> For the complete documentation index, see [llms.txt](https://knowledge.illumina.com/llms.txt). Markdown versions of documentation pages are available by appending `.md` to page URLs; this page is available as [Markdown](https://knowledge.illumina.com/instrumentation/nextseq-500-550/instrumentation-nextseq-500-550-reference_material-list/000002064.md).

# How to denature and dilute libraries on the NextSeq 500/550

**Loading Volume and Concentration**

Denature and dilute libraries to a final loading volume of 1.3 ml at the recommended concentration:

* For the PhiX control library, Illumina recommends a final loading concentration of 1.8 pM for high output kits and 1.5 pM for mid output kits.
* For sequencing libraries, ideal loading concentrations can vary depending on library preparation and quantification methods. If sequencing non-Illumina libraries, users must optimize their loading concentrations internally.

**Best Practices**

* Always prepare freshly diluted NaOH for denaturing libraries for cluster generation. This step is essential to the denaturation process.
* To prevent pipetting errors from affecting the final NaOH concentration, prepare at least 1 ml of freshly diluted NaOH.
* For best results, begin thawing reagents before denaturing and diluting libraries. For instructions, see the [NextSeq 500/550 Sequencing System Guide](https://support-docs.illumina.com/IN/NextSeq_550-500/Content/IN/FrontPages/NextSeq-550-500.htm).

For the complete NextSeq 500/550 denature and dilution protocol, see the [NextSeq 500/550 Denature and Dilute Libraries Guide](https://support-docs.illumina.com/IN/dnd-wizard/Content/dnd/landing.htm?ins=nextseq550).

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| *For any feedback or questions regarding this article (Illumina Knowledge Article #2064), contact Illumina Technical Support* [*techsupport@illumina.com*](mailto:techsupport@illumina.com?subject=Question%2FFeedback%20Regarding%20Illumina%20Knowledge%20Article%20#000002064%20-%20Instrumentation%20\&body=Dear%20Illumina%20Technical%20Support,%0D%0A%0D%0A)*.* |


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