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NEBuilder HiFi DNA Assembly versus Gibson Assembly


NEBuilder HiFi DNA Assembly from New England BioLabs (NEB) sets a new standard for seamless and fast cloning. The kit is developed to improve the efficiency and accuracy of DNA assembly. It allows easy seamless assembly of multiple DNA fragments, regardless of fragment length or end compatibility.

"From now on we will use the NEBuilder HiFi instead of Gibson Assembly."
- Mr. Nils Schoovaerts, lab technician at VIB Center for Biology of Disease -

Mr Nils Schoovaerts, a lab technician working at the VIB Center for Biology of Disease/KULeuven Center for Human Genetics, Laboratory of Neuronal Communication, shares his positive experience with the NEBuilder HiFi Assembly Master Mix for cloning different constructs with multiple fragments:

“To generate viral vectors for stem cell applications we need assembly of multiple fragments as well as 100% correct clones. The main advantage of using assembly cloning over regular cloning is that we can assemble the multiple fragments in one single reaction. We already use the NEB Gibson assembly and we wanted to test the new NEBuilder HiFi assembly in a side-by-side comparison.

Here we assembled in total 4 fragments to generate the viral vectors: one vector fragment and 3 inserts. The inserts were obtained by PCR (with the Q5 High Fidelity DNA polymerase, NEB). The vector was cut with two different restriction enzymes (NEB). All fragments have overlapping regions of 21 bp.

Equimolar ratios (0.0085 pmol/fragment) of vector and fragments were used. The reaction was set up on ice and incubated for 1h at 50°C. Thereafter cells were transformed according to each manual: 1µl of the NEBuilder HiFi assembled products and in parallel 2µl of 1/3 diluted Gibson assembled products.

Figure 1A.
Gel image of colony PCR from NEBuilder HiFi (A) and Gibson (B) assembly reactions for construct 1.

Figure 1B.
Gel image of colony PCR from NEBuilder HiFi (C) and Gibson (D) assembly reactions for construct 2.

Figure 2A. Peak pattern of clone 3 assembled with the Gibson Assembly Master Mix. Indicated with the arrow is the extra base pair, which leads to an unwanted frame shift.

Figure 2B. Peak pattern of clone 3 assembled with the NEBuilder HiFi Master Mix. Sequence was 100% correct.

A colony PCR was first performed to establish the assembly into one construct. This PCR revealed more assembled clones by size for the NEBuilder HiFi Master Mix, for construct 1. Fourteen correct clones versus 8 correct clones with the Gibson assembly (Fig. 1A). While the number of positive clones were equal for construct 2 with both master mixes (Fig. 1B), sequencing one of the clones obtained with the Gibson assembly revealed a base pair insertion, causing an unwanted frame shift, adjacent to one of the overlap regions (Fig. 2A). All sequenced clones derived with the NEBuilder HiFi Master Mix were correct (Fig. 2B).

In conclusion, the NEBuilder is the better choice for complex assembly reactions as described above. We clearly show two different advantages of the NEBuilder master mix. It can generate more correct clones when using multiple fragments and it has a greater accuracy. From now on we will use the NEBuilder HiFi Master Mix instead of the Gibson Assembly Master Mix.”

Ordering Information

Please find the ordering information below. For more information about one of the listed products, please click here.

Cat. # Description Quantity
E2621S NEBuilder HiFi DNA Assembly Master Mix 10 rxns
E2621L NEBuilder HiFi DNA Assembly Master Mix 50 rxns
E2621X NEBuilder HiFi DNA Assembly Master Mix 250 rxns
E5520S NEBuilder HiFi DNA Assembly Cloning Kit 10 rxns
E2623S NEBuilder HiFi DNA Assembly Bundle for Large Fragments 20 rxns


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