Why did my PCR / Gel fail?

Playbooks

• PCR / Gel: no bandsAmplification failure — no product formed in any lane.
• PCR / Gel: faint bandsLow-yield amplification — product present but under-loaded.
• PCR / Gel: primer dimersPrimers annealing to each other instead of (or in addition to) template.
• PCR / Gel: non-specific smearOff-target amplification across a broad size range.
• PCR / Gel: unexpected band sizeAmplicon runs at the wrong molecular weight versus the expected/ladder reference.

Calculators

• Molarity converterConvert between nM, µM, mM, and M.
• Dilution (C1V1)Solve C1V1 = C2V2 and plan serial dilutions.
• Master mix scalerScale PCR/qPCR master mix with overage.
• Buffer recipesStandard bench buffers with per-liter amounts.

Protocols

• Standard PCRMaster mix assembly, thermal cycling, and a gel check.

FAQ — PCR / Gel

No bands

Amplification failure — no product formed in any lane. Drop annealing temperature by 3–5°C below the lower primer Tm — A 5°C gap above true Tm is the single most common cause of total dropout. Re-run with a verified positive control template — Isolates instrument/reagent failure from a template problem. Check primer Tm difference between forward and reverse — A >3°C Tm mismatch biases annealing toward one primer and can silence the pair entirely. Fresh-thaw a new polymerase aliquot — Repeated freeze-thaw denatures Taq/Q5 activity after ~8–10 cycles.

Faint bands

Low-yield amplification — product present but under-loaded. Increase cycle count by 3–5 cycles — Each extra cycle roughly doubles product in the exponential phase. Load 2× more PCR product per lane — Cheapest first check before touching chemistry. Re-quantify template input by Qubit/Nanodrop before the next run — Confirms whether the issue is amplification efficiency or starting material. Swap to a fresh master-mix / polymerase aliquot within expiry — Rules out lot activity drop after storage or freeze-thaw.

Primer dimers

Primers annealing to each other instead of (or in addition to) template. Halve primer concentration (e.g. 500 nM → 250 nM) — Directly reduces primer-primer collision frequency. Raise annealing temperature to 55°C — Suppresses the weaker, non-specific primer-dimer interaction while template binding stays strong. Add a hot-start polymerase if not already in use — Prevents primer-dimer extension during setup at room temperature. Re-design the 3′ ends of both primers to remove complementarity — Fixes the root design issue if dimers persist after chemistry changes.

Non-specific smear

Off-target amplification across a broad size range. Increase annealing temperature to 55°C (or +3–5°C over current) — Tightens primer specificity and is the highest-yield first fix. Reduce cycle count by 3–5 cycles — Stops the reaction before non-specific products accumulate. Titrate Mg²⁺ down in 0.5 mM steps — Lower Mg²⁺ increases annealing stringency. DNase-treat the RNA prep before RT — Removes genomic DNA carryover that amplifies alongside the intended amplicon.

Unexpected band size

Amplicon runs at the wrong molecular weight versus the expected/ladder reference. Sequence or digest the product to confirm identity — Fastest way to distinguish mispriming from a real isoform. BLAST both primers against the reference genome for secondary sites — Identifies mispriming before repeating the run. DNase-treat template and re-run to rule out genomic contamination — If the band shrinks to expected size, gDNA was the cause. Re-verify primer tube labels against the plate map — Rules out a labeling mismatch at setup.