How to thaw and store sequencing reagents for optimal performance
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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.
iSeq 100 Reagent Kits ()
After receiving reagent shipment, store reagent cartridge in the freezer at -20°C to -25°C with the arrow pointing up 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°C to 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 to 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
iSeq 100 i1 Reagent v2 kits can be thawed at 4°C for 36 hours and are stable at 4°C for up to one week prior to use
MiniSeq Reagent Kits ()
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 at 2°C to 8°C for 18 hours, it is stable up to 72 hours
MiSeq v2 and v3 Reagent Kits ()
There are several ways to thaw the reagent cartridge:
If thawed 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
If thawed overnight at 2°C to 8°C, store at 2°C to 8°C for up to 1 week
There are several ways to thaw the reagent cartridge:
If thawed 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
If thawed overnight at 2°C to 8°C, store at 2°C to 8°C for up to 1 week - Note: Reagents require a minimum of 18 hours to thaw
XLEAP-SBS Sequencing Kits
Water Bath: Thaw in temperature controlled 25°C with a minimum of 9.5 cm water depth for 8 hours. Do not exceed 10 hours.
Refrigerator (2°C to 8°C): One day prior to anticipated run, remove cartridge from -25°C to -15°C storage. Thaw at room temperature for 6 hours. Transfer to refrigerator at 2°C to 8°C, then continue to thaw for a minimum of 16 hours. The cartridge is stable up to 72 hours in the refrigerator. During the room temperature thawing time, cartridges must have air on all sides except the bottom and must not be stacked. In the refrigerator, cartridges may be stacked but must be given space on all other sides.
Room temperature (20°C to 25°C): Place cartridges on wire rack for 12 hours and do not exceed 16 hours. During the room temperature thawing, cartridges must have air on all sides and must not be stacked.
Standard SBS Sequencing Kits
Water bath: Thaw in temperature controlled 25°C water bath, with a minimum of 9.5 cm water depth for 6-8 hours for 300 cycle or smaller kits; and 600 cycle kits require 8-10 hours.
Refrigerator (2°C to 8°C): One day prior to anticipated run, remove cartridge from -25°C to -15°C storage. Thaw at room temperature for 6 hours. Transfer to refrigerator 2°C to 8°C, and continue to thaw for a minimum of 12 hours for 300 cycle or smaller kits; or continue to thaw for a minimum of 16 hours for 600 cycle kits. The cartridge is stable up to 72 hours in the refrigerator. When thawing at room temperature, cartridge must have air on all sides except the bottom and must not be stacked. In the refrigerator, cartridges may be stacked but must be given space on all other sides.
Room temperature (20°C to 25°C) for a minimum of 9-16 hours for 300 cycle or smaller kits; 12 hours (16 hour maximum) for 600 cycle kit.
HiSeq 1000/1500/2000/2500 Cluster and SBS Kits
TruSeq SR Cluster Kit v3, TruSeq PE Cluster Kit v3, HiSeq SR Cluster Kit v4, and HiSeq PE Cluster Kit v4
There are several ways to thaw the reagent plate:
If the cBot reagent plate is thawed in a room temperature water bath for 60 minutes, use within the same day
If the cBot reagent plate is thawed at 2°C to 8°C overnight, no longer than 16 hours, use the plate within the 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
There are several ways to thaw the reagent plate:
If the cBot reagent plate is thawed in a room temperature water bath for 30 minutes, use within the same day
If the cBot reagent is plate thawed at 2°C to 8°C overnight, no longer than 16 hours, use the plate within the 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 v4 High Output SBS Reagents and HiSeq Rapid v2 SBS Reagents
There are several ways to thaw the reagents:
If the reagents are thawed in a room temperature deionized water bath for about 90 minutes, use within the same day
If the reagents are thawed at 2°C to 8°C overnight, 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/X Cluster and SBS Kits
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.
HiSeq 3000/4000 SBS Reagents and HiSeq X Five/Ten SBS Reagents
There are several ways to thaw the reagents:
If the reagents are thawed in a room temperature deionized water bath for about 90 minutes, use within the same day
If the reagents are thawed at 2°C to 8°C overnight, 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.
NovaSeq Cluster and SBS cartridges
Thaw SP, S1, and S2 cluster cartridges in a room temperature deionized water bath for 2 hours
Thaw S4 cluster cartridges in a room temperature water bath for 4 hours
Thaw SP, 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.
NovaSeq XP Workflow 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 refreezing, do so immediately after thawing.
Reagent Cartridge
Water Bath: Remove cartridge from box, then remove cartridge from the bag. Submerge reagent cartridge in laboratory-grade water bath at 15°C to 30°C until the water reaches the bottom of the cartridge cover. Thaw for 4 hours and do not exceed 24 hours. Load cartridge into instrument within 24 hours. If reagents cannot be used within 24 hours, store at 2°C to 8°C for up to 72 hours or return to -25°C to -15°C storage for up to 7 days. After thawing, do not refreeze more than one time.
Refrigerator: Remove cartridge from box, then remove cartridge from the bag. Thaw in a 2°C to 8°C refrigerator for 48 hours. If reagent cartridge cannot be loaded into the instrument within 24 hours, store at 2°C to 8°C for up to 72 hours or return to -25°C to -15°C storage for up to 7 days. After thawing, do not refreeze more than one time.
Lyo Insert
Remove from -25°C to -15°C storage and thaw at room temperature for 10 minutes.
If Lyo Insert cannot be used within 24 hours, return to -25°C to -15°C storage. After thawing, do not refreeze more than one time.
Pre-load and Custom Primer Buffers
Remove from -25°C to -15°C storage and thaw at room temperature for 10 minutes.
Invert 5 times.
If buffers cannot be used within 8 hours, return to -25°C to -15°C storage. After thawing, do not refreeze more than one time.
Flow Cell
Remove flow cell from 2°C to 8°C storage.
Set the sealed flow cell package aside for 10-15 minutes to allow the flow cell to reach room temperature.
Leave flow cell in package until ready to load flow cell into instrument. Use the flow cell within 2 hours of removing it from 2°C to 8°C storage.
If flow cell cannot be used within 2 hours, return to 2°C to 8°C storage and use within 24 hours.
NextSeq 500/550 Reagent Kits ()
NextSeq 1000/2000 Reagent Kits ()
NovaSeq 6000 Reagent Kits ()
NovaSeq X Plus ()
For any feedback or questions regarding this article (Illumina Knowledge Article #1533), contact Illumina Technical Support .