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.