Gibson assembly

Question

Gibson assembly requires careful calculation of the molar amounts of each piece being assembled. What would happen if there was significantly more insert than plasmid present?

Answer 1

If the insert concentration is massively higher than the concentration of plasmid this can result in "swamping" of the reaction. In the ideal reaction a single insert molecule is assembled into the plasmid, however, if the insert concentration is high enough, 2 separate inserts can assemble with each homology region of the plasmid. The ends of the 2 inserts cannot assemble with each other because they have different sequences, however, they could react with another plasmid resulting in a concatenated plasmid.