FAQ: Why do many of the published Golden Gate Assembly articles feature precloned inserts as opposed to inserts generated by PCR?

Precloned inserts allow stable storage of inserts and make it easy to calculate the molarity of inserts, while using amplicon inserts saves time. Stable storage of amplicon inserts is important. Single insert cloning/assemblies can use unpurified amplicons, while multiple insert amplicons should be purified, for example by spin column protocols such as the NEB Monarch PCR and DNA Cleanup Kit (5 μg), NEB #T1030. For long term storage at -20°C, store DNA in 10 mM Tris (pH 8.5), 1 mM EDTA (TE) or short-term storage in 10 mM Tris (pH 8.5), 0.1 mM EDTA (modified TE). EDTA at these levels will not significantly lower the 10 mM MgCl2 present in the T4 DNA Ligase Buffer.