qPCR: flat amplification curve

1. Ranked causes

   1. Probe or SYBR omitted / degraded in the master mixGel of the same reaction shows product; fluorescence channel stays flat.
   2. No template or failed RT (empty input)Positive-control well curves normally; sample wells stay flat.
   3. Wrong filter / reporter channel selected on the instrumentRaw data in another channel shows the curve; assay chemistry is intact.
   4. Cycling program never reaches extension / read stepProtocol file missing plate-read; fixing the program restores curves.

2. Recovery steps

   1. Verify reporter channel / filter matches dye (e.g. FAM vs HEX) in the run setupWrong channel is a frequent flat-curve cause after copying a template run.
   2. Re-make mix with fresh dye/probe; include a kit positive controlSeparates detection chemistry from template problems.
   3. Confirm the cycling file includes a plate-read at the end of extensionMissing read steps produce empty datasets that look like chemistry failure.

3. Controls first

   • Did the positive-control well also stay flat in the selected channel?If yes: Channel, dye/probe, or cycling program — not a single sample.

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