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Golden Gate cloning has become one of the most popular DNA assemblytechniques. Its modular and hierarchical structure allows the construction ofcomplex DNA fragments. Over time, Golden Gate cloning allows for the creationof a repository of reusable parts, reducing the cost of frequent sequencevalidation. However, as the number of reactions and fragments increases, sodoes the cost of consumables and the potential for human error. Typically,Golden Gate reactions are performed in volumes of 10 to 25 microlitre. Recenttechnological advances have led to the development of liquid handling robotsthat use sound to transfer liquids in the nL range from a source plate to atarget plate. These acoustic dispensers have become particularly popular in thefield of synthetic biology. The use of this technology allows miniaturizationand parallelization of molecular reactions in a tip-free manner, making itsustainable by reducing plastic waste and reagent usage. Here, we provide astep-by-step protocol for performing and parallelizing Golden Gate cloningreactions in 1 microlitre total volume.

Automation and miniaturization of Golden Gate DNA assembly reactions using acoustic dispensers