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DNA Assembly & Synthetic Biology Now includes NEBExpress® Cell-free E. coli Protein Synthesis System DNA Assembly & Synthetic Biology TOOLS TO SUPPORT DESIGN AND DNA ASSEMBLY 1 0 01 0 1 SYNTHETIC BIOLOGY DNA Assembly & Synthetic Biology – Tools to support your design and assembly The goal of synthetic biology, in which genes and proteins are viewed as parts or devices, is redesigning and/or assembling them in novel ways to create a new and useful functionality. These projects often rely on the ordered assembly of multiple DNA sequences to create large, artificial DNA structures, and methods have evolved to simplify this process. New England Biolabs now offers several products that can be used for DNA assembly and cloning. Use this chart to determine which product would work best to assemble your DNA. NEBuilder HiFi Gibson NEB Golden Gate USER® Enzyme DNA Assembly Assembly® Assembly Kit (NEB #M5505) (BsaI-HF®v2, (NEB #E2621) (NEB #E5510) BsmBI-v2) Thermolabile (NEB #E5520) (NEB #E2611) USER II Enzyme (NEB #E2623) (NEB #E1601) (NEB #E1602) (NEB #M5508) PROPERTIES Removes 5´ or 3´ End Mismatches *** * N/A N/A Assembles with High Fidelity at Junctions *** ** *** *** Tolerates Repetitive Sequences at Ends * * *** *** Generates Fully Ligated Product *** *** *** NR Joins dsDNA with Single-stranded Oligo *** ** NR NR Assembles with High Efficiency with Low Amounts of DNA *** ** ** ** Accommodates Flexible Overlap Lengths *** *** * ** APPLICATIONS Simple Cloning (1-2 Fragments) *** *** *** *** 4-6 Fragment Assembly (one pot) *** *** *** *** 7-11 Fragment Assembly (one pot) *** ** *** *** 12-50+ Fragment Assembly (one pot)(1) * * *** NR Template Construction for In vitro Transcription *** *** *** *** Synthetic Whole Genome Assembly *** * * * Multiple Site-directed Mutagenesis *** ** ** ** Library Generation *** *** *** ** KEY Metabolic Pathway Engineering Optimal, recommended product for *** ** *** *** *** selected application TALENs ** ** *** ** ** Works well for selected application Short Hairpin RNA Cloning (shRNA) Will perform selected application, *** ** * * * but is not recommended gRNA Library Generation *** ** * * (1) Please visit neb.com/GoldenGate for Large Fragment (> 10 kb) Assembly *** *** *** ** more information Small Fragment (< 100 bp) Assembly N/A Not applicable to this application *** * *** *** NR Not recommended Use in Successive Rounds of Restriction Enzyme Assembly *** * NR * Enzymes for Innovation Enzymes for Innovation (EFI) is a unique program initiated by NEB to provide enzymes with interesting activities and unique properties to the scientific community, in hopes of enabling the discovery of new and innovative applications. Learn more at www.neb.com/enzymesforinnovation 2 1 0 01 SYNTHETIC BIOLOGY 0 1 NEBuilder® HiFi DNA Assembly NEBuilder HiFi DNA Assembly enables virtually error-free joining of DNA fragments, RECOMMENDED PRODUCTS even those with 5´- and 3´-end mismatches. Available with and without competent E. coli, this flexible kit enables simple and fast seamless cloning utilizing a proprietary NEBuilder HiFi DNA Assembly Cloning Kit high-fidelity polymerase. Make NEBuilder HiFi your first choice for DNA assembly (NEB #E5520) and cloning. NEBuilder HiFi DNA Assembly Master Mix (NEB #E2621) Overview of the NEBuilder HiFi DNA Assembly cloning method NEBuilder HiFi DNA Assembly Bundle for Large Fragments DNA Preparation (NEB #E2623) From: C • Simple and fast seamless cloning in as little as 15 minutes • PCR B Linear A • Restriction enzyme vector + • Use one system for both "standard-size" cloning and larger digestion DNA inserts with 15-30 bp • Synthetic DNA overlapping ends (PCR-amplied) gene assembly products (up to 11 fragments and 22 kb) (e.g., gBlocks) • Single-stranded oligo • DNA can be used immediately for transformation or as template for PCR or RCA NEBuilder HiFi DNA B • Adapts easily for multiple DNA manipulations, Assembly Master Mix A C including site-directed mutagenesis Single-tube reaction • Exonuclease chews Assembled • Enjoy less screening/re-sequencing of constructs, back 5´ ends to create DNA single-stranded 3´ overhangs with virtually error-free, high-fidelity assembly • DNA polymerase lls in gaps within Incubate at 50°C each annealed fragment for 15-60 minutes • Use NEBuilder HiFi in successive rounds of assembly, • DNA ligase seals nicks as it removes 5´- and 3´-end mismatches in the assembled DNA • Bridge two ds-fragments with a synthetic ssDNA oligo for simple and fast construction (e.g., linker insertion Transformation or gRNA library) • No licensing fee requirements from NEB for NEBuilder products DNA Analysis • NEBuilder HiFi DNA Assembly Cloning Kit includes OR OR the NEBuilder HiFi DNA Assembly Master Mix and NEB 5-alpha Competent E. coli RE Digest Colony PCR Sequencing • NEBuilder HiFi DNA Assembly Bundle for Large Fragments includes the NEBuilder HiFi DNA Assembly Master Mix and NEB 10-beta Competent E. coli for assemblies larger NEBuilder HiFi DNA Assembly than 15 kb. can be used for a variety of DNA assembly methods. TOOLS & RESOURCES 2-fragment assembly ssOligo & dsDNA assembly Visit NEBuilderHiFi.com to find: • Online tutorials to help with assembly and primer design • Application notes utilizing NEBuilder HiFi 4-fragment assembly Annealed-oligo assembly 15-bp overlap 25-bp overlap • Access to NEBuilder Assembly Tool, our online Sticky end Blunt end primer design tool High efficiency DOWNLOAD THE NEB AR APP* 3´- and 5´-end mismatch assembly Site-directed mutagenesis Multiple sites How does NEBuilder HiFi DNA Assembly work? NEBuilder HiFi DNA Assembly can be used for a variety of DNA assembly methods. *see back cover for details 3 1 0 01 0 1 SYNTHETIC BIOLOGY Gibson Assembly Gibson Assembly enables multiple, overlapping DNA fragments to be joined in a single- RECOMMENDED PRODUCTS tube isothermal reaction, with no additional sequence added (scar-less). The Gibson Assembly Master Mix includes three different enzymatic activities that perform in a Gibson Assembly Cloning Kit (NEB #E5510) single buffer. The assembled, fully-sealed construct is then transformed into NEB 5-alpha competent E. coli. The entire protocol, from assembly to transformation, takes just under Gibson Assembly Master Mix (NEB #E2611) two hours. • Visit NEBGibson.com to learn more Cell-free Expression ® NEBExpress Cell-free E. coli Protein Synthesis System RECOMMENDED PRODUCTS The NEBExpress Cell-free E. coli Protein Synthesis System is a coupled transcription/ translation system designed to synthesize proteins encoded by a DNA or mRNA NEBExpress Cell-free E. coli Protein template under the control of a T7 RNA Polymerase promoter. The system offers high Synthesis System expression levels, the ability to produce high molecular weight proteins, scalability, and (NEB #E5360) is cost-effective for high throughput expression applications. The speed and robustness • Synthesize high yields of protein (typically 0.5 mg/ml) of the system facilitates protein synthesis in applications such as protein engineering, • Protein can be synthesized and visualized in approximately mutagenesis studies and enzyme screening. 2–4 hours The NEBExpress Cell-free E. coli Protein Synthesis System contains all the components • Synthesize target proteins ranging from 17 to 230 kDa required for protein synthesis, except for the target template DNA. It is a combination of a highly active cell extract from a genetically engineered strain, a reaction buffer, and • Templates can be plasmid DNA, linear DNA, or mRNA an optimized T7 RNA Polymerase, which together yield robust expression of a wide • RNase contamination can be inhibited by the supplied variety of protein targets ranging from 17 to 230 kDa. RNase inhibitor, eliminating clean-up steps The NEBExpress Cell-free E. coli Protein Synthesis System can be used to express a • Flexible reaction conditions achieve maximum yield; protein wide range of proteins synthesis can be sustained for 10 hours at 37°C or up to 24 hours at lower temperatures M neg bGalS bGal SP6 FLuc BglA CitS vGFP GMK DHFR CALM 200 • Reactions can be miniaturized or scaled up to yield milligram quantities of protein 100 PURExpress In Vitro Protein Synthesis Kit (NEB #E6800) 60 • Suitable for circular or linear DNA template • Visualize synthesized protein directly on a Coomassie stained gel 25 50 µl reactions containing 250 ng template • Protein expression in approximately 2 hours DNA were incubated at 37°C for 3 hours. The red dot indicates the protein of interest. • Templates can be plasmid DNA, linear DNA or mRNA 15 M = Unstained Protein Standard, Broad Range (NEB #P7717), “neg” = negative • Transcription/translation components can be removed by control, no DNA affinity chromatography PURExpress® In Vitro Protein Synthesis Kit A rapid method for gene expression analysis, PURExpress is a novel cell-free transcription/translation system reconstituted from purified components necessary for E. coli translation. Synthesize a wide range of proteins free of modification or degradation by simply mixing two tubes followed by the addition of template DNA. With results available in only a few hours, PURExpress saves valuable laboratory time and is ideal for high throughput technologies. Product selection includes the original kit, with all components in two tubes, as well as options for protein translation experiments, protein synthesis/ribosomal display experiments and synthesis with modified amino acids. 4 1 0 01 SYNTHETIC BIOLOGY 0 1 Golden Gate Assembly The efficient and seamless assembly of DNA fragments, commonly referred to as Golden RECOMMENDED
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