Real lab failures, root causes, and fixes — curated and bilingually annotated by our team.
Partial digestion observed with both expected fragments and uncut substrate visible on gel; incomplete linearization of plasmid DNA.
Restriction digest product appears as a smear rather than discrete bands on agarose gel, indicating DNA degradation or enzyme-DNA complex formation.
Extra DNA bands appear on gel beyond expected digest fragments, indicating cleavage at non-canonical recognition sequences due to relaxed enzyme specificity.
Incomplete digestion persists despite adequate enzyme units and incubation time; some restriction enzymes require two recognition sites on the same DNA molecule for efficient cleavage.
DNA bands migrate slower than expected molecular weight on gel, appearing larger due to restriction enzyme remaining bound to digested DNA fragments.
No cleavage observed in restriction digest reaction; DNA remains uncut on gel, resulting in few or no transformants in downstream cloning applications.
DNA substrate is not fully cleaved after restriction enzyme incubation. Uncut or partially cut DNA bands persist on agarose gel alongside expected digestion products.
Extra bands appear on gel beyond expected digestion products. Enzyme cleaves at non-canonical sites with relaxed sequence specificity under suboptimal conditions.
Larger molecular weight bands than expected appear on gel. Restriction enzyme remains bound to cleaved DNA substrate, retarding migration.
Gel shows mixture of fully digested, partially digested, and uncut DNA. Some recognition sites cleaved while others remain intact in the same molecule.
Restriction enzyme fails to cleave recognition sites located within 6 bp of linear DNA ends, such as PCR product termini. Full-length uncut product observed.
Bacterial transformation yields few or no colonies after restriction digest-based cloning. Vector may not be linearized or insert ends incompatible.
Digested DNA appears as a smear rather than discrete bands on agarose gel. No sharp bands are visible across a wide molecular weight range.
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