> ## Documentation Index
> Fetch the complete documentation index at: https://docs.ospribrain.com/llms.txt
> Use this file to discover all available pages before exploring further.

# Assay Design

> AI-powered PCR primer and probe design with multiplex validation.

## Overview

The Assay Design tool uses AI to design PCR primers and probes for your targets. It supports multiplex assay development with dimer screening, fluorophore compatibility, and protocol generation.

## Design Workflow

<Steps>
  <Step title="Create a project">
    Click **New Project**, enter a name, and select your instrument (Bio-Rad CFX96, ABI 7500, LightCycler 480, etc.).
  </Step>

  <Step title="Add targets">
    For each target, specify:

    * **Organism** — Target species
    * **Gene** — Target gene
    * **Accession** — NCBI accession number
    * **Channel** — Fluorophore channel (FAM, HEX, VIC, ROX, Cy5, etc.)
  </Step>

  <Step title="Generate designs">
    Click **Generate** and the AI designs primer/probe sets for each target.
  </Step>

  <Step title="Review designs">
    Each target gets a ranked list of design candidates.
  </Step>
</Steps>

<Note>
  **Alex's screenshot note:** Take a screenshot of the design project page showing the target list with organism, gene, and channel columns.
</Note>

## Design Results

For each target, the AI generates multiple primer/probe candidates ranked by score:

| Column             | Description                    |
| ------------------ | ------------------------------ |
| **Rank**           | Overall ranking                |
| **Forward Primer** | Forward primer sequence and Tm |
| **Reverse Primer** | Reverse primer sequence and Tm |
| **Probe**          | Probe sequence and Tm          |
| **Amplicon Size**  | Expected amplicon length       |
| **Score**          | Overall design quality score   |

## Visualizations

### Amplicon Map

A D3.js visualization showing primer and probe binding positions on the target gene.

### Radar Chart

Multi-axis chart comparing design candidates across:

* Tm optimality
* GC content
* Amplicon size
* Penalty score
* Dimer safety

### Fluorophore Spectra

Emission curves for each channel showing spectral overlap between fluorophores.

<Note>
  **Alex's screenshot note:** Take a screenshot of the amplicon map showing primer positions on the gene. Take another of the radar chart comparing 2-3 design candidates.
</Note>

## Multiplex Check

For multi-target assays, the **Multiplex Check** screens for:

* Primer-dimer formation between all primer/probe combinations
* Compatibility matrix showing safe/problematic pairs
* DimerZipper visualization for detailed dimer analysis

## Protocol Builder

Generate a ready-to-use PCR protocol:

* Reaction volume and components
* Primer/probe concentrations
* Master mix recipe
* Cycling conditions (denaturation, annealing, extension)

## Order Sheet

Export your selected designs for ordering:

* Oligo table with all sequences
* **Copy FASTA** — Copy sequences in FASTA format
* **IDT Bulk CSV** — Export for IDT ordering
* **Export CSV** — General CSV export