FAQ: Can PCR amplicons be used directly in assembly reactions without purification?

Yes, although efficiencies will be decreased by 3 to 8-fold. BsaI digestion generates 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. Tests  done with unpurified PCR material amplified by Taq, Hot-Start Taq, Q5 and Hot-Start Q5 confirmed single insert cloning worked well if the volume of material brought into the 20 µl Golden Gate Assembly reaction was no more than 1 µl. For multiple insert Golden Gate assemblies, amplicons must be purified (we recommend Monarch PCR & DNA Cleanup kit, NEB# T1030), and if non-specific products are present, amplicons must be gel purified (we recommend Monarch DNA Gel Extraction Kit, NEB# T1020).