HomeFAQsUsing purified amplicons directly without precloning seems much easier, but is the assembly efficiency decreased?
FAQ: Using purified amplicons directly without precloning seems much easier, but is the assembly efficiency decreased?
No. While in general DNA is more stable in circular form than in linear form due to the absence of free ends, amplicons are a viable and easy way to build assemblies as long as they have been purified and are stored in the appropriate buffer (see FAQ). The suggested 2:1 molar ratio of amplicon inserts : destination vector brings the assembly efficiency to that of precloned inserts (using 75 ng of each plasmid) for most assemblies.