FAQ: When using a polymerase that doesn't contain a 3'-5' exonuclease activity (such as Taq DNA Polymerase) to amplify fragments to be used in a Gibson Assembly reaction, should I be concerned about the potential 3' mismatch generated by the addition of a non-templated nucleotide?

This type of 3' mismatch can affect the efficiency of the assembly, especially when attempting to assemble a high number of fragments (4-6). In those cases, it may be necessary to screen more colonies to obtain the correctly assembled clone. To prevent this, we recommend the use of polymerases that produce blunt-ended amplicons, such as Q5 High-Fidelity DNA Polymerase .