Target amplification using cDNA as the template is the second step in the two-step RT-PCR. In this step, the DNA polymerase amplifies target cDNA using sequence-specific primers and cleaves the TaqMan® probe to generate a fluorescent signal that is measured by the real-time PCR system.
Keep the TaqMan® assays in the freezer, away from light, until you are ready to use them. Excessive exposure to light may affect the fluorescent probes.
The rtPCR step must be performed on a real-time PCR system.
When preparing samples for PCR amplification:
- Use a positive-displacement pipette or aerosol-resistant pipette tips.
- Follow proper pipette-dispensing techniques to prevent aerosols.
- Wear clean gloves and a clean lab coat (not previously worn while handling amplified PCR products or used during sample preparation).
- Change gloves whenever you suspect that they are contaminated.
- Maintain separate areas and dedicated equipment and supplies for:
- Analysis of PCR products
- Never bring amplified PCR products into the PCR setup area.
- Open and close all sample tubes carefully. Centrifuge tubes before opening. Try not to splash or spray PCR samples.
- Keep reactions and components capped as much as possible.
- Clean lab benches and equipment periodically with 10% bleach solution.