Parallel DNA assembly is the assembly of multiple fragments of DNA in a single reaction. Assembly techniques can be grouped into those that use restriction endonucleases, including BioBrick and Type IIS (e.g. Golden Gate), and those based on overlaps between adjacent parts such as Gibson Assembly, CPEC, SLIC, Ligase Cycling etc. There are several recent reviews that compare various parallel DNA assembly techniques. e.g. Patron (2014) and Casini et al (2015).

In the lab we most frequently use TypeIIS mediated cloning and have published a toolkit for plants and established standards for plants. For sequence-independent cloning we recommend Gibson Assembly.

• Type IIS Cloning utilises type IIS restriction enzyme to allow the assembly of large, complex constructs from constitutive standard parts in just a few reactions. The disadvantages are (a) that it is not sequence-independent; BsaI/BpiI/BsmBI recognition sequences must be removed from all parts, and (b) that the defining of standard parts mean that scars of up to 4 base pairs may be left between parts. If you are screening a large number of genes that you will not synthesise then it may not the best choice, but if you often work with the same set of sequences, assembling them in different combinations, then it is a powerful and extremely efficient method. To help you get started with Type IIS Cloning we have developed a short online course that explains how TypeIIS restriction enzymes enable the easy and reliable assembly of multiple standard parts in a single reaction, and how to make your own standard parts. You can also download printed materials to help with primer design and wet-lab protocols from that page.

• Gibson Assembly can seamlessly assemble multiple fragments into any plasmid backbone in a sequence-independent, one-step protocol. Although you can buy Gibson reagents as pre-mixed ‘kit’, the master mix is non-proprietary and the same mix can very easily be made more economically by purchasing the regents and enzymes. The main disadvantage of all overlap-depenednt methods is that they require linear PCR products and therefore the cost and time required for primer design, amplification, PCR purification and sequence verification can be significant. However, if you need a truly seamless protocol (e.g. for domain shuffling or recoding), or if you are cloning a large number of sequences into a the same vector, then Gibson Assembly is an easy and efficient method. Learn more about the reaction and obtain a lab protocol.

• USER (Uracil-specific excision reagent) cloning is another overlap-dependent method. The plasmid backbone must be converted by the addition of a USER cassette into which a few fragments can be assembled in a parallel reaction. The plasmid (but not the fragments that will be assembled into it) must be free of recognition sites used in the USER cassette. It is somewhat like old-fashioned Gateway® cloning in that there will always be a large scar between the USER cassette and the rest of the plasmid. A further disadvantage is the requirement for primers with uracil bases and the use of specific proofreading polymerases that can read and extend through uracil bases. Additionally, the standard USER cassette cannot be inserted into every plasmid as it uses specific restriction enzymes for vector conversion. However, in some projects where you (a) wish to avoid synthesis/domestication of a large number of sequences (b) wish to clone them in a single backbone and do not mind a large scar between the inserted sequence(s) and the backbone and (c) Gibson Assembly is proving problematic (perhaps because of short fragments or secondary structure), then it might be a reasonable alternative. Learn more about USER.