DNA Assembly and Cloning

DNA assembly refers to a molecular cloning method that physically links together multiple fragments of DNA, in an end-to-end fashion, to achieve a designed, higher-order assembly prior to joining to a vector. This process is the cornerstone of the synthetic biology field and allows the construction of novel biological systems and devices using defined components. These techniques are carried out in vitro and are typically enzymatically driven with the final constructions being maintained in microbial host cells.

NEBuilder^® HiFi DNA Assembly and NEBridge^® Golden Gate Assembly products are the next-generation of tools for seamless cloning, assembly, and the synthetic biology field. These DNA assembly methods are optimized for quality, speed, flexibility, accuracy, scalability, and the ability to automate.

Which Assembly Method Should I Choose?

To help select the best DNA assembly method for your needs, please use the Product Summary Chart below and our Synthetic Biology/DNA Assembly Selection Chart for more details. If you are new to molecular cloning, you can review the advantages and disadvantages of each method by visiting the Which Molecular Cloning Technique Is Best For You page.

	NEBuilder® HiFi DNA Assembly Learn More	NEBridge® Golden Gate Assembly Learn More
STEPS IN PROTOCOL	Single tube (one-pot), only one reaction step	Single tube (one-pot), only one reaction step
CLONING EFFICIENCY	>95%	>95%
FRAGMENT SIZE	<100 bp to >10kb(1)	<50 bp to >10 kb(2)
REACTION TIME(S)	From 15 minutes	From 5 minutes
FRAGMENT NUMBER	Up to 12	Up to 50+(3)
FORMATS	Available as Master Mix or full kit with chemically competent cells	Available as Master Mix or kits with optimized destination plasmid
IDEAL APPLICATION	Single insert cloning to medium complexity assemblies of 2-6 fragments; single-stranded oligo(s) to dsDNA bridge assembly; single or multi-site mutagenesis	Single insert cloning to highly complex assemblies of up to 30 fragments; sequences with high GC content and areas of repeats; dsDNA fragments < 100 bp
PRIMER DESIGN	15-30 bp overlaps for all fragment lengths and number of fragments. Supported by online primer design tool.	3-4 bp(4) unique overhangs/overlaps with Type IIS recognition site. Supported by online primer design and ligase fidelity tools.

(1) The minimum recommended size for assembly of dsDNA fragments is 120 bp. For <100 bp fragments, single-stranded DNA oligos can be used.
(2) 50 bp to 10 kb have been validated internally. Sizes outside this range are possible.
(3) 50+ fragment assemblies require significant optimization. See Pryor et al (2022). Up to 30 fragments are recommended for routine assemblies and optimal performance.
(4) Overhang length depends on choice of Type IIS restriction enzyme.