Few or No Transformants After Cloning (Restriction Enzyme Digest)

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Bacterial transformation yields few or no colonies after restriction digest-based cloning. Vector may not be linearized or insert ends incompatible. Common causes: 1) Recognition site blocked by Dam, Dcm, or CpG methylation preventing cleavage 2) Incorrect buffer used reducing enzyme activity to <10% 3) DNA contaminants (salts, proteins, polysaccharides) inhibiting enzyme 4) PCR fragment has <6 nucleotides between recognition site and DNA end, preventing cleavage Solutions: 1) Check enzyme methylation sensitivity; use dam⁻/dcm⁻ strain (NEB #C2925) for methylation-sensitive enzymes 2) Use recommended NEBuffer supplied with restriction enzyme; verify 100% activity 3) Clean up DNA with spin column (NEB #T1030) to remove inhibitors before digestion 4) Design PCR primers with ≥6 nucleotides flanking the recognition site at DNA ends