# Best practices for storage of bead normalized Nextera XT libraries

The Nextera XT protocol includes an optional bead-based normalization step for libraries after the PCR clean-up step in the protocol.

Because the bead-based normalized libraries are single-stranded after elution from the normalization beads, the libraries are not as stable as double-stranded DNA libraries, are more susceptible to degradation, and can adhere to the walls of tubes.

After bead-based normalization, **libraries can be stored for up to 1 week at -25°C to -15°C**.

To make sure that all library strands are completely denatured, **perform a heat denaturation of the libraries immediately before sequencing**, as follows, with the diluted library pool just prior to loading into the sequencing cartridge.

1. Incubate the denatured and diluted library at 96°C for 2 minutes (using a pre-heated heat block).
2. After the heat incubation, invert the tube 1-2 times to mix.
3. Quickly move the library to an ice water bath for 5 minutes. The quick cooling step helps to lock the sample in its single-stranded form.
4. Proceed immediately to cluster generation.

**Note:** For longer term storage of libraries, stop immediately after the PCR clean-up step when the libraries are still double-stranded, and store the SGP plate at -25°C to -15°C. Aliquot as needed to avoid multiple freeze-thaw cycles.

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| *For any feedback or questions regarding this article (Illumina Knowledge Article #1146), contact Illumina Technical Support* [*techsupport@illumina.com*](mailto:techsupport@illumina.com?subject=Question%2FFeedback%20Regarding%20Illumina%20Knowledge%20Article%20#000001146%20-%20Library%20Preparation%20\&body=Dear%20Illumina%20Technical%20Support,%0D%0A%0D%0A)*.* |


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