Product Class: Kit

NEBuilder® HiFi DNA Assembly Cloning Kit

NEBuilder HiFi DNA Assembly Master Mix has been reformulated with components containing Recombinant Albumin (rAlbumin) beginning with Lot #10238675.

Product Introduction

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 (15–30 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. Visit NEBuilderHifi.com for more information.

  • Assembly and transformation in just under two hours
  • Enjoy less screening/re-sequencing of constructs, with virtually error-free, high-fidelity assembly
  • Flexible sequence design (scar-less cloning)
  • No PCR clean-up step required
  • Join DNA fragments together more efficiently, even with larger fragments or low DNA inputs
  • Use 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 requirements
  • Flexible sequence design (scar-less cloning)
  • Easily adapted for multiple DNA manipulations, including site-directed mutagenesis
  • Supplied with 10 x 50 μl vials of NEB® 5-alpha Competent E. coli (NEB #C2987) which are chemically competent
Catalog # Size Concentration
E5520S 10.0 reactions

Featured Videos

View Video Library
  • Construction

    Construction of an sgRNA-Cas9 Expression Vector via an ssOligo Bridge

  • nebuilder_overview_thumb

    Introduction to NEBUILDER HIFI DNA ASSEMBLY®

  • NEBioCalculator Thumbnail

    NEBioCalculator® - Using the ds: mass < — > moles module to plan an NEBuilder® HiFi DNA Assembly Reaction

  • NEBTVwebinar_NEBuilderHiFi_720

    NEBUILDER® HiFi: the next generation of DNA Assembly

  • nebuilder_3primemismatchvideo_thumb

    NEBUILDER HIFI DNA ASSEMBLY® Removal of 3´ end Mismatches

  • NEBuilder_BridgingVideo_Thumb

    NEBUILDER® HIFI DNA ASSEMBLY® : Bridging dsDNA with a ssDNA Oligo

  • PrimerDesignFragmentAssembly_720

    Primer Design and Fragment Assembly Using NEBuilder HiFi DNA Assembly® or Gibson Assembly®

  • NEBTV_Ep8_video_thumb

    NEB® TV Ep. 8 – Cloning and DNA Assembly

  • NEBTV 12

    NEB® TV Ep. 12 – Applications of DNA Assembly

  • NEBTV_Ep1_VideoThumb

    NEB® TV Ep. 1 – Synthetic Biology

  • NEBTVToolsThumbnailNEBuilderAssemblyTool20WhatsNew1920

    NEBUILDER® Assembly Tool 2.0 What’s New?

  • NEBTVToolsThumbnailNEBuilderAssemblyTool20Fragments Amplified by PCR

    NEBUILDER® Assembly Tool 2.0 Fragments Amplified by PCR

  • NEBTVToolsNEBuilderAssemblyTool20RestrictionEnzymeDigest1920

    NEBUILDER® Assembly Tool 2.0 Restriction Enzyme Digest

Product Information

Description



NEBuilder HiFi DNA Assembly Cloning Kit 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 (15–30 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 fragment
  • The DNA ligase seals nicks in the assembled DNA 
The 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 Cloning Kit is supplied with NEB 5-alpha High Efficiency Competent 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 routine assemblies of 15kb 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 15kb. 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:
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: Not your average DNA assembly reagent



Figure 2: Overview of the NEBuilder HiFi DNA Assembly method



Figure 3: NEBuilder HiFi DNA Assembly offers improved efficiency in 4-fragment assembly reactions


Four fragments (~20 fmol) with 20 bp overlap were assembled using NEBuilder HiFi DNA Assembly Master Mix (NEB #E2621), GeneArt® Gibson Assembly® Mix (Thermo Fisher #A46627) and In-Fusion® Snap Assembly Master Mix (Takara Bio USA #638947) to create a pUC19 vector. Assembly reactions were performed at 50°C for 60 min or 15 min. 2 μl of each assembled mix was transformed into NEB 5-alpha Competent E.coli (NEB #C2987) and spread on LB/Amp plates with IPTG and X-Gal. Blue colonies ( >95% of total colonies) that indicated correct assembly were counted. NEBuilder HiFi DNA Assembly Mix yields more colonies than both competitors.

View additional performance data compared to GeneArt Gibson Assembly and In-Fusion Snap Assembly
This product is related to the following categories:
DNA Assembly, Cloning and Mutagenesis Kits Products
This product can be used in the following applications:
NEBuilder® HiFi DNA Assembly,
Genome Editing Applications

Kit Components

Kit Components

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration

Properties & Usage

Materials Required but not Supplied

DNA 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.

Features


Benefits over GeneArt Gibson Assembly and In-Fusion Snap Assembly:

  • 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 inputs
  • Use 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 requirements
  • Switch from other systems easily, because NEBuilder 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.

Product Notes

  1. Upon arrival, store the kit components at –80°C. Before use, thaw and vortex the master mix thoroughly and keep on ice. After first use, store the HiFi DNA Assembly Master Mix and postive controls at –20°C, store the SOC Outgrowth Medium at room temperature. Store the competent cells at –80°C.
  2. 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.
  3. 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.

  4. 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), Nico21(DE3), and SHuffle®. When using competent E. coli from a vendor other than NEB, we have seen decreased robustness of transformation with high-fidelity DNA assembled products.
  5. Electroporation: Electroporation can increase transformation efficiency by several logs. When using the NEBuilder HiFi DNA Assembly Master Mix, use 1 μl 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"

  6. 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.

Protocols, Manuals & Usage

Protocols

  1. NEBuilder® HiFi DNA Assembly Electrocompetent Transformation Protocol
  2. NEBuilder® HiFi DNA Assembly Chemical Transformation Protocol (E2621, E5520, E2623)
  3. NEBuilder HiFi DNA Assembly Reaction Protocol
  4. NEBuilder Assembly of a PCR Fragment
  5. Protocol for cloning DNA containing repeat elements (C3040)
  6. Protocol for Bridging double-stranded DNA with a single-stranded DNA oligo using NEBuilder HiFi DNA Assembly (NEB #E2621)
  7. Protocol for assembling annealed DNA oligonucleotides and a double-stranded DNA vector using NEBuilder HiFi DNA Assembly (NEB #E2621)

Manuals

The Product Manual includes details for how to use the product, as well as details of its formulation and quality controls.

Usage & Guidelines

Application Notes

Tools & Resources

Selection Charts

Web Tools

FAQs & Troubleshooting

FAQs

  1. I am not sure whether to choose NEBuilder HiFi DNA Assembly or NEB Gibson Assembly? How are the products different?
  2. What are the advantages of this method compared to traditional cloning methods?
  3. Are there any differences between NEBuilder HiFi DNA Assembly Master Mix and NEBuilder HiFi DNA Assembly Cloning Kit ?
  4. What is the difference between NEBuilder HiFi DNA Assembly Master Mix/DNA Assembly Cloning kit/Bundle for Large Fragments kit and the current NEB Gibson Assembly Master Mix/Cloning Kit?
  5. What is the largest single fragment that has been assembled with NEBuilder HiFi DNA Assembly Master Mix?
  6. How many fragments of DNA can be assembled in one reaction?
  7. Is this method applicable to the assembly of repetitive sequences?
  8. What are the shortest overlaps that can be used with this assembly method?
  9. What are the longest overlaps that can be used with this method?
  10. Can ≤ 200 bp dsDNA fragments be assembled by this method?
  11. Can multiple ssDNA oligonucleotides be assembled with dsDNA fragments?
  12. Can longer or shorter incubation times be used?
  13. Will the reaction work at other temperatures?
  14. Is it necessary to purify PCR products when doing DNA assemblies using either NEBuilder HiFi DNA Assembly Master Mix or Gibson Assembly Master Mix?
  15. Is it necessary to inactivate restriction enzymes after vector digestion?
  16. I would like to produce overlapping dsDNA fragments by PCR. Do I need to use PCR primers that have been purified by PAGE or HPLC?
  17. I would like to assemble ssDNA oligonucleotides into dsDNA fragments. Do I need to use oligonucleotides that have been purified by PAGE or HPLC?
  18. Can I use a 15-nt overlap that is entirely composed of His-tag repeats (i.e., CACCACCACCACCAC)?
  19. Can I PCR-amplify the assembled product?
  20. The NEBuilder Positive Control reaction is not resulting in any colonies. Why?
  21. What should I do if my assembly reaction yields no colonies, a small number of colonies, or clones with the incorrect insert size following transformation into E. coli?
  22. How can I reduce the number of vector-only background colonies?
  23. What type of competent cells are suitable for transformation of DNA constructs created using NEBuilder HiFi DNA Assembly Master Mix?
  24. Can I use electroporation instead of chemical transformation?
  25. Are there any differences between the requirements for 2–3 fragment assemblies versus 4+?
  26. Can I use PCR product amplified from Taq DNA polymerase?
  27. Can I Use other primer design tools such as SnapGene for Gibson Assembly, to design primers for NEBuilder HiFi DNA Assembly?
  28. Do you have any suggestions on how to improve DNA assembly when using synthesized DNA (e.g. gBlocks) and NEBuilder HiFi DNA Assembly Master Mix (E2621/E5520/E2623)?
  29. I want to join two fragments, but the overlap sequence is 500bp from the end of the vector. Can NEBuilder HiFi DNA Assembly Master Mix accomplish this?
  30. How can I improve the efficiency of very complex assembly reactions when using NEBuilder HiFi DNA Assembly Master Mix?
  31. How should fragments be prepared for assembly using NEBuilder HiFi?
  32. I would like to miniaturize DNA assembly with NEBuilder HiFi DNA Assembly Master Mix using the Labcyte Echo Liquid Handler. Do you have any information that I can refer to help me automate DNA assembly?
  33. How does overlap length affect NEBuilder HiFi DNA Assembly efficiency?
  34. What antibiotic resistance is encoded in the NEBuilder Positive Control (assembly control)?

Tech Tips

Interested in how DNA assembly works? Learn more in this short animation.
nebuilder_overview_thumb
When assembling DNA, you can bridge two dsDNA fragments with a ssDNA oligo using NEBuilder HiFi DNA Assembly. Learn more in this short animation.
NEBuilder_BridgingVideo_Thumb
When designing your DNA assembly experiment, we suggest designing the experiment in silico to help with primer design. Learn more in this short animation.
PrimerDesignFragmentAssembly_720

Quality, Safety & Legal

Quality Assurance Statement

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.

Specification Change Notifications

NEBuilder HiFi DNA Assembly Master Mix has been reformulated with components containing Recombinant Albumin (rAlbumin) beginning with Lot #10238675. All subsequent (higher number) lots will contain rAlbumin.

NEBuilder HiFi DNA Assembly Mix has been reformulated to be more stable starting with Lot #10111279 and all subsequent (higher number) lots.

Specifications

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]

Certificate Of Analysis

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

Safety DataSheets

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

Legal and Disclaimers

Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@neb.com.

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.

New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.

Licenses

Limited Warranty: The NEBuilder® HiFi Assembly Master Mix and NEBuilder® HiFi DNA Assembly Cloning Kit are warranted 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.

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