How to thaw and store sequencing reagents for optimal performance

Sep 24, 2018


Sequencing reagent performance depends on thawing technique and thawed reagent storage time and conditions. It is important to understand and follow the recommendations for reagent thawing and storage because some reagents contain enzymes that are sensitive to temperature and time. Unless specified below, refreezing thawed reagents can negatively affect enzyme activity and run performance.

Clustering Reagents and Kits

TruSeq SR Cluster Kit v3, TruSeq PE Cluster Kit v3, HiSeq SR Cluster Kit v4, and HiSeq PE Cluster Kit v4

Thaw the cBot reagent plate in a room temperature water bath for 60 minutes and use within the same day. Alternatively, thaw at 2°C to 8°C overnight, no longer than 16 hours, and use within that same day. Store the thawed clustering kit on ice or at 2°C to 8°C until use. For best results, use as soon as possible after thawing.

TruSeq Rapid v1 Cluster Kit and HiSeq Rapid v2 Cluster Kit

Thaw the cBot reagent plate in a room temperature water bath for 30 minutes and use within the same day. Alternatively, thaw at 2°C to 8°C overnight, no longer than 16 hours, and use within that same day. Store the thawed clustering kit on ice or at 2°C to 8°C until use. For best results, use as soon as possible after thawing.

HiSeq 3000/4000 SR Cluster Kit, HiSeq 3000/4000 PE Cluster Kit, and HiSeq X Five/Ten Cluster Kit

Thaw the cBot reagent plate in a room temperature water bath for 60 minutes. Thaw the EPX1, EPX2, EPX3, and RSP reagents at room temperature for 10 minutes, then set on ice. For best results, use as soon as possible within the same day. Prepare the ExAmp reaction master mix immediately before use and load onto the cBot within 15 minutes of preparation.

NovaSeq Xp Reagents

Thaw DPX1, DPX2 and DPX3 at room temperature for 10 minutes, then set aside on ice. For best results, use as soon as possible. For the best performance, prepare the ExAmp master mix immediately before use. Store the ExAmp master mix up to 1 hour on ice, or 30 minutes at room temperature. After loading the libraries and ExAmp, load flow cells onto the instrument within 30 minutes. If ExAmp reagents cannot be used, they can be refrozen one time only. If you must refreeze, do so immediately after thawing.

SBS Reagents

HiSeq v4 High Output SBS Reagents and HiSeq Rapid v2 SBS Reagents

Thaw reagents in a room temperature deionized water bath for about 90 minutes and use within the same day. Alternatively, thaw at 2°C to 8°C overnight and use the next day. Store thawed CRM on ice in a separate container to avoid cross-contamination. Store the thawed SBS reagents on ice or at 2°C to 8°C until use.

HiSeq 3000/4000 SBS Reagents and HiSeq X Five/Ten SBS Reagents

Thaw reagents in a room temperature deionized water bath for about 90 minutes and use within the same day. Alternatively, thaw at 2°C to 8°C overnight and use the next day. Store thawed HCM on ice in a separate container to avoid cross-contamination. Store thawed SBS reagents on ice or at 2°C to 8°C until use.

Clustering and SBS Reagent Cartridges

iSeq 100 Reagent Kits

After receiving reagent shipment, store reagent cartridge in the freezer at -20 to -25°C for at least 24 hours before thawing for use. There are several ways to thaw the reagent cartridge:

  • If thawed in a room temperature water bath (20-25°C) for a minimum of 6 hours, the cartridge is stable up to 18 hours in the water bath.
  • If thawed in a refrigerator (2°C -8°C) for a minimum of 36 hours, the cartridge is stable up to 72 hours in the refrigerator.
  • If thawed in room temperature air (20°C to 25°C) for a minimum of 9 hours, the cartridge is stable up to 18 hours in room temperature air.

MiSeq v2 and v3 Reagent Kits

Thaw reagent kit in a room temperature deionized water bath for 60 minutes. Place on ice or set aside at 2°C to 8°C and use within 6 hours. Alternatively, thaw reagents overnight at 2° to 8°C, then store 2° to 8°C for up to 1 week.

NextSeq v2 Reagent Kit

Thaw reagent kit in a room temperature deionized water bath for 60 minutes. Place on ice or set aside at 2°C to 8°C, and use as quickly as possible or within the same day. Alternatively, thaw overnight at 2° to 8°C, then store 2° to 8°C for up to 1 week.

MiniSeq Reagent Kit

There are several ways to thaw the reagent cartridge:

  • If thawed in a 37°C water bath for 35 minutes, it is stable up to 2 hours.
  • If thawed in a room temperature water bath (19°C to 25°C) for 90 minutes, it is stable up to 24 hours.
  • If thawed at room temperature air (19°C to 25°C) for 5 hours, it is stable up to 24 hours.
  • If thawed by chilling at 2°C to 8°C for 18 hours, it is stable up to 72 hours.

NovaSeq Reagent Kits

Thaw S1 and S2 cluster cartridges in a room temperature deionized water bath for 2 hours. Thaw S4 cluster cartridges, and S1, S2 and S4 SBS cartridges in a room temperature water bath for 4 hours. Store thawed reagents at 2°C to 8°C until use for up to 24 hours. Cluster and SBS cartridges can be refrozen once at -20°C if run cannot be set up right away.