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tooluniverse-primer-design

Designs and validates PCR primer pairs with Tm calculation and secondary-structure screening.

1.7k|254|Updated Mar 3, 2025
Search Tags:#molecular biology#pcr#primer design#oligo#melting temperature#qpcr#primer-dimer
One-click install
npx skills add https://github.com/mims-harvard/ToolUniverse --skill tooluniverse-primer-design
Or copy as Structured Prompt for Agent▼
Please help me install this Agent Skill.
Skill: tooluniverse-primer-design
Source: https://github.com/mims-harvard/ToolUniverse/tree/main/plugins/tooluniverse/skills/tooluniverse-primer-design
Command: npx skills add https://github.com/mims-harvard/ToolUniverse --skill tooluniverse-primer-design

SYSTEM DOCUMENTATION & REQUIREMENTS

💡 This Skill includes scripts (resource) components.

What problem does it solve?

Designing PCR or qPCR primers by hand is error-prone: mismatched melting temperatures, GC clamp violations, hairpins, and primer-dimers silently cause failed amplifications. This Skill automates primer pair design, Tm/Ta calculation, and quality-control screening so you get thermodynamically sound primers with clear PASS/WARN diagnostics.

Core Features & Use Cases

  • Primer Pair Design: Generate forward/reverse primers for a target region using SantaLucia nearest-neighbor thermodynamics, with control over product size and target Tm.
  • Tm and Annealing Temperature Calculation: Compute polymerase-specific Tm and recommended annealing temperature via NEB and IDT calculators, plus GC content checks.
  • Primer QC Screening: Run the bundled primer_qc.py script to check length, GC%, GC clamp, mononucleotide runs, 3' self/cross-dimers, and pair Tm matching.
  • Use Case: Given a gene sequence and a target region, design an 80-140 bp amplicon primer pair at 60 °C Tm, verify the pair passes all QC rules, then BLAST each primer for genome specificity before ordering.

Quick Start

Ask the agent to design forward and reverse primers for your target DNA region with a 60 °C melting temperature and then QC the resulting pair for dimers and Tm match.

Frequently Asked Questions about tooluniverse-primer-design

High-intent search queries and answers about installing and using this skill.

FAQPage Schema
How do I design PCR primers for a target DNA region?▼

Provide the full template sequence with target_start and target_end coordinates, plus a target Tm and product size range. The tool returns forward and reverse primer sequences with Tm, GC content, length, position, and product size.

How do I calculate primer Tm and annealing temperature?▼

Use the NEB Tm calculator with your primer sequence, polymerase type, primer concentration, and salt concentration to get Tm and a recommended annealing temperature. Pick one calculator and condition set for the whole experiment, since methods differ.

What makes a good PCR primer pair?▼

Good primers are 18-24 nt long with 40-60% GC content, Tm of 58-62 °C matched within 3 °C across the pair, a 1-2 G/C clamp at the 3' end, no runs of 4+ identical bases, and no 3' complementarity causing dimers.

Why does primer design fail with a target region error?▼

The target span must fit within the product size window with enough flanking sequence on both sides. If the target is narrower than product_size_min, lower that minimum; if the product cannot cover the target, widen product_size_max or add flanking sequence.

Does primer design check genome specificity?▼

No, these tools only check thermodynamics and secondary structure, not genome specificity. Always BLAST each primer or use Primer-BLAST against the target genome to confirm a single intended product before ordering.

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