PCR / Gel: unexpected band size

1. Ranked causes

   1. Splice variant or isoform amplified (RT-PCR)Size difference matches a known exon in size.
   2. Mispriming at a secondary binding siteSingle sharp band, just at the wrong size — not a smear.
   3. Genomic DNA contamination spanning an intronBand is larger than expected by roughly one intron length.
   4. Wrong primer pair loaded (tube labels vs plate map)Correlates with a specific well or plate row.

2. Recovery steps

   1. Sequence or digest the product to confirm identityFastest way to distinguish mispriming from a real isoform.
   2. BLAST both primers against the reference genome for secondary sitesIdentifies mispriming before repeating the run.
   3. DNase-treat template and re-run to rule out genomic contaminationIf the band shrinks to expected size, gDNA was the cause.
   4. Re-verify primer tube labels against the plate mapRules out a labeling mismatch at setup.

3. Controls first

   • Did the positive control band run at the expected size on the ladder?If no: Ladder, control template, or primer identity issue — check materials before sample interpretation.
   • Is the band a single sharp line, not smeared?If yes: Specific amplification of the wrong target — check primer specificity and template identity, not PCR chemistry.

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