  Golden Gate cloning has become one of the most popular DNA assembly
techniques. Its modular and hierarchical structure allows the construction of
complex DNA fragments. Over time, Golden Gate cloning allows for the creation
of a repository of reusable parts, reducing the cost of frequent sequence
validation. However, as the number of reactions and fragments increases, so
does the cost of consumables and the potential for human error. Typically,
Golden Gate reactions are performed in volumes of 10 to 25 microlitre. Recent
technological advances have led to the development of liquid handling robots
that use sound to transfer liquids in the nL range from a source plate to a
target plate. These acoustic dispensers have become particularly popular in the
field of synthetic biology. The use of this technology allows miniaturization
and parallelization of molecular reactions in a tip-free manner, making it
sustainable by reducing plastic waste and reagent usage. Here, we provide a
step-by-step protocol for performing and parallelizing Golden Gate cloning
reactions in 1 microlitre total volume.
