Explore qPCR using your existing PCR machine!

• Techniques: Micropipetting, fluorescence detection, quantitative PCR
• Topics: Gene expression, fluorescence, biotechnology
• Time required: 120 minutes; can be split over 2 or 3 class periods
• Level: Advanced high school through college

Perform quantitative polymerase chain reaction (qPCR) in a directly observable format and understand molecular diagnostics. qPCR monitors the amount of PCR product produced in real-time using fluorescent dyes or probes, and it is the gold standard for the detection of viral infections including COVID-19. In this lab, students use low-cost equipment to observe the change in fluorescence first hand, without the need for a qPCR machine. Students directly follow the increases in fluorescence that accompany PCR cycles, understanding how exponential DNA amplification works. They then use their measurements to quantify nucleic acids. This lab grounds students in the principles and practice of qPCR, the gold standard technique in the molecular diagnosis of infections including SARS-CoV-2. An optional extension allows students to compare results of real-time qPCR and end-point PCR using gel electrophoresis.

Required equipment (one per lab group):
- Micropipettes and tips.
- Thermal cycler.
- P51™ Molecular Fluorescence Viewer or other blue light transilluminator (460 to 480 nm, e.g. blueBox or blueGel).

Optional extension requirements (end-point gel electrophoresis):
- Learning Lab Companion Kit (catalog no. 470352-026).
- Gel electrophoresis and visualization system.

Storage notes:
- Reagents require freezer storage.
- Reagents are stable for 12 months when stored in the freezer.

Note: Reagents are compatible with P51™ Molecular Fluorescence Viewer or other blue light transilluminator (460 to 480 nm, e.g. blueBox™ or blueGel™).