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.
Our testing indicates that the choice of competent cells is critical. We recommend the use of high efficiency chemically competent cells such as NEB 5-alpha Competent E. coli (High Efficiency) (NEB #C2987). The reaction can be added directly to the cells without any dilution, although further dilution of the reaction mix may improve transformation efficiency. However when using high efficiency chemically competent cells from some other vendors, if you did not get any colonies, we recommend a 1:4 dilution of the reaction prior to transformation. For transformation into all high efficiency electrocompetent cells, including NEB's, we recommend a 1:3 dilution of the reaction.