Time for ChangeMonarch Nucleic Acid Purification Kits are optimized for maximum performance and minimal environmental impact. Kits are available for total RNA purification, plasmid miniprep, gel extraction, and DNA RNA cleanup. For maximum convenience and value, columns and buffers are also available separately. Learn more and request a sample!You ll be Thrilled to PiecesDo you need a faster, more reliable solution for DNA fragmentation and library construction? Our new NEBNext Ultra II FS DNA Library Prep Kitwith novel fragmentation reagent meets the dual challenge of generating high quality next gen sequencing libraries from ever-decreasing input amounts AND simple scalability. Learn more and request a sample!Time for ChangeMonarch Nucleic Acid Purification Kits are optimized for maximum performance and minimal environmental impact. Kits are available for total RNA purification, plasmid miniprep, gel extraction, and DNA RNA cleanup. For maximum convenience and value, columns and buffers are also available separately. Learn more and request a sample!You ll be Thrilled to PiecesDo you need a faster, more reliable solution for DNA fragmentation and library construction? Our new NEBNext Ultra II FS DNA Library Prep Kitwith novel fragmentation reagent meets the dual challenge of generating high quality next gen sequencing libraries from ever-decreasing input amounts AND simple scalability. Learn more and request a sample!New England Biolabs is working diligently to ensure we keep our employees and their families safe, while maintaining our business continuity. Despite a \"Stay At Home\" advisory being put in place in Massachusetts, USA, we are deemed an essential business, and our manufacturing and distribution teams continue to be fully operational. We are continuing to work with our suppliers and distribution partners to ensure uninterrupted access to our products and technical support.Did you know this product can be purchased in larger volumes? Submit an inquiry to find out more.

NEBuilder HiFi DNA Assembly Master Mix allows for seamless assembly of multiple DNA fragments, regardless of fragment length or end compatibility. It has utility for the synthetic biology community, as well as those interested in one-step cloning of multiple fragments due to its ease of use, flexibility and simple master-mix format. Visit NEBuilderHifi.comfor more information.

Also available with competent cellsat a discounted price.Enjoy less screening/re-sequencing of constructs, with virtually error-free, high-fidelity assemblyJoin DNA fragments together more efficiently, even with larger fragments or low DNA inputsUse NEBuilder HiFi in successive rounds of assembly, because it removes 5 and 3 end mismatches. (Save time by avoiding time-consuming PCR amplification steps.)Bridge two ds-fragments with a synthetic ss-DNA oligo for simple and fast construction (e.g., linker insertion or gRNA library)Use with lower DNA input requirementsNo licensing fee requirements from NEB for NEBuilder productsTo find out how to order this product from your current location, click the button below: International Customer Order Support NEBuilder HiFi DNA Assembly Master Mix was developed to improve the efficiency and accuracy of DNA assembly. This method allows for seamless assembly of multiple DNA fragments, regardless of fragment length or end compatibility. This method has been used to assemble either single-stranded oligonucleotides or different sizes of DNA fragments with varied overlaps (15 80 bp). It has utility for the synthetic biology community, as well as those interested in one-step cloning of multiple fragments due to its ease of use, flexibility and simple master-mix format. The reaction includes different enzymes that work together in the same buffer (see Figure 1):The exonuclease creates single-stranded 3 overhangs that facilitate the annealing of fragments that share complementarity at one end (the overlap region)The polymerase fills in gaps within each annealed fragmentThe DNA ligase seals nicks in the assembled DNAThe end result is a double-stranded fully sealed DNA molecule that can serve as template for PCR, RCA or a variety of other molecular biology applications, including direct transformation of E coli.NEBuilder HiFi DNA Assembly kits are available in various formats: with NEB 5-alpha chemically competent cells (Cloning Kit, NEB #E5520), as a bundle with NEB 10-beta chemically competent cells (Bundle for Large Fragments, NEB #E2623) and without competent cells (Master Mix, NEB #E2621). NEB 5-alpha chemically competent cells are excellent for routineassemblies of 15 kb or less. NEB recommends NEB 10-beta Competent E. coli(High Efficiency, NEB #C3019 ) or NEB 10-beta Electrocompetent E. coli (NEB #C3020) for assemblies larger than 15 kb. If the assembled genes contain repetitive sequences, NEB Stable Competent E. coli (NEB #C3040) should be used.NEBuilder has been used in various applications, including:Site-directed mutagenesis Construction of an sgRNA-Cas9 expression vector | Animation Assembly of linear yeast expression cassettes To help select the best DNA assembly method for your needs, please use our Synthetic Biology/DNA Assembly Selection Chart.For help designing your primers for use with NEBuilder, please view our primer design video.Figure 1: Overview of the NEBuilder HiFi DNA Assembly MethodFigure 2: NEBuilder HiFi DNA Assembly offers improved efficiency and accuracy over NEB Gibson AssemblyReactions were set up in a 2- and 6- fragment assembly reaction according to recommended reaction conditions. NEBuilder HiFi DNA Assembly results in larger numbers of colonies over NEB Gibson Assembly, for both 2- and 6-fragment assemblies. View additional performance data compared to NEB Gibson AssemblyFigure 3: NEBuilder HiFi delivers higher colony yield than In-Fusion HD Two-fragment reactions were set up using the positive control from theIn-Fusion HD Cloning Kit (Clontech Takara Bio USA, Inc), according torecommended protocols. 2 l of assembly reaction was transformed intosupplied competent cells. 1/50 of outgrowth was spread on an ApR plate.View additional performance data compared to In-Fusion HDKit Components

The following reagents are supplied with this product:

Features

Benefits over NEB Gibson Assembly Master Mixand/or In-Fusion HD:Enjoy less screening/re-sequencing of constructs, with virtually error-free, high-fidelity assembly.Join DNA fragments together more efficiently, even with larger fragments or low DNA inputsUse NEBuilder HiFi in successive rounds ofassembly, because it removes 5 and 3 end mismatches. (Save time byavoiding time-consuming PCR amplification steps.) Bridge two ds-fragments with a synthetic ss-DNAoligo for simple and fast construction (e.g. linker insertion or gRNAlibrary)Use with lower DNA input requirementsSwitch from other systems easily, becauseNEBuilder HiFi is compatible with Gibson Assembly - and In-Fusion-designed fragments No licensing fee requirements from NEB for the NEBuilder products

Benefits over Traditional Cloning:

Save time with Simple and Fast Seamless Cloning Use one system for both \"standard-size\" cloning and larger gene assembly products, up to 11 fragments.Move on with your workflow, because DNA can be used immediately for transformation or as template for PCR or RCA.Adapts easily for multiple DNA manipulations, including site-directed mutagenesis.Materials Required but not SuppliedDNA Polymerase (for generating PCR products): For generating PCR Products, we recommend Q5 High-Fidelity DNA Polymerase (NEB #M0491) or related products, such as Q5 Hot Start High-Fidelity DNA Polymerase (NEB #M0493) or Q5 Hot Start High-Fidelity 2X Master Mix (NEB #M0494). LB (Luria-Bertani) plates with appropriate antibiotic. For selection of transformed competent cells, we recommend LB plates with appropriate antibiotic.Storage Temperature-20 C To ensure the successful assembly and subsequent transformation of assembled DNAs, NEB recommends the following:DNA: PCR product purification is not necessary if the total volume of all PCR products is 20% or less of the assembly reaction volume. Higher volumes of PCR products may reduce the efficiency of high-fidelity DNA assembly and transformation due to the elevated carryover amounts of PCR reaction buffer and unused primers present in the PCR product. Column purification of PCR products may increase the efficiency of both high-fidelity DNA assembly and transformation by 2 10 fold and is highly recommended when performing assemblies of three or more PCR fragments or assembling longer than 5 kb fragments. Purified DNA for assembly can be dissolved in ddH2O (Milli-Q water or equivalent is preferable), TE or other dilution buffers. Insert: When directly assembling fragments into a cloning vector, the concentration of assembly fragments should be at least 2 times higher than the concentration of vector. For assembly of 4 or more fragments into a vector, we recommend using an equimolar ratio of fragments. Transformation: NEB 5-alpha Competent E. coli (High Efficiency, NEB #C2987) provided with the NEBuilder HiFi DNA Assembly Cloning Kit are recommended for use for assembled products of less than 15kb. It is also possible to use other NEB competent E. coli strains, with the exception of BL21, BL21(DE3), Lemo21(DE3), Nico(DE3), and SHuffle . When using competent E. coli from a vendor other than NEB, we have been decreased robustness of transformation with high-fidelity DNA assembled products. Electroporation: Electroporation can increase transformation efficiency by several logs. When using the NEBuilder HiFI DNA Assembly Master Mix, use 1 l of the assembled product for electroporation, and plate multiple dilutions.Should you require the use of Electrocompetent cells, please use the \"Electrocompetent Cells Transformation Protocol\" Biology: Some DNA structures, including inverted and tandem repeats, are selected against by E. coli. Some recombinant proteins are not well tolerated by E. coli and can result in poor transformation or small colonies. The Product Manual includes details for how to use the product, as well as details of its formulation and quality controls. manualE2621 Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version] E2621S_X_L_v1 This product is covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). While NEB develops and validates its products for various applications, the use of this product may require the buyer to obtain additional third party intellectual property rights for certain applications. For more information about commercial rights, please contact NEB\'s Business Development team at [email protected]. This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.  LicensesLimited Warranty: The NEBuilder HiFiAssembly Master Mix and NEBuilder HiFi DNA Assembly Cloning Kit arewarranted to perform according to specifications stated on the certificate of analysis. No other warranty is made, whether express or implied, including any warranty of merchant ability or fitness for a particular purpose. This warranty limits NEB s and its licensors liability to only the price of the product. Neither NEB nor its licensors shall have any responsibility or liability for any special, incidental, indirect or consequential loss or damage whatsoever. Trademarks NEW ENGLAND BIOLABS , NEBUILDER , Q5 , HF , NEBIOCALCULATOR and SHUFFLE are registered trademarks of New England Biolabs, Inc. CUTSMART is a trademark of New England Biolabs, Inc. GIBSON ASSEMBLY is a registered trademark of Synthetic Genomics, Inc. MILLI-Q is a registered trademark of Millipore, Inc. Nanodrop is a registered trademark of Nanodrop Technologies, Inc.Ineligible item added to cart

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