qPCR Primer and Probe Design - Usage Guide

qPCR Primer and Probe Design - Usage Guide

Overview

This skill covers designing primers and probes for quantitative PCR (real-time PCR) using primer3-py. Supports TaqMan hydrolysis probes and SYBR Green primer-only assays.

Prerequisites

pip install primer3-py biopython pandas

Quick Start

Ask your AI agent:

• "Design qPCR primers with a TaqMan probe for my gene"
• "Design SYBR Green primers for this sequence"
• "Find qPCR primers that span exon 2-3 junction"

Example Prompts

TaqMan Assay

"Design primers and a TaqMan probe for quantifying GAPDH"

"Create a qPCR assay with probe Tm around 70C"

SYBR Green Assay

"Design SYBR Green primers for a 100bp amplicon"

"Find qPCR primers with minimal dimer potential"

cDNA-Specific

"Design primers that span the exon junction so they won't amplify genomic DNA"

"Create exon-spanning primers for my mRNA target"

Multiplex

"Design qPCR primers for both my target gene and housekeeping gene with matching Tms"

What the Agent Will Do

1. Load your template sequence
2. Configure primer3 for qPCR-specific parameters
3. Design primers with optimal Tm (~60C)
4. Design probe with elevated Tm (~70C) if requested
5. Return primer/probe sets ranked by quality

Tips

• Short amplicons - 70-150bp is ideal for qPCR efficiency
• Probe Tm - Should be 8-10C higher than primer Tm
• Avoid G at probe 5' - G adjacent to FAM quenches fluorescence
• Exon spanning - Prevents genomic DNA amplification in RNA quantification
• Tm matching - Keep primer Tms within 1-2C for consistent annealing
• SYBR specificity - Use strict complementarity settings to minimize primer dimers