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.
·Assembly and transformation in just under two hours.
·Enjoy less screening/re-sequencing of constructs, with virtually error-free, high-fidelity assembly.
·Flexible sequence design (scar-less cloning).
·No PCR clean-up step required.
·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.
·Flexible sequence design (scar-less cloning).
·Easily adapted for multiple DNA manipulations, including site-directed mutagenesis.
·Supplied with 10 x 50 μl vials of NEB® 5-alpha Competent E. coli (NEB #C2987) which are chemically competent.