Technical Manual for Promega GoTaq Probe 1-Step RT-qPCR System

Quick guide from the manual

The GoTaq Probe 1-Step RT-qPCR System is designed for quantitative PCR assays. The workflow involves preparing a reaction mix by combining the GoScript RT Mix, GoTaq Probe qPCR Master Mix with dUTP, primers, and the hydrolysis probe, followed by the addition of the RNA template. The reaction is then performed on a real-time PCR instrument using either standard or fast cycling conditions.

Product Description

This system enables detection and relative quantification of RNA expression levels using a one-step RT-qPCR method. It combines GoScript Reverse Transcriptase and GoTaq Probe qPCR Master Mix. The master mix is a ready-to-use 2X formulation containing GoTaq Hot Start Polymerase, MgCl2, dNTPs, and a proprietary reaction buffer. It is formulated with dUTP to allow for carryover contamination control using UNG.

Storage Conditions

• Temperature: Store all components between -30°C and -10°C.
• Light: Protect components from light at all times.
• Handling: Mix thawed solutions gently to minimize aeration and foaming. Store on ice during use.
• Short-term: The master mix can be stored at +2°C to +10°C for up to 3 months if protected from light.

General Considerations

• Contamination Prevention: Use designated work areas and pipettes for pre- and post-amplification steps. Wear gloves and change them often. Use aerosol-resistant pipette tips. Do not open reaction plates after amplification.
• RNA Template: For unknown expression levels, start with 100ng of total RNA per reaction. Ensure the template is free of genomic DNA contamination.
• CXR Reference Dye: The system includes a separate tube of CXR Reference Dye. Add this to the master mix if your real-time PCR instrument requires normalization.

Protocol: Adding CXR Reference Dye

If your instrument requires normalization, add the CXR Reference Dye to the master mix before assembling the reaction:

1. Thaw the GoTaq Probe qPCR Master Mix with dUTP.
2. Vortex for 3–5 seconds.
3. Add the appropriate volume of CXR Reference Dye based on your instrument requirements (Low-dye: 30nM; High-dye: 500nM).
4. Vortex again for 3–5 seconds to mix.
5. Mark the tube/bottle to indicate the dye has been added.

Assembling the Reaction Mix

The final reaction volume is 20µl. Prepare the reaction mix (minus RNA template) by combining:

• GoTaq Probe qPCR Master Mix with dUTP: 10µl
• GoScript RT Mix: 0.4µl
• Forward Primer (20X): 1µl
• Reverse Primer (20X): 1µl
• Hydrolysis Probe (20X): 1µl
• Nuclease-Free Water: To a final volume of 20µl

Add the RNA template (2–5µl) to the reaction mix in the PCR plate. Seal the tubes/plate and centrifuge briefly before cycling.

Thermal Cycling Conditions

Standard Cycling

• Reverse Transcription: 45°C for 15 minutes (1 cycle)
• RT Inactivation/Polymerase Activation: 95°C for 2 minutes (1 cycle)
• Denaturation: 95°C for 15 seconds (40 cycles)
• Annealing/Extension: 60°C for 1 minute (40 cycles)

FAST Cycling

• Reverse Transcription: 45°C for 5 minutes (1 cycle)
• RT Inactivation/Polymerase Activation: 95°C for 2 minutes (1 cycle)
• Denaturation: 95°C for 3 seconds (40 cycles)
• Annealing/Extension: 60°C for 30 seconds (40 cycles)