PCR Technologies Protocols Table of Contents
Although quantitative PCR uses the same basic concept as traditional PCR, the reactions differ in that the amplicons are generally smaller and are detected indirectly using an additional dye or labeled probe or primer.
In the PCR Technologies Guide, the requisite components and quality control requirements for qPCR experiments were described in detail. With those in mind, the following is a protocol that can be used as a basic template for qPCR incorporating up to four detection probes. In these reactions, primers and probe are included at a final concentration of 200 nM and are run using LuminoCt® ReadyMix. However, multiplex experiments require optimization and it is advisable to test the assay combinations by adding each to the multiplex sequentially. A detailed assay optimization protocol for each single assay is described in Primer Concentration Optimization and Primer Optimization Using Temperature Gradient.
4. Prepare a master mix for all reactions according to Table P6‑14 (calculate volumes for each reaction and add 10% to
allow for pipetting error). Mix well, avoiding bubbles.
5. Add 5 μL of water to the NTC reaction tubes.
6. Add 5 μL of cDNA/gDNA solution to the appropriate tubes/wells.
7. Aliquot 15 μL template master mix remaining from step 4 into the PCR tubes.
8. Cap tubes or seal the PCR plate and label (according to instrument requirements). (Make sure the labeling
does not obscure instrument excitation/detection light path.)
9. Centrifuge briefly and visually check that all tubes/wells contain sample at the bottom at the correct volume.
10. Run samples according to the two-step protocol (Table P6‑15), repeating steps 1–2 through 40 cycles.
(Note: These conditions are specific for FAST cycling protocols).
11. For reactions containing Scorpions® Probes or Molecular Beacons, a three-step protocol (Table P6‑16) may
result in more efficient/sensitive detection. When adopting this protocol, the annealing temperature of step 2
can be optimized. Cycle steps 1–3 through 40 cycles.