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 NEBuilder HiFi DNA Assembly Master Mix/Cloning Kit. 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.