PCR / Gel: no bands

1. Ranked causes

   1. Annealing temperature too high for primer TmPrimers never bind template; every lane is blank including the positive control.
   2. Missing or degraded template / no reverse transcriptionBlank across all lanes but master mix band (primer-dimer) still present at the dye front.
   3. Mg²⁺ or dNTP concentration off in the master mix (lot / formulation)Common after a fresh master-mix lot swap.
   4. Polymerase inactive (freeze-thaw cycled too many times)Positive control also fails; enzyme aliquot has many thaw cycles.
   5. Thermocycler block temperature out of calibrationControls fail on one instrument but succeed on another with the same mix.

2. Recovery steps

   1. Drop annealing temperature by 3–5°C below the lower primer TmA 5°C gap above true Tm is the single most common cause of total dropout.
   2. Re-run with a verified positive control templateIsolates instrument/reagent failure from a template problem.
   3. Check primer Tm difference between forward and reverseA >3°C Tm mismatch biases annealing toward one primer and can silence the pair entirely.
   4. Fresh-thaw a new polymerase aliquotRepeated freeze-thaw denatures Taq/Q5 activity after ~8–10 cycles.

3. Controls first

   • Did the positive control amplify as expected?If no: Master mix, enzyme, or thermocycler fault — not this template alone.
   • Is a primer-dimer band visible at the dye front?If yes: Primers are intact and functional — the block is annealing temperature or template.

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