LimsPulse
Bioanalytical

Real-time PCR (qPCR) system

A thermal cycler with a built-in fluorescence reader. It copies a chosen DNA sequence cycle by cycle and measures the fluorescence in every well after each cycle, so the starting amount of DNA can be calculated from a standard curve.

  • Biotechnology
  • Biopharmaceutical
  • Cell and gene therapy
  • Academic research
  • Agricultural biotech
  • Food authenticity
Photo of a Real-time PCR (qPCR) system
Photo: İnformatic, CC BY-SA 3.0 (cropped), via Wikimedia Commons
Follow your site SOP
This is a typical operating sequence for learning. Your instrument model and approved SOP take precedence.
STAGE 1

Before you start

  1. 1Check the instrument is within its calibration (block temperature and optical calibration) and maintenance due dates.
  2. 2Work in separate areas for reagent preparation, sample handling and amplification to avoid contamination.
  3. 3Thaw reagents on ice, mix gently and spin down. Keep enzymes cold.
  4. 4Prepare the plate layout: standards (often 5 levels, 10-fold steps), samples with spikes, NTCs and controls, all in replicate.
  • Never open a sealed plate after amplification in the pre-PCR area. Amplified product is the most common source of contamination.
STAGE 2

Plate set-up and run

  1. 1Dispense master mix first, then samples and standards, changing tips between wells.
  2. 2Seal with optical film using a roller or applicator so every well is fully sealed.
  3. 3Centrifuge the plate briefly to remove bubbles and bring liquid to the bottom.
  4. 4Load the plate in the right orientation, select the dyes, passive reference and thermal profile, and start the run.
  • Do not write on the optical film over the wells; ink and fingerprints block the light path.
STAGE 3

Data analysis

  1. 1Check amplification curves look sigmoidal and NTCs show no amplification (or Cq well beyond the lowest standard).
  2. 2Set baseline and threshold as defined in the method (automatic or fixed).
  3. 3Check standard curve slope, efficiency and r², and replicate Cq agreement.
  4. 4Calculate sample quantities and spike recovery; report as per the method.
STAGE 4

Shutdown

  1. 1Remove the plate and discard it sealed into the designated waste.
  2. 2Close the software, back up data and sign the instrument log.
  3. 3Wipe the work area with a DNA-decontaminating solution as per site procedure.
In LimsPulse · Calibration Management

Keep every qPCR in control: usage logs, calibration and maintenance schedules, and automatic blocking of overdue instruments.

See how