Unexpected or Incorrect Quantification Value (Nucleic Acid Quantification)

Question

Accepted Answer

Qubit Fluorometer shows unexpected concentration values that do not match expected sample concentration Common causes: 1) Misunderstanding between assay tube concentration vs. stock concentration 2) 260 nm absorbing contaminants in sample 3) Lid not completely closed during reading 4) Improper dye dilution (not 1:199 dye to buffer) 5) Tube labeling error 6) Expired kit or degraded reagents 7) Dye not protected from light during storage 8) Buffer and dye not at room temperature (stored at 4°C) 9) Air bubbles in solution affecting reading 10) Pipetting errors with concentrated or viscous samples Solutions: 1) Use 『Calculate Stock Concentration』 feature to convert assay tube reading to sample concentration 2) Check assay kit manual for list of interfering contaminants 3) Ensure lid is completely closed during all readings 4) Prepare fresh dye working solution (1 µL dye to 199 µL buffer) 5) Verify correct tube labeling 6) Check kit expiration date and replace if necessary 7) Store dye protected from light 8) Ensure buffer and dye are at room temperature (takes hours to warm from 4°C) 9) Remove air bubbles from solution before reading 10) For concentrated/viscous samples: dilute and use larger volume to reduce pipetting error