---
title: "How do I make an equimolar pool for sequencing?"
slug: "how-do-i-create-an-equimolar-pool"
updated: 2025-11-25T20:56:01Z
published: 2025-11-25T20:56:01Z
canonical: "support.epicypher.com/how-do-i-create-an-equimolar-pool"
---

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

# How do I make an equimolar pool for sequencing?

When preparing sequencing-ready libraries, it is important to create an **equimolar pool** so that each library contributes equally to the final sequencing run. Below is the recommended workflow using the CUTANATM CUT&RUN Library Prep Kit ([EpiCypher 14-1001](https://www.epicypher.com/product/cutana-cutrun-library-prep-kit/)).

1. **Quantify each library and assess library size**
  - Calculate the concentration of each library using the 200 - 700 bp range on the Agilent TapeStation® or Bioanalyzer®. Note that in most cases you will need to manually set this window.
  - Record the average fragment size (in base pairs) as determined by the TapeStation/BioAnalyzer.
2. **Calculate molar concentration**
  - Convert concentration (ng/µL) into molarity (nM) using the following formula (where 660 g/mol/bp is the approximate molecular weight of double-stranded DNA):

nM = (concentration in ng/µL × 1,000,000) ÷ (660 × average fragment size in bp)

1. **Normalize libraries**
  - Dilute each library to a common molarity using nuclease-free water or a low-EDTA buffer (such as 0.1X TE).
2. **Pool normalized libraries**
  - Combine equal volumes (e.g., 5 µL) of each normalized library into a single tube. This creates the equimolar pool.
3. **Validate pooled libraries**
  - Run the pool on a TapeStation or Bioanalyzer to confirm pooled fragment distribution. If needed, perform clean up for small fragments on the entire pooled library at this time.
  - Quantify the pool using Qubit.
4. **Dilute to the concentration required for your sequencing platform.**

### Key Notes

- Always normalize based on **molarity**, not mass concentration, to ensure equal sequencing representation.
- If libraries vary widely in concentration, do NOT dilute to the lowest concentration sample.