NEBuilder® HiFi DNA Assembly Master Mix
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.com for more information.
- Also available with competent cells at a discounted price.
- 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
- No licensing fee requirements from NEB for NEBuilder products
Construction of an sgRNA-Cas9 Expression Vector via an ssOligo Bridge
Introduction to NEBuilder HiFi DNA Assembly
NEBuilder HiFi: the next generation of DNA Assembly
NEBuilder® HiFi DNA Assembly Removal of 3´ end Mismatches
NEBuilder® HiFi DNA Assembly: Bridging dsDNA with a ssDNA Oligo
Primer Design and Fragment Assembly Using NEBuilder HiFi DNA Assembly or Gibson Assembly®
NEB TV Ep. 8 – Cloning and DNA Assembly
NEB TV Ep. 12 – Applications of DNA Assembly
NEB TV Ep. 1 – Synthetic Biology
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
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 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
For help designing your primers for use with NEBuilder, please view our primer design video.
The following reagents are supplied with this product:
Store at (°C) Concentration NEBuilder® HiFi DNA Assembly Master Mix -20 NEBuilder® Positive Control -20 2 X
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