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

See below for an overview of the denature and dilution process for the NextSeq 500/550.

**Loading Volume and Concentration**

Denature and dilute libraries to a final loading volume of 1.3 ml at a 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.
* In practice, loading concentration can vary depending on library preparation and quantification methods. Library loading concentrations must be optimized within the lab.

**Protocol Variations**

Follow the appropriate denature and dilute protocol depending on the procedure used during library prep.

* Standard normalization (Protocol A): Libraries are normalized using standard library quantification and quality control procedures recommended in the library prep documentation.
* Bead-based normalization (Protocol B): Libraries are normalized using a bead-based procedure described in the library prep documentation for methods that support bead-based normalization.
* AmpliSeq for Illumina normalization (Protocol C): For all AmpliSeq for Illumina panel libraries, follow Protocol C.

**Best Practices**

* Always prepare freshly diluted NaOH for denaturing libraries for cluster generation. This step is essential to the denaturation process.
* To prevent small 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 and NextSeq 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 Denature and Dilute Libraries Guide](https://support-docs.illumina.com/IN/dnd-wizard/Content/dnd/landing.htm?ins=nextseq550).

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