qPCR Using a Single Detection Probe Protocol

PCR Technologies Protocols Table of Contents

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

• DNA/cDNA template: cDNA reaction diluted 1:10 to detect a medium to highly expressed targets or 1:2 to 1:5 for rare transcripts. 10 ng to 100 ng gDNA.
  
• LuminoCt^® ReadyMix™ (L6669)
  
• PCR grade water: PCR grade water (W1754 or W4502) as 20 mL aliquots; freeze; use a fresh aliquot for each reaction.
  
• Forward and reverse primers diluted to working concentration (10 μM working stocks are suitable for single probe reactions but more concentrated stocks are needed for multiplex).
  
• Specific target detection probe (see Chapter 10) diluted to working concentration (10 μM working stocks are suitable for single probe reactions but more concentrated stocks are needed for multiplex).
  • Custom oligos can be ordered here
  
• Sterile filter pipette tips
• Sterile 1.5 mL screw-top microcentrifuge tubes (CLS430909)
• PCR tubes and plates, select one to match desired format:
  • Individual thin-walled 200 μL PCR tubes (Z374873 or P3114)
  • Plates
  - 96-well plates (Z374903)
  - 384-well plates (Z374911)
  • Plate seals
  - ThermalSeal RTS™ Sealing Films (Z734438)
  - ThermalSeal RT2RR™ Film (Z722553)
  

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.

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.