High-throughput cloning and automation solutions
High-throughput cloning is a molecular biology method of assembling large numbers of DNA sequences, such as genes, open reading frames (ORFs) or highly repetitive gRNAs, to create libraries and enable screening of constructs, protein expression or protein function. With the integration and adoption of automation, researchers can scale up and increase throughput to hundreds or thousands of reactions, save time and money with rapid workflows and miniaturized volumes, and improve reproducibility with automated complex mixing that reduces manual errors.
Applications including efficient and accurate DNA Assembly and mutagenesis, sequencing, and cell-free protein synthesis and purification are amenable to high-throughput workflows and automation devices. These tools enable the rapid interrogation of a more expansive set of custom designs.
High-throughput DNA assembly
NEBuilder® HiFi DNA Assembly and NEBridge® Golden Gate Assembly are leading the way in the next generation of cloning. Both methods are amenable to high-throughput workflows and scale up using automation platforms such as the Echo® 525 Liquid Handler from Labcyte®, Inc. and the mosquito® LV from sptlabtech. High efficiency (> 95%) and accuracy with both cloning methods ensures you can confidently and quickly progress with accurate constructs.
NEBuilder HiFi is recommended when working with 2–11 fragment assemblies, while NEBridge Golden Gate Assembly is appropriate for more complex designs, as well as designs containing regions of high GC content or areas of repeat. Please refer to the DNA Assembly Selection Chart for additional help determining which product is best suited for your needs.
NEBuilder HiFi DNA Assembly
- Perform less sequencing and screening of constructs with high-fidelity, virtually error-free assembly
- Enjoy compatibility with synthetic dsDNA fragments, such as gBlocks™, and ssDNA oligos
- Save time by avoiding PCR clean-up, simplifying your workflow
- Supports miniaturization with nanoliter scale volumes
- Technical note: Modular DNA Assembly of PIK3CA Using Acoustic Liquid Transfer in Nanoliter Volumes
- Technical note: Miniaturized Multi-Piece DNA Assembly Using the Echo 525 Liquid Handler
- Technical note: Nanoliter Scale DNA Assembly Utilizing the NEBuilder HiFi Cloning Kit with the Labcyte Echo 525 Liquid Handler
- Application note: Automated high-density sample storage and miniaturized liquid handling for high-throughput synthetic biology construct assembly
- Easily adaptable to multiple site-directed mutagenesis
- Design primers and assemblies quickly and easily with our free online tool, NEBuilder Assembly Tool
NEBridge Golden Gate Assembly
- Experience high efficiency within regions of high GC content and areas of repeats
- Supports miniaturization
- Enjoy compatibility with synthetic dsDNA fragments, such as gBlocks
- Find flexibility with NEBridge Ligase Master Mix (NEB #M1100) and your choice of Type IIS restriction enzymes
- Design complex, high-fidelity Golden Gate Assemblies easily with our free online tools
- NEBridge™ Golden Gate Assembly Tool – design primers, predict overhang fidelity and find optimal junctions
- NEBridge Ligase Fidelity Tools – visualize overhang ligation preferences, predict high fidelity junction sets and split DNA sequences for scarless high-fidelity assembly
NEB Cloning Competent Cells
- Experience high transformation efficiencies
- Enjoy compatibility with 96-well and 384-well formats
- Find the right fit with bulk formats and custom packaging options
- Choose from:
- NEB 5-alpha Competent E. coli (High Efficiency) (NEB #C2987)
- NEB 10-beta Competent E. coli (High Efficiency) (NEB #C3019)
- NEB Stable Competent E. coli (High Efficiency) (NEB #C3040)
- NEB Turbo Competent E. coli (High Efficiency) (NEB #C2984)
- dam–/dcm– Competent E. coli (NEB #C2925)
- NEB 5-alpha F'Iq Competent E. coli (High Efficiency) (NEB #2992)
Reliably create mutant libraries in a high-throughput workflow, whether making single or multiple point mutations, or performing combinatorial mutagenesis.
For single point mutations, site directed mutagenesis using Q5® Hot Start DNA Polymerase and KLD Enzyme Mix is recommended, and for multiple sites, or combinatorial mutagenesis, we recommend NEBuilder HiFi DNA Assembly.
Single site-directed mutagenesis
- Quickly create mutant libraries with primers designed with our free online tool, NEBaseChanger®
- Scale-up, or miniaturize with individual master mix formats compatible with automation
- Use Q5 Hot Start High-Fidelity 2X Master Mix (NEB #M0494) for PCR amplification
- Use KLD Enzyme Mix (NEB #M0554) for kinase, ligase and DpnI enzymatic activities in single mix
- Reduce screening of correct mutants with extremely accurate and robust Q5 High-Fidelity DNA Polymerase
- Take advantage of automation compatible room temperature reaction set up using a Hot Start Polymerase
Multi site-directed mutagenesis
Using NEBuilder HiFi DNA Assembly (NEB #E2621), perform multi-site mutagenesis or combinatorial mutagenesis for diverse multi-site mutant library creation and screening.
High-throughput protein expression and purification
Traditional cell-based protein expression methods are challenged by limitations in speed, throughput and automation complexity. Synthesizing proteins using cell-free protein synthesis (CFPS), can overcome these shortcomings and provide a rapid and reproducible method, even for toxic proteins. CFPS components are readily dispensable by automated liquid handling devices, thus enabling high-throughput workflows and miniaturization, as described in our application note. High-throughput protein purification and separation can also be accomplished using a variety of high affinity magnetic beads and racks.
NEBExpress® Cell-free E. coli Protein Synthesis System (NEB #E5360)
- E. coli extract-based transcription/translation engineered for high in vitro synthesis performance
- Synthesize high yields of target proteins ranging from 17 to 230 kDa (typically 0.5 mg/ml)
- Complete synthesis and visualization in approximately 2–4 hours
- Templates can be plasmid DNA, linear DNA or mRNA
PURExpress® In Vitro Protein Synthesis Kit (NEB #E6800)
- Reconstituted protein synthesis system includes all necessary components for in vitro transcription and translation, purified from E. coli
- Defined system with all his-tagged proteins for coupled transcription/translation; Ribosome is not his-tagged
- T7 RNA Polymerase drives in vitro transcription
- Minimal nuclease and protease activity ensure stability of synthesized protein and encoding target
- Compatible templates include plasmid DNA, linear DNA or mRNA
NEBExpress Ni-NTA Magnetic Beads (NEB #S1423)
- An affinity matrix for the small-scale isolation and purification of polyhistidine-tagged (His-tagged) fusion proteins in manual or automated formats
- Prepared with agarose based, super-paramagnetic microparticles which provide high binding capacity and fast magnetic response permitting high throughput and scalable purification strategies of His-tagged fusion proteins
Need bulk sizes or a customized solution?
Contact NEB's Customized Solutions Team, and we will work with you to meet your specific needs.
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Introduction to NEBUILDER HIFI DNA ASSEMBLY®
Find out how NEBuilder® HiFi DNA Assembly can reliably join DNA fragments in a single tube, isothermal reaction, with advantages over NEB Gibson Assembly®.
NEBUILDER® HIFI DNA ASSEMBLY® : Bridging dsDNA with a ssDNA Oligo
Learn how NEBuilder® HiFi DNA Assembly bridges dsDNA with a ssDNA oligo.
Construction of an sgRNA-Cas9 Expression Vector via an ssOligo Bridge
Learn how you can use NEBuilder HiFi to generate an sgRNA-Cas9 expression vector with a single-stranded oligo bridge.
Primer Design and Fragment Assembly Using NEBuilder HiFi DNA Assembly® or Gibson Assembly®
Watch an interactive tutorial on primer design to see how simple it really is to clone with either NEBuilder HiFi DNA Assembly or the Gibson Assembly Cloning Kit.
NEBUILDER HIFI DNA ASSEMBLY® Removal of 3´ end Mismatches
Did you know that one of the advantages of NEBuilder HiFi is that it removes 3’ and 5’ -end mismatch sequences prior to fragment assembly? Learn how!
Golden Gate Assembly Workflow
Find out how Golden Gate Assembly can be used to quickly join multiple DNA fragments.
Golden Gate Assembly Tool Tutorial
This video demonstrates how to use the Golden Gate Assembly Tool, we will walk through selecting insert and plasmids, primer design to make amplicon inserts.
Listen to DAD (Data-optimized Assembly Design) when constructing high-complexity Golden Gate Assembly targets
This webinar introduces insights and modified protocols for Golden Gate Assembly enabling 50+ DNA fragments to be faithfully assembled in a single reaction.
Introduction to the NEBExpress® Cell-free Protein Synthesis System
Watch this tutorial explaining the streamlined workflow for our new NEBExpress® Cell-free Protein Synthesis System to learn how you can easily synthesize your protein in as little as 2 to 4 hours.
Which molecular cloning
technique is best for you?
For the new cloner, NEB suggests choosing one of three cloning methods. Find the method that works for your application.
Try our free online tools for
high-throughput DNA assembly
NEBuilder Assembly Tool
Design primers for your NEBuilder HiFi DNA Assembly or Gibson Assembly® reactions
NEBridge Golden Gate Assembly Tool
Design primers, predict overhang fidelity and find optimal junctions for Golden Gate Assembly.
NEBridge Ligase Fidelity Tools
Visualize overhang ligation preferences, predict high fidelity junction sets and split DNA sequences for scarless high-fidelity assembly.
Try our free online tool for
Use NEBaseChanger to generate primer sequences and annealing temperatures for mutagenesis. Now able to upload batches.
Need bulk sizes or
a customized solution?
Contact NEB's Customized Solutions Team, and we will work with you to meet your specific needs.