HomeFAQsCan PCR amplicons be used directly in assembly reactions without purification?
FAQ: Can PCR amplicons be used directly in assembly reactions without purification?
Yes, as long as insert volume is 1 µl or less and single insert (cloning) is being done. Although efficiencies will be decreased. Most Type IIS restriction enzymes used for Golden Gate Assembly generate 5´-four base overhangs that can be filled-in by the carryover DNA polymerase used in PCR when using unpurified amplicons, producing blunt ends. This will lead to nonspecific assembly. For single insert cloning/assembly, the ligase successfully competes with the carryover DNA polymerase such that unpurified PCR amplicon inserts can be used but will result in lower assembly performance. For multiple insert Golden Gate assemblies, purify the amplicons and if non-specific products are present, optimize the PCR or gel purify.