Performing Gibson Assembly with fragments that are already digested
Our Gibson tool is designed to help facilitate the fragment digestion as part of the assembly wizard. If you are looking to set up an assembly from digested fragments, we suggest that you use our copy/paste method.
Ideal annealing Temperature and primer Tm
The conventionally suggested annealing temperature is 50˚C (which is why our Tm min default is 50). Generally, you'd set your annealing temperature below (or just below) the primers Tm.
Maintaining Restriction Sites
Unfortunately our tool at the moment does not allow you to specify whether restriction sites are maintained in the assembly or not. We apologize and hope to build out this feature sometime in the future.
Chewing Back 5' and 3' Overhangs
The Gibson assembly wizard performs assemblies based on the sense strand cuts (rather than overhang directionality) and designs primers accordingly, essentially combining the two sense ends. Depending on which kit/exonuclease is used, there would be different exonuclease activity (potentially 5' -> 3' or 3' -> 5') so we picked a common solution to cover both of them.