Apr 10, 2023

Public workspaceIllumina denature and dilute

DOI
dx.doi.org/10.17504/protocols.io.6qpvr4ex2gmk/v1
  • 1Vanderbilt University
Morad C Malek
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DOI: dx.doi.org/10.17504/protocols.io.6qpvr4ex2gmk/v1
Protocol Citation: Angela R.S. Kruse, Morad C Malek, Jamie Allen, Melissa Farrow, Jeff Spraggins 2023. Illumina denature and dilute. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr4ex2gmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 07, 2022
Last Modified: October 18, 2023
Protocol Integer ID: 69710
Abstract
This protocol describes the preparation of a library for Illumina NGS sequencing.
Materials
Consumables:

The following consumables are required to prepare DNA libraries for sequencing on the MiSeq.




Standard Normalization Method
Standard Normalization Method
Use the following steps to denature and dilute libraries that have been normalized using standard library quantification and quality control procedures recommended in the library prep documentation.

Follow the steps most appropriate for your library and the version of Illumina Reagent Kit you are using.
Loading concentration can also vary depending on library type and quantification methods.


The denaturation steps described in this guide make sure that the concentration of NaOH is not more than 0.001 (1 mM) in the final solution after diluting with HT1. Higher concentrations of NaOH in the library inhibit library hybridization to the flow cell and decrease cluster density.

Prepare Reagents
Prepare Reagents
Prepare a Fresh Dilution of NaOH

1: Combine the following volumes in a microcentrifuge tube
  • Laboratory-grade water (800 μl)
  • Stock 1.0 N NaOH (200 μl)

The result is 1 ml of 0.2 N NaOH.

2: Invert the tube several times to mix.

NOTE
Use the fresh dilution within 12 hours.

Prepare HT1

1 Remove HT1 from -25°C to -15°C storage and thaw at room temperature.

2 Store at 2°C to 8°C until you are ready to dilute denatured libraries.
Denature a 4 nM Library
Denature a 4 nM Library
1 Combine the following volumes in a microcentrifuge tube.
  • 4 nM library (5 μl)
  • 0.2 N NaOH (5 μl)
Vortex briefly and then centrifuge at 280 × g for 1 minute.
Incubate at room temperature for 5 minutes.
Add 990 μl prechilled HT1 to the tube containing denatured library.
The result is 1 ml of a 20 pM denatured library.

Note that the denatured library concentration may vary by Illumina platform.