Why did my qPCR fail?
Amplification absent or Ct far later than expected for the input. No-template control shows amplification above the lab’s Ct cutoff. Melt analysis shows more than one product peak for a supposed single-amplicon assay. Standard-curve efficiency outside the lab’s accepted window. Technical replicate Cts disagree beyond
Playbooks
- qPCR: late / no ctAmplification absent or Ct far later than expected for the input.
- qPCR: ntc amplificationNo-template control shows amplification above the lab’s Ct cutoff.
- qPCR: multiple melt peaksMelt analysis shows more than one product peak for a supposed single-amplicon assay.
- qPCR: poor efficiencyStandard-curve efficiency outside the lab’s accepted window.
- qPCR: high replicate ct cvTechnical replicate Cts disagree beyond the lab’s usual spread.
- qPCR: flat amplification curveFluorescence stays at baseline across cycles despite expected template.
Calculators
FAQ — qPCR
- Late / no Ct
- Amplification absent or Ct far later than expected for the input. Re-run with a verified positive-control template and fresh master-mix aliquot — Splits template vs reagent vs instrument in one plate. Drop annealing temperature 3 °C and re-check amplification curves — Common fix when primers are intact but binding is marginal. Quantify and dilute input into the validated dynamic range — Over-dilute or inhibited templates present as late/no Ct.
- NTC amplification
- No-template control shows amplification above the lab’s Ct cutoff. Replace water, primers, and master-mix aliquots; set up NTC first — Removes the most common contamination reservoirs. Inspect melt curves; raise anneal temp or redesign if primer-dimer dominates — Distinguishes true contamination from SYBR primer-dimer signal. Re-check Ct with manual threshold above NTC noise floor — Avoids calling flat NTCs as positive from auto-baseline artefacts.
- Multiple melt peaks
- Melt analysis shows more than one product peak for a supposed single-amplicon assay. Raise annealing temperature 2–3 °C and re-check melt — Often removes non-specific peaks without losing the true product. Run NTC melt; if low-Tm peak matches samples, redesign or lower primer concentration — Primer-dimer is diagnosed when NTC shares the extra peak. Spot-check on a gel or switch to a hydrolysis-probe chemistry — Confirms whether extra fluorescence is a real second amplicon.
- Poor efficiency
- Standard-curve efficiency outside the lab’s accepted window. Remake the standard curve with fresh serial dilutions (new tips each step) — Dilution error is the most common efficiency failure. Dilute samples/standards further to outrun inhibitors; compare efficiency — Inhibition presents as efficiency << 100% that recovers on dilution. Re-analyse with threshold set in the exponential phase for all wells — Analysis artefacts can mimic chemistry problems.
- High replicate Ct CV
- Technical replicate Cts disagree beyond the lab’s usual spread. Re-plate with reverse pipetting; discard bubbled wells — Volume inconsistency is the top driver of technical Ct CV. Vortex master mix immediately before every dispense round — Prevents settling across a long plating session. Replace optical seal; avoid leaving edge wells empty without buffer moat — Reduces evaporation-driven perimeter outliers.
- Flat amplification curve
- Fluorescence stays at baseline across cycles despite expected template. Verify reporter channel / filter matches dye (e.g. FAM vs HEX) in the run setup — Wrong channel is a frequent flat-curve cause after copying a template run. Re-make mix with fresh dye/probe; include a kit positive control — Separates detection chemistry from template problems. Confirm the cycling file includes a plate-read at the end of extension — Missing read steps produce empty datasets that look like chemistry failure.