FAQ: What should I do if my assembly reaction yields no colonies, a small number of colonies, or clones with the incorrect insert size following transformation into E. coli?

Assemble and transform the positive control provided with the Gibson Assembly Master Mix. Successful assembly of a positive control will demonstrate that the assembly mixture is functional and the transformation conditions are suitable.

Analyze the reaction on an agarose gel. An efficient assembly reaction will show assembled products of the correct size and the disappearance of fragments.

Check the primer design of the overlapping DNA fragments to ensure that there is sufficient overlap to facilitate assembly.

Consider whether the cloned insert may be toxic to E. coli and a low-copy vector, such as a BAC, should be used. Because the assembled product is a covalently closed molecule, it may be alternatively amplified by PCR or RCA.