Low Fluorescence Due to Inadequate Probe Labeling or Quenching (PCR (Sigma Guide))

Question

Accepted Answer

Low or absent fluorescence in both test sample and positive control; correct PCR product is visible on gel; one probe in multiplex shows consistently high background with no amplification signal; background fluorescence equivalent to water control Common causes: 1) Probe is inadequately labeled with fluorophore 2) Probe has inadequate quenching (quencher not functioning properly) 3) Degraded probe 4) Probe was not added to the reaction (setup error) 5) Incorrect probe concentration in reaction Solutions: 1) Perform DNase I digestion test: digest 5 fmoles of probe with DNase I and measure fluorescent yield (should increase at least 10,000 units) 2) Purchase a new probe with verified labeling 3) Verify probe concentration and adjust if necessary 4) Check multicomponent/raw data plot to verify probe was included in reaction 5) Compare to a functioning probe with same label and quencher 6) Test probe in SYBR Green I reaction to verify primers work correctly