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NEBuilder® HiFi DNA Assembly – Benefits Over GeneArt Gibson Assembly® and In-Fusion® Snap Assembly

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NEBuilder HiFi DNA Assembly offers several advantages over GeneArt Gibson Assembly and In-Fusion Snap Assembly. These include: higher accuracy due to the use of a high-fidelity polymerase, the ability to assemble both 5´- and 3´-end mismatches, lower DNA input requirements and the ability to bridge two dsDNA fragments with a ssDNA oligo. NEBuilder HiFi is the clear choice for efficient and accurate DNA assembly.


Performance Data

See how NEBuilder HiFi outperforms GeneArt Gibson Assembly and In-Fusion Snap Assembly

 

Figure 1: NEBuilder HiFi DNA Assembly offers improved thermostability



Two-fragment assembly reactions were performed using a positive control* according to recommended protocols for NEBuilder HiFi DNA Assembly Master Mix (NEB #E2621), GeneArt Gibson Assembly Mix (Thermo Fisher Scientific #A46627) and In-Fusion Snap Assembly Master Mix (Takara Bio USA #638947). 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 an LB/Amp plate. After 34 days of incubation at 25°C, the number of colonies from NEBuilder HiFi DNA Assembly Mix are 4–8 times higher than from the GeneArt Gibson Assembly Mix and 3-5 times higher than In-Fusion Snap Assembly Mix.

* Positive control from In-Fusion Snap Assembly Master Mix (Takara Bio USA)  

Figure 2: NEBuilder HiFi DNA Assembly offers improved efficiency in 2-fragment assembly reactions



Two-fragment assembly reactions were performed using a positive control*, according to recommended protocols for 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). GeneArt Gibson Assembly reaction was performed for 60 min at 50°C. In-Fusion Assembly reaction was performed for 15 min at 50°C. 2 μl of each assembled mix was transformed into NEB 5-alpha Competent E.coli (NEB #C2987) and spread on an LB/Amp plate.

* Positive control from In-Fusion Snap Assembly Master Mix (Takara Bio USA)  

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 that indicated correct assembly were counted. NEBuilder HiFi DNA Assembly Mix yields more colonies than both competitors.  

Figure 4: NEBuilder HiFi DNA Assembly offers improved efficiency in 6-fragment assembly reactions



Six-fragment (5 x 1 kb + 3.3 kb vector) assembly reactions were performed according to recommended protocols for 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). Reactions were performed in a 20 μl (NEBuilder HiFi and GeneArt) or 10 μl (In-Fusion) reaction volume. Assembly reactions were performed at 50°C for 60 min (NEBuilder HiFi and GeneArt) or 15 min (In-Fusion). 2 μl of each assembled mix was transformed into NEB 5-alpha Competent E.coli (NEB #C2987) and spread on a Rich/Cam plate with IPTG and X-Gal. Blue colonies that indicated correct assembly were counted. NEBuilder HiFi DNA Assembly Master Mix yields more colonies and is more efficient than GeneArt Gibson Assembly and In-Fusion Snap Assembly.

Figure 5: NEBuilder HiFi DNA Assembly offers improved efficiency in assembly of a ssDNA oligo with a linearized vector



One pmol of ssDNA oligos were assembled with a linearized vector (30 ng of CRISPR Nuclease Reporter DNA) using NEBuilder HiFi DNA Assembly Master Mix (NEB #E2621), GeneArt Gibson Assembly Mix (Thermo Fisher #A46627) and In-Fusion Snap Assembly Mix (Takara Bio USA #638947). Assembly reactions were performed at 50°C for 60 min or 15 min. 2 μl of the assembled mix was transformed into NEB 5-alpha Competent E.coli NEB #C2987). 20 colonies were further screened by PCR to confirm the presence of inserts. Greater than 95% of colonies tested from NEBuilder HiFi and GeneArt Gibson Assembly reactions contained proper inserts, although GeneArt Gibson Assembly yielded fewer colonies. In-Fusion Snap did not yield any successful colonies. NEBuilder HiFi DNA Assembly Master Mix outperformed both GeneArt Gibson Assembly and In-Fusion Snap Assembly.  

Figure 6: NEBuilder HiFi DNA Assembly offers improved efficiency in assembly of annealed ssDNA oligos with 3´ and 5´ overhangs to a linearized vector



One pmol of seven annealed ssDNA oligos, which yielded dsDNA with nicks and 3´and 5´ overhangs, were assembled with a linearized vector (20 fmol pUC19) using NEBuilder HiFi DNA Assembly Master Mix (NEB #E2621), GeneArt Gibson Assembly Mix (Thermo Fisher #A46627) and In-Fusion Snap Assembly Mix (Takara Bio USA #638947) with incubation at 50°C for either 60 min or 15 min. 2 μl of the assembled mix was transformed into NEB 5-alpha Competent E.coli (NEB #C2987) and spread on an LB/Amp plate with IPTG and X-Gal. Blue colonies that indicated correct assembly were counted. NEBuilder HiFi DNA Assembly Master Mix outperforms both GeneArt Gibson Assembly and In-Fusion Snap Assembly.  

Figure 7: Amplification of 12-fragment DNA assembly product demonstrates efficiency of NEBuilder HiFi DNA Assembly



1 μl was removed from 12-fragment assembled DNA reactions respectively (NEBuilder HiFi DNA Assembly Master Mix (NEB #E2621), GeneArt Gibson Assembly Mix (Thermo Fisher #A46627) and In-Fusion Snap Assembly Mix (Takara Bio USA #638947)) and used as templates in PCR. In vitro amplification of 12-fragment assembled product can only be found using NEBuilder HiFi but not from GeneArt Gibson Assembly and In-Fusion Snap Assembly, suggesting NEBuilder HiFi DNA Assembly Mix has higher assembly efficiency.  

Figure 8: In vitro enrichment of 20 kb assembled DNA by phi-29 DNA Polymerase



A 3-fragment assembly reaction was setup to construct a 20 kb dsDNA. 1 μl was removed from assembled DNA reactions respectively (NEBuilder HiFi DNA Assembly Master Mix (NEB #E2621), GeneArt Gibson Assembly Mix (Thermo Fisher #A46627) and In-Fusion Snap Assembly Mix (Takara Bio USA #638947) and used as templates in rolling circle amplification (RCA). Amplified DNA was further purified by SPRIselect™ beads followed by restriction enzyme digestion to confirm the insert. Red star indicates the presence of insert when the amplified DNA was digested with EcoRI-HF (NEB #R3101) and SrfI (NEB #R0629). Amplified DNA can be detected from all three assembly mixes followed by phi-29 amplification protocol. However, only the amplified product using templates from NEBuilder HiFi mix yielded a specific DNA band corresponding to the size of insert (~12 kb) after EcoRI-HF/SrfI digestion. This suggests that only NEBuilder HiFi DNA Assembly yields an adequate amount of covalently sealed 20 kb plasmid that can be efficiently amplified in vitro by phi-29 DNA Polymerase.  



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