Hybridization Buffer (HT1) and Resuspension Buffer (RSB) alternatives and purchasing availability

**Hybridization Buffer (HT1) is used for the final library dilution(s) following denaturation, prior to sequencing on multiple Illumina instruments (though not all), and Resuspension Buffer (RSB) is a commonly used buffer in Illumina library preparation kits.

What can be used as a substitute for RSB when diluting libraries prior to sequencing?**

• 10 mM Tris-HCl, pH 8.5 with 0.1% Tween 20.

• 10 mM Tris-HCl, pH 8.5.

• Nuclease free water can also be used.

• Note: the final library dilution for sequencing on the NextSeq 1000/2000 requires the RSB with Tween reagent, which is critical for clustering. If RSB with Tween is not available, use the first option above as a substitute for RSB with Tween.

What can be used as a substitute for HT1? The HT1 buffer formulation is proprietary. Illumina has not tested or validated any reagent substitutions for HT1.

• Note that HT1 and RSB compositions are different and these reagents are not interchangeable. There are no alternatives for HT1.

Is RSB sold individually? RSB is not available for purchase individually at this time.

How can RSB with Tween be ordered, and when should it be used? RSB with Tween must be used when diluting libraries for sequencing on the NextSeq 1000/2000. It is included in the NextSeq 1000/2000 sequencing reagent kits.

Is HT1 sold individually? Yes, it can be purchased here.

Is the HT1 included in the Nextera XT library prep kits the same as the HT1 that is included in sequencing reagent kits? Yes, the composition of the HT1 is the same in both, but the part numbers are different.

For any feedback or questions regarding this article (Illumina Knowledge Article #5040), contact Illumina Technical Support [e-mail].