﬂIII 2409 NdeI 208 competent cells) contains an optimized Cloning NruI 226 DrdI 2307 SbfI - PstI 464 BsaJI 2249 PmeI 473 Mix containing a proprietary ligation enhancer and Constitutive PspXI - XhoI 510 • Clone faster, with low/ Promoter BamHI 516 Sites EcoRI 522 a linearized vector that uses a novel mechanism BseYI 2105 PacI 561 Flanking ZraI 567 no background Toxic Minigene AatII 569 for background colony suppression to give a low AlwNI 2000 ori Cloning Site EcoRI 572 pMiniT™ 2.0 XhoI 577 NotI- EagI 584 background. It allows simple and quick cloning 2,588 bp BsmBI 602 • Get the colonies you need, with SspI 716 of any PCR amplicon, whether the amplification high transformation efficiency reactions are performed with proofreading DNA R Ap polymerases, such as Q5 which produce blunt BanI 1568 XmnI 921 AhdI 1521 BcgI 1017 • No need for end-modification steps BmrI 1481 ScaI - RsaI 1040 ends; or nonproofreading DNA polymerases, such BpmI 1452 FspI 1298 BsrFI 1436 PvuI 1152 as Taq which produce single base overhangs. BglI 1403 NmeAIII 1373 • No blue/white screening required This is possible due to “invisible” end polishing Features within Sequence Flanking the Toxic Minigene/Cloning Site

Cloning Analysis Forward Primer components in the master mix that are active SbfI/PstI PmeI 5´ ACC TGC CAA CCA AAG CGA GAA CAA AAC ATA ACA TCA AAC GAA TCG ACC GAT TGT TAG GTA ATC GTC ACC TGC AGG AAG GTT 3´ TGG ACG GTT GGT TTC GCT CTT GTT TTG TAT TGT AGT TTG CTT AGC TGG CTA ACA ATC CAT TAG CAG TGG ACG TCC TTC CAA • New superior Vector pMiniT 2.0 during the ligation step only if needed. The kit also +1 SP6 Promoter PmeI PspXI/XhoI BamHI EcoRI 5´ TAA ACG CAT TTA GGT GAC ACT ATA GAA GTG TGT ATC GCT CGA GGG ATC CGA ATT CAG GAG GTA AAA ACC including SP6 and T7 promotors allows direct cloning from amplification reactions 3´ ATT TGC GTA AAT CCA CTG TGA TAT CTT CAC ACA TAG CGA GCT CCC TAG GCT TAA GTC CTC CAT TTT TGG without purification, and works well whether or not ...ATG AT INSERT C TGA TAA TAA... the primers used in the PCR possess 5´-phosphate ...TAC TA G ACT ATT ATT... Met Ile (interrupted) ** *

groups. Two Amino Acid Toxic Minigene with Cloning Site Shown (As diagrammed, minigene inactivated if insert cloned into site) PCR cloning has never SPECIAL PacI ZraI/AatII EcoRI/XhoI NotI/EagI BsmBI been easier! 5´ TTA ATT AAG ACG TCA GAA TTC TCG AGG CGG CCG CAT GTG CGT CTC CCT ATA GTG AGT CGT ATT AAT TTC GCG GGC * 3´ AAT TAA TTC TGC AGT CTT AAG AGC TCC GCC GGC GTA CAC GCA GAG GGA TAT CAC TCA GCA TAA TTA AAG CGC CCG +1 PRICE T7 Promoter until 31.01.2018 5´ GGA ACC CCT ATT TGT TTA TTT TTC TAA ATA CAT TCA AAT ATG TAT CCG CTC ATG AGA CAA TAA CCC TGA 3´ ORDERING INFORMATION 3´ CCT TGG GGA TAA ACA AAT AAA AAG ATT TAT GTA AGT TTA TAC ATA GGC GAG TAC TCT GTT ATT GGG ACT 5´ Cloning Analysis Reverse Primer

PRODUCT NEB # SIZE PCR cloning with low/no background Map shown above displays the construct formed if no insert is present. Unique restriction sites are shown in black. Additional A 500 bp PCR product was cloned using the NEB PCR Cloning NEB PCR Cloning Kit (without kit (with competent E. coli). The left plate serves as the control, E1203S 20 rxns restriction sites that can be used for subcloning are shown in competent cells) red. Expanded box below shows location of sequencing primers, with vector backbone only. The right plate shows colonies NEB PCR Cloning Kit (incl. NEB10- restriction sites for subcloning, and placement of insertion site containing the PCR insert. beta competent cells) E1202S 20 rxns within the toxic minigene.

* No Witchcraft! Special prices on selected NEB Cloning Products incl. all Restriction Enzymes (Art.# Rnnnn). All other eligible discounted products are marked with the "Special Price" icon within this newsletter pages 8-13. 12 Campaign ends Jan. 31st, 2018. Please ask your local distributor for details. LUNA® Universal qPCR & RT-qPCR Reagents

New England Biolabs is pleased to introduce a bright, new choice for your qPCR and RT-qPCR. Luna products have been optimized for robust performance on Visible tracking dye diverse sample sources and target types. Available for dye-based or probe-based detection, Luna products can be used across a wide variety of instrument platforms. With so many qPCR and RT-qPCR options available, why not make a simpler, more cost-effective choice that delivers the sensitivity and precision you expect for your qPCR and RT-qPCR.

EXPERIENCE BEST-IN-CLASS PERFORMANCE A blue visible dye assists in tracking the reagents when pipetting into • All Luna products have undergone rigorous testing to clear, multi-welled PCR plates. optimize specificity, sensitivity, accuracy and reproducibility.

• Products perform consistently across a wide variety of Luna Universal One-Step RT-qPCR Kit offers exceptional sample sources. sensitivity, reproducibility & RT-qPCR performance • A comprehensive evaluation of commercially-available qPCR p p and RT-qPCR reagents demonstrates superior performance 10 p of Luna products. n = 8 1 μg 0.1 μg 10 ng ® 1 ng OPTIMIZE YOUR RT-qPCR WITH LUNA WARMSTART 1 REVERSE TRANSCRIPTASE ∆Rn 0.1 ng 10 pg • Novel, thermostable reverse transcriptase (RT) improves 1 pg 0.1 pg performance. NTC 0.1 • WarmStart RT paired with Hot Start Taq increases reaction 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 Cycle specificity and robustness.

RT-qPCR targeting human GAPDHr was performed re using the Luna Universal One-Stepe RT-qPCR re Kit (Input: 32.5 1.4 1 μg – 0.1 pg Jurkat total RNA); NTC =e non-template control MAKE A SIMPLER CHOICE 30.0 1.2 27.5 25.0 1.0 see all details in new catalog: • One product per application simplifies selection. 22.5 q 0.8

C 20.0 p. 91-92 & 352-355 0.6 • Convenient master mix formats and user-friendly protocols 17.5

15.0 Derivative 0.4

simplify reaction setup. 12.5 Reporter (-Rn´) 0.2 10.0 R2 = 0.998 0.0 • Non-interfering, visible tracking dye helps to eliminate 0.01 Request0.1 1 10 10 a2 free103 104 sample*!105 106 107 65 70 75 80 85 90 95 pipetting errors. Quantity (pg) Temperature (°C) Free Sample Visit LUNAqPCR.eu

Find the right Luna product for your application: Ideally for high throughput users: Select your 2 detection method Now also available in Extra-Large Dye-based Probe-based economic "E-Pack" sizes!

Genomic DNA Luna Universal Luna Universal Probe qPCR Master Mix qPCR Master Mix or cDNA PRODUCT NEB # SIZE (NEB #M3003) (NEB #M3004) Select Luna Universal qPCR Master Mix M3003S/L/X/E 200/500/1.000/2.500 rxns your target 1 Luna Universal Probe qPCR Master Mix M3004S/L/X/E 200/500/1.000/2.500 rxns Luna Universal Luna Universal Probe Luna Universal One-Step RT-qPCR Kit E3005S/L/X/E 200/500/1.000/2.500 rxns RNA One-Step RT-qPCR Kit One-Step RT-qPCR Kit (NEB #E3005) (NEB #E3006) Luna Universal Probe One-Step RT-qPCR Kit E3006S/L/X/E 200/500/1.000/2.500 rxns

*Limit one per customer, while supplies last. 13 Choose NEB Competent Cells Advantages for your cloning • High transformation efficiencies NEB’s growing line of competent cells includes several popular strains for cloning • Compatible with NEBuilder® and protein expression, in addition to strains with unique properties, including fast HiFi DNA Assembly and Gibson colony growth, tight control of expression and disulfide bond formation. Our cloning Assembly® reactions, as well as strains include derivatives of the industry standards, DH5 ™ and DH10B™. NEB α ligation reactions Turbo is unique to NEB, and produces visible colonies after only 6.5 hours of growth. NEB’s dam–/dcm– strain enables Dam and Dcm methylation-free plasmid growth. • Strains also available for cloning NEB Stable is recommended in most difficult cloning experiments. Our cells are all toxic genes extensively tested for phage resistance, antibiotic resistance and sensitivity, blue/white • All strains are free of animal screening and transformation efficiency. High efficiency, 5-minute transformation and products and T1 phage resistant electroporation protocols are provided, when applicable. • Media and control plasmid

NEB 5-ALPHA NEB TURBO NEB 5-ALPHA F´ NEB 10-BETA dam –/dcm – NEB STABLE is included COMPETENT COMPETENT I q COMPETENT COMPETENT COMPETENT COMPETENT E. coli E. coli E. coli E. coli E. coli E. coli • Choose from a variety of (#C2987) (#C2984) (#C2992) (#C3019) (#C2925) (#C3040) convenient formats FEATURES Versatile l l • Bulk formats and custom packaging Fast growth (< 8 hours) l are available Toxic gene cloning l l l Large plasmid/BAC cloning l Dam/Dcm-free plasmid growth l Retroviral/lentiviral l vector cloning RecA l l l l Special Offer FORMATS Chemically competent l l l l l l Through January 31st, Electrocompetent l l l receive a free Subcloning l NEB Tube Opener 96-well format* l with every competent 384-well format* l cell purchase.** 12 x 8-tube strips* l

* Other strains are available upon request. For more information, contact [email protected].

FIGURE 1: FIGURE 2: Benefit from high transformation efficiencies Take advantage of the low cost per transformation Transformation efficiencies were compared using manufacturers’ recommended protocols. Calculations were based on published list prices as of 08/2017 and recommended transformation Values shown are the average of triplicate experiments. volumes. Prices may differ in various European countries.

) 30 9 2.5 25 26 25 24 2.0 22 20 1. 5 16 15 13 1. 0 12 10 10 (cfu/µg pUC19) 0.5 5 Cost/transformation () Cost/transformation 0 ® ® ® ®

Transformation Efficiency (x10 Efficiency Transformation ® ® ® ® ® ® ®

R R ® 5-alpha -T1 ® 5-alpha NEB Turbo One Shot One Shot -T1 One Shot NEB Stable Stbl3 One Shot One Shot NEB Turbo One Shot One Shot NEB α NEB 10-beta Mach1 NEB Stable Stbl3 NEB NEB 10-beta Mach1 MA Efficiency MA EfficiencyDH10B DH10B DH5 MA DH5aEfficiency MA Efficiency

**Limit one per customer, while supplies last. Please contact your local distributor for details 14 Enhancing Transformation Efficiency

Transformation efficiency is defined as the number of colony forming units (cfu) that would be produced by transforming 1 µg of plasmid into a given volume of competent cells. However, 1 µg of plasmid is rarely transformed. Instead, efficiency is routinely calculated by transforming 100 pg–1 ng of highly purified supercoiled plasmid under ideal conditions. Transformation Efficiency (TE) is calculated as: TE = Colonies/µg/Dilution. Efficiency calculations can be used to compare cells or ligations. Our recommended protocols and tips to help you achieve maximum results are presented here:

TRANSFORMATION TIPS Thawing • Use NEB 10-beta/Stable Outgrowth Medium • The optimal amount of DNA is lower than • Cells are best thawed on ice for 10-beta and Stable Competent E. coli. Use commonly recognized. Using clean, super- • DNA should be added as soon as the last trace SOC for all other strains. coiled pUC19, the efficiency of transformation of ice in the tube disappears • Outgrowth medium gives 2-fold higher TE is highest in the 100 pg–1 ng range. However, than LB medium the total colonies which can be obtained from • Cells can be thawed by hand, but warming a single transformation reaction increase up to above 0°C decreases efficiency • Incubation with shaking or rotation results in about 100 ng. 2-fold higher TE Incubation of DNA with Cells on Ice DNA CONTAMINANTS TO AVOID • Incubate on ice for 30 minutes. Expect a 2-fold Plating loss in TE for every 10 minutes this step is • Selection plates can be used warm or cold, CONTAMINANT REMOVAL METHOD shortened. wet or dry with no significant effects on TE Detergents Ethanol precipitate • Warm, dry plates are easier to spread and Heat Shock Phenol Extract with chloroform and ethanol • Both temperature and time are specific to the allow for the most rapid colony formation precipitate transformation volume and vessel. Typically, DNA 30 seconds at 42°C is recommended. Ethanol or Dry pellet before resuspending • DNA should be purified and resuspended in Isopropanol Outgrowth water or Tris-EDTA Buffer PEG Column purify or phenol/chloroform • Outgrowth at 37°C for 1 hour is best for • Up to 10 µl of DNA from a ligation mix can extract and ethanol precipitate cell recovery and for expression of antibiotic be used with only a 2-fold loss of efficiency resistance. Expect a 2-fold loss in TE for every • Purification by either a spin column or DNA binding Column purify or phenol/ chloroform 15 minutes this step is shortened. proteins extract and ethanol precipitate phenol/chloroform extraction and ethanol (e.g., ligase) precipitation is ideal

See all details in new catalog: p. 230 – 241

Featured Videos Online Tools Find these and other helpful tips for using NEB competent cells at NEBcloner® www.neb.com/CloningCompCells Visit NEBcloner.neb.com for help with choosing the right competent cells for your experiment

NEBioCalculator® Visit NEBioCalculator.neb.com for help with conversions and How to perform a transformation with Tips for successful transformations with calculations NEB competent cells NEB competent cells 15 New to Molecular Biology? Your local NEB distributor: BeNeLux: POLAND: BIOKÉ Lab-JOT Ltd. Sp.z o.o. Sp.k. Start your career off on the right path. Tel: (+31) 71 720 0220 Stacjonarny: +48 22 335 988 4 (BE: 0800 - 71640) Komórkowy: +48 606 338 879 Fax: (+31) 71 891 0019 Fax: +48 22 335 981 9 Get your free NEB Starter Pack*! [email protected] [email protected] www.bioke.com www.labjot.com

BULGARIA PORTUGAL: The NEB Starter Pack contains: ELTA 90M Ltd. Werfen Tel: (+359) 2 983 96 49 Tel. +351 214 247 312 • NEB Catalog & Technical Reference • Free Sample of OneTaq HS DNA Polymerase Fax: (+359) 2 9832211 Fax +351 21 425 52 80 [email protected] [email protected] 2017/18 incl. Buffer Poster • Free Sample of Quick-Load DNA Ladders www.elta90.com http://pt.werfen.com

• Voucher for 20% discount on your first • Year planning calendar poster 2018 CROATIA: ROMANIA: DIAGNOSTICA SKALPELI D.O.O. ELTA 90 MEDICAL RESEARCH SRL purchase of any NEB product incl. free • Tech-Guide "Molecular Cloning" Free call: 800-315911 Tel: +40 21 2322694 NEBCool lab bench cooler • NEB Pencil Tel: (+385) 1 377 8484 Fax: +40 21 2322696 Fax: (+385) 1 377 8585 [email protected] • NEB notepad • Water bath floatie [email protected] www.elta90mr.ro www.skalpeli.hr RUSSIA: CZECH REPUBLIC: SkyGen LLC BIOTECH A.S. Tel: +7 495 215 02 22 Tel: (Toll Free) 0800 124683 [email protected] Fax: 02 72701742 www.skygen.com [email protected] www.ibiotech.cz SERBIA: ALFA GENETICS D.O.O. DENMARK: Tel: 00381 11 2084252 BioNordika Denmark A/S Fax: 00381 11 2084253 DISCOUNT DENMARK [email protected] 20% VOUCHER Tel: (39) 56 20 00 www.alfagenetics.rs Fax: (39) 56 19 42 [email protected] SPAIN: www.bionordika.dk Werfen + FREE NEBCool Tel: +34 902 20 30 90 LAB BENCH COOLER FINLAND: Fax: +34 902 22 33 66

be drive stay INSPIRED DISCOVERY GENUINE BioNordika Oy [email protected] Tel: (+358) 207 410 270 http://es.werfen.com [email protected]

FREE RESOURCES: NEB Tools & www.bionordika.fi Video Tutorials SLOVENIA: www.neb.com/tools Molecular Cloning Mikro+Polo d.o.o. TECHNICAL GUIDE GREECE: Tel: +386 2 614 33 00 BIOLINE SCIENTIFIC Fax: +386 2 614 33 20 Tel: (0030) 210 522 6547 [email protected] Fax: (0030) 210 524 4744 www.mikro-polo.com [email protected] www.bioline.gr SOUTH AFRICA: INQABA BIOTEC HUNGARY: Tel: +27 123 43 58 29 KVALITEX SCIENTIFIC Fax: +27 86 677 8409 Phone: (1) 340 4700 [email protected] Fax: (1) 339 8274 www.inqababiotec.co.za [email protected] www.kvalitex.hu SWEDEN, ESTONIA, LATVIA, be INSPIRED drive DISCOVERY stay GENUINE LUTHUANIA:

Cloning_Tech_Guide_D_F_EU_1609.indd 1 22.12.16 15:05 ITALY: BIONORDIKA SWEDEN AB EUROCLONE S.P.A. Tel: +46 8 30 60 10 Free call: 800-315911 Fax: +46 8 30 60 15 New England Biolabs, Inc. Toll Free: (USA Orders) 1-800-632-5227 Tel: +39 02 38195.1 [email protected] (USA Tech) 1-800-632-7799 Fax: (978) 921-1350 Fax.: +49/(0)69/305-23149 Fax: +39 02 3810145 www.bionordika.se [email protected] international.neb.com [email protected] www.euroclonegroup.it SWITZERLAND: 2018JANUARY FEBRUARY MARCH APRIL MAY JUNE JULY AUGUST SEPTEMBER OCTOBER NOVEMBER DECEMBER 01 M 01 01 D 01 D 01 01 DI 01 01 01 MI 01 A 01 M 40 01 D 01 A BIOCONCEPT E S

02 DI 02 02 02 M 14 02 MI 02 A 02 M 27 02 D 02 02 DI 02 02 E ISRAEL: Tel: +41 (0)61 486 80 80 03 MI 03 A 03 A 03 DI 03 D 03 03 DI 03 03 M 36 03 MI 03 A 03 M 49 04 D 04 04 04 MI 04 04 M 23 04 MI 04 A 04 DI 04 D 04 04 DI ORNAT LTD. Fax: +41 (0)61 486 80 00 05 05 M 06 05 M 10 05 D 05 A 05 DI 05 D 05 05 MI 05 05 MO 45 05 MI Tel: (+972) 8 947 7077 [email protected] 06 A 06 DI 06 DI 06 06 06 MI 06 06 M 32 06 D 06 A 06 DI 06 D 07 07 MI 07 MI 07 A 07 M 19 07 D 07 A 07 DI 07 07 07 MI 07 Fax: (+972) 8 936 3034 www.bioconcept.ch 08 M 02 08 D 08 D 08 08 DI 08 08 08 MI 08 A 08 M 41 08 D 08 A

09 DI 09 09 09 M 15 09 MI 09 A 09 M 28 09 D 09 09 DI 09 09 [email protected] 10 MI 10 A 10 A 10 DI 10 D 10 10 DI 10 10 M 37 10 MI 10 A 10 M 50 www.ornat.co.il TURKEY: 11 D 11 11 11 MI 11 11 M 24 11 MI 11 A 11 DI 11 D 11 11 DI

12 12 M 07 12 M 11 12 D 12 A 12 DI 12 D 12 12 MI 12 12 M 46 12 MI SACEM HAYAT TEKNOLOJILERI 13 A 13 DI 13 DI 13 13 13 MI 13 13 M 33 13 D 13 A 13 DI 13 D NORWAY: Gebze Plastikçiler Organize Sanayi 14 14 MI 14 MI 14 A 14 M 20 14 D 14 A 14 DI 14 14 14 MI 14 15 M 03 15 D 15 D 15 15 DI 15 15 15 MI 15 A 15 M 42 15 D 15 A BioNordika AS Bölgesi (GEPOSB) 16 DI 16 16 16 M 16 16 MI 16 A 16 M 29 16 D 16 16 DI 16 16 Tel +47 23 03 58 00 Tel: 0262 751 02 74 17 MI 17 A 17 A 17 DI 17 D 17 17 DI 17 17 M 38 17 MI 17 A 17 M 51 18 D 18 18 18 MI 18 18 M 25 18 MI 18 A 18 DI 18 D 18 18 DI Fax +47 23 03 58 01 Fax: 0262 751 02 75 19 19 M 08 19 M 12 19 D 19 A 19 DI 19 D 19 19 MI 19 19 M 47 19 MI

20 A 20 DI 20 DI 20 20 20 MI 20 20 M 34 20 D 20 A 20 DI 20 D [email protected] [email protected] 21 21 MI 21 MI 21 A 21 M 21 21 D 21 A 21 DI 21 21 21 MI 21 www.bionordika.no www.sacem.com.tr 22 M 04 22 D 22 D 22 22 DI 22 22 22 MI 22 A 22 M 43 22 D 22 A

23 DI 23 23 23 M 17 23 MI 23 A 23 M 30 23 D 23 23 DI 23 23

24 MI 24 A 24 A 24 DI 24 D 24 24 DI 24 24 M 24 MI 24 A 24 M 39 C E 52

25 D 25 25 25 MI 25 25 M 25 MI 25 A 25 DI 25 D 25 25 DI 26 C

26 26 M 26 M 26 D 26 A 26 DI 26 D 26 26 MI 26 26 M 26 MI 09 13 48 C

27 A 27 DI 27 DI 27 27 27 MI 27 27 M 35 27 D 27 A 27 DI 27 D

28 28 MI 28 MI 28 A 28 M 22 28 D 28 A 28 DI 28 28 28 MI 28

29 M 05 29 D 29 29 DI 29 29 29 MI 29 A 29 M 44 29 D 29 A

30 DI 30 30 M 30 MI 30 A 30 M 30 D 30 30 DI 30 30 F 18 31

31 MI 31 A 31 D 31 DI 31 31 MI 31 M 01 Für Druckfehler keine Haftung. Stand 09/17. © New England Biolabs GmbH 2017 Stand 09/17. Für Druckfehler keine Haftung.

Register at www.neb-online.eu/starterpack *Free Starter Packs are available to all research students, PhD students, PostDocs, PIs etc. who are new to Molecular Biology and who are willing to join the mailing list. neb.comneb.com Offer available while stocks last through to Jan. 31st, 2018. Limited to one pack/person. Content may vary from those shown. 20% discount voucher/offer void where prohibited by regional or institutional laws or reglementations. Ask your local distributor for details.