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qPCR Using a Single Detection Probe Protocol

Quantitative PCR Protocols

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 a detection probe that is specific to a single target. In these reactions, primers and probe are included at a final concentration of 200 nM and are run using LuminoCt® ReadyMix™. Detailed optimization protocols for primer concentration and annealing temperature are presented separately (see Primer Concentration Optimization and Primer Optimization Using Temperature Gradient).

Equipment

  • Quantitative PCR instrument
  • Microcentrifuge
  • Laminar flow hood for PCR set up (optional)

Supplies

Method

1.    Defrost all reaction components on ice, taking care to protect the probe from exposure to light.

2.    Calculate the number of reactions required to enable samples and controls to be run in duplicate. Include two No Template
       Controls (NTC).

3.    Prepare a master mix for all reactions according to Table P5‑10 (calculate volumes for each reaction and add 10% to
       allow for pipetting error). Mix well, avoiding bubbles.

Table P5-10. Master Mix for Single Probe qPCR.

Reagent Volume (μL) per Single
20 μL Reaction
2× LuminoCt qPCR ReadyMix 10
Forward primer (10 μM stock) 0.4
Reverse primer (10 μM stock) 0.4
Probe (10 μM stock) 0.4
PCR grade water 3.8

 

4.    Add 5 μL of water to the NTC reaction tubes.

5.    Add 5 μL of cDNA/gDNA solution to the appropriate tubes/wells.

6.    Visually check that all tubes/wells contain sample at the bottom at the correct volume.

7.    Aliquot 15 μL template master mix remaining from step 3 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 and at the
       correct volume.

10.   Run samples according to the two-step protocol (Table P5‑11), repeating steps 1–2 through 40 cycles
        (Note:These conditions are specific for FAST cycling protocols).

11.   Note: For reactions containing Scorpions® Probes or Molecular Beacons, a three-step protocol (Table P5-12)
        may result in more efficient/sensitive detection. When adopting this protocol, the annealing temperature of
        step 2 can be optimized (see Primer Optimization Using Temperature Gradient). Cycle steps 1–3 through 40 cycles.

12.   See Data Analysis and the instrument manual for guidance on data analysis.

Table P5-11. Fast PCR Cycling Conditions.

FAST Cycling Conditions Temp (°C) Time (sec)
Initial Hot Start/denaturation 95 30
Steps 1 and 2 are repeated through 40 cycles
Step 1 95 5
Step 2
60 30

 

Table P5-12. Three-step PCR Cycling Conditions for Use with Scorpions® Probes or Molecular Beacons.

3-step Cycling Conditions (for Use with Scorpions®
Probes or Molecular Beacons)
Temp (°C) Time (sec)
Initial denaturation 95 30
Steps 1–3 are repeated through 40 cycles
Step 1 95 5
Step 2 (select optimized Ta) 58 15
Step 3 72 10

 

Materials