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, 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?**

1. 10 mM Tris-HCl, pH 8.5 with 0.1% Tween 20. This is documented as a substitute for RSB in several Denature and Dilute guides, including for example the NextSeq 550 Denature and Dilute Guide (pg 3).2. 10 mM Tris-HCl, pH 8.5.

2. Nuclease free water can also be used\*. (\*Unless sequencing on NextSeq 1000/2000, in which case the RSB with Tween reagent is critical for clustering; if RSB with Tween is not available, use option #1 above as a substitute for RSB with Tween.) What can be used as a substitute for HT1? There are no officially recommended substitutes. Use 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 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].