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