NEBuilder® HiFi DNA Assembly Master Mix


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 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 kits are available in various formats: with NEB 5-alpha competent cells (Cloning Kit, NEB #E5520), as a bundle with NEB 10-beta competent cells (Bundle for Large Fragments, NEB #E2623) and without competent cells (Master Mix, NEB #E2621) . NEB 5-alpha competent cells 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: Overview of the NEBuilder HiFi DNA Assembly MethodFigure 1: Overview of the NEBuilder HiFi DNA Assembly Method

Figure 2: NEBuilder HiFi DNA Assembly offers improved efficiency and accuracy over
NEB Gibson Assembly

Reactions 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 Assembly
Figure 3: NEBuilder HiFi delivers higher colony yield than In-Fusion HD
Two-fragment reactions were set up using the positive control from the In-Fusion HD Cloning Kit (Clontech Takara Bio USA, Inc), according to recommended protocols. 2 μl of assembly reaction was transformed into supplied competent cells. 1/50 of outgrowth was spread on an ApR plate.

View additional performance data compared to In-Fusion HD

table icon Comparison of DNA Assembly Reaction Types


Kit Components

The following reagents are supplied with this product:

Store at (°C)Concentration
NEBuilder® High-Fidelity Master Mix-20
NEBuilder® Positive Control-202X

Advantages and Features


Benefits over NEB Gibson Assembly Master Mix and/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 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 6 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.

Properties and 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.

Storage Temperature



  1. 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.

  2. 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.

  3. 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.

  4. Electroporation: Electroporation can increase transformation efficiency by several logs. When using the NEBuilder HiFI DNA Assembly Master Mix, use 1μl 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"

  5. 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.


  1. What are the advantages of this method compared to traditional cloning methods?
  2. Are there any differences between NEBuilder® HiFi DNA Assembly Master Mix and NEBuilder HiFi DNA Assembly Cloning Kit ?
  3. 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?
  4. What is the largest single fragment that has been assembled with NEBuilder® HiFi DNA Assembly Master Mix?
  5. How many fragments of DNA can be assembled in one reaction?
  6. Is this method applicable to the assembly of repetitive sequences?
  7. What are the shortest overlaps that can be used with this assembly method?
  8. What are the longest overlaps that can be used with this method?
  9. Can ≤ 200 bp dsDNA fragments be assembled by this method?
  10. Can ssDNA oligonucleotides be assembled with dsDNA fragments?
  11. Can longer or shorter incubation times be used?
  12. Will the reaction work at other temperatures?
  13. Is it necessary to purify PCR products?
  14. Is it necessary to inactivate restriction enzymes after vector digestion?
  15. 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?
  16. I would like to assemble ssDNA oligonucleotides into dsDNA fragments. Do I need to use oligonucleotides that have been purified by PAGE or HPLC?
  17. Can I use a 15-nt overlap that is entirely composed of His-tag repeats (i.e., CACCACCACCACCAC)?
  18. Can I PCR-amplify the assembled product?
  19. The control reaction is not resulting in any colonies. Why?
  20. 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?
  21. How can I reduce the number of vector-only background colonies?
  22. What type of competent cells are suitable for transformation of DNA constructs created using NEBuilder® HiFi DNA Assembly Master Mix?
  23. Can I use electroporation instead of chemical transformation?
  24. Are there any differences between the requirements for 2–3 fragment assemblies versus 4+?
  25. Can I use PCR product amplified from Taq DNA polymerase?
  26. Can I Use other primer design tools such as SnapGene for Gibson Assembly, to design primers for NEBuilder® HiFi DNA Assembly?
  27. 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)?
  28. 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?
  29. How can I improve the efficiency of very complex assembly reactions when using NEBuilder® HiFi DNA Assembly Master Mix?

Tech Tips


  1. NEBuilder® HiFi Electrocompetent Transformation Protocol (E2621)
  2. NEBuilder® HiFi DNA Assembly Chemical Transformation Protocol (E2621)
  3. NEBuilder® HiFi DNA Assembly Reaction Protocol


The Product Manual includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name these document files: manual[Catalog Number].

Selection Charts

Interactive Tools

Application Notes

Safety Data Sheet

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