PCR / Gel: non-specific smear

1. Ranked causes

   1. Annealing temperature too lowSmear collapses to a single band when Ta is raised in a gradient test.
   2. Too many amplification cycles (over-cycling)Smear worsens toward the plateau cycles.
   3. Mg²⁺ concentration too high in the mix lotExcess Mg²⁺ stabilizes mismatched primer binding.
   4. Degraded / genomic DNA contamination in an RNA prepSmear appears only in RT-PCR, not in DNA-template controls.

2. Recovery steps

   1. Increase annealing temperature to 55°C (or +3–5°C over current)Tightens primer specificity and is the highest-yield first fix.
   2. Reduce cycle count by 3–5 cyclesStops the reaction before non-specific products accumulate.
   3. Titrate Mg²⁺ down in 0.5 mM stepsLower Mg²⁺ increases annealing stringency.
   4. DNase-treat the RNA prep before RTRemoves genomic DNA carryover that amplifies alongside the intended amplicon.

3. Controls first

   • Did the no-template control stay clean (no smear)?If no: Contamination or primer/mix issue — clean reagents before adjusting Ta.
   • Does the smear tighten to one band on a gradient PCR at higher Ta?If yes: Confirms an annealing-specificity problem, not a template or enzyme fault.

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