Skip to content

usage-guide.md

Latest commit

 

History

History
76 lines (51 loc) · 2.06 KB

usage-guide.md

File metadata and controls

76 lines (51 loc) · 2.06 KB

PCR Primer Design - Usage Guide

Overview

This skill covers designing PCR primers using primer3-py, the Python binding for the Primer3 primer design software. Use this for basic PCR, cloning, sequencing primers, and amplicon design.

Prerequisites

pip install primer3-py biopython

Quick Start

Ask your AI agent:

  • "Design primers for this sequence"
  • "Find primers that amplify a 200bp region around position 500"
  • "Design primers with Tm around 60C"

Example Prompts

Basic Design

"Design PCR primers for my gene sequence"

"Find primers for a 150-250bp amplicon"

Target Specific Regions

"Design primers flanking positions 100-200"

"Find primers that amplify exon 3"

With Constraints

"Design primers with Tm 58-62C and 45-55% GC"

"Find primers avoiding the SNP at position 150"

"Design primers with no more than 3 consecutive identical bases"

For Cloning

"Design primers for Gibson assembly with 20bp overlaps"

"Find primers for restriction cloning with EcoRI sites"

What the Agent Will Do

  1. Load your template sequence
  2. Configure primer3 parameters based on your requirements
  3. Run primer design
  4. Return ranked primer pairs with Tm, GC%, and product size
  5. Optionally check for secondary structures

Tips

  • Tm matching - Aim for <2C difference between forward and reverse primers
  • GC content - 40-60% is ideal; avoid GC-rich 3' ends
  • Product size - For standard PCR, 100-500bp works well
  • 3' end - Should have 1-2 G/C bases (GC clamp) but avoid >3
  • Poly-X runs - Avoid >4 consecutive identical bases
  • Secondary structure - Check hairpin and dimer Tm are well below annealing temp