> For the complete documentation index, see [llms.txt](https://knowledge.illumina.com/llms.txt). Markdown versions of documentation pages are available by appending `.md` to page URLs; this page is available as [Markdown](https://knowledge.illumina.com/instrumentation/general/instrumentation-general-reference_material-list/000001533.md).

# How to thaw and store sequencing reagents for optimal performance

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

**iSeq 100 Reagent Kits (**[**Reagent stability and thawing for the iSeq 100**](https://github.com/illumina-swi/illumina-knowledge/tree/master/articles/Knowledge/Reagent-Stability-and-Thawing-for-the-iSeq/README.md)**)**

* 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:
  * Thaw 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.\
    \- It is important to store the reagent cartridge at -20°C for at least one day before use. If the reagent cartridge is moved directly from dry ice shipment to a water bath thaw, this can heat shock the fluidics systems and cause total run failure.
  * Thaw in a refrigerator (2°C to 8°C) for a minimum of 36 hours, not exceeding 1 week.
  * Thaw at 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.
* Reagents must be used immediately after thawing (within the recommended time ranges described above), and re-freezing reagents is not recommended or supported. The longest thaw time stability is 1 week at 4°C.

**MiSeq v2 and v3 Reagent Kits (**[**Reagent Stability and Thawing Guidelines for MiSeq Sequencing Kits**](https://github.com/illumina-swi/illumina-knowledge/tree/master/articles/Knowledge/Instrumentation-MiSeq-Reagent-Stability-and-Thawing/README.md)**)**

* After receiving reagent shipment, store reagent cartridge in the freezer at -20°C.
* There are several ways to thaw the reagent cartridge:
  * **v2 cartridges**: Thaw in a room temperature deionized water bath with enough water to submerge the base of the reagent cartridge for 60 minutes. Place on ice or set aside at 2°C to 8°C and use within 6 hours.
  * **v3 cartridges:** Thaw in a room temperature deionized water bath with enough water to submerge the base of the reagent cartridge for 60-90 minutes. Place on ice or set aside at 2°C to 8°C and use within 6 hours.
  * **v2 and v3 cartridges:** thawed overnight at 2°C to 8°C, store at 2°C to 8°C for up to 1 week.
* Thawing the reagent cartridge overnight in the refrigerator allows for more flexibility as the reagents are then able to be stored at 4°C for up to one week.

###

**MiniSeq Reagent Kits (**[**Thawing and Stability Guidelines for MiniSeq Reagent Kits**](https://github.com/illumina-swi/illumina-knowledge/tree/master/articles/Knowledge/Thawing-and-Stability-Guidelines-for-MiniSeq-Reagent-Kits/README.md)**)**

* After receiving reagent shipment, store reagent cartridge in the freezer at -20°C.
* There are several ways to thaw the reagent cartridge:
  * Thaw in a 37°C water bath for 35 minutes, it is stable up to 2 hours.
  * Thaw in a room temperature water bath (19°C to 25°C) for 90 minutes, it is stable up to 24 hours.
  * Thaw at room temperature air (19°C to 25°C) for 5 hours, it is stable up to 24 hours.
  * Thaw at 2°C to 8°C for 18 hours, it is stable up to 72 hours.
* If reagents need to be stored during troubleshooting, store them at 4°C until ready to run within the stability time frames listed above.
*

**NextSeq 500/550 Reagent Kits (**[**Instructions for thawing and storing NextSeq 500/550 reagents**](https://github.com/illumina-swi/illumina-knowledge/tree/master/articles/Knowledge/Instrumentation-NextSeq-550-Reagents-and-Stability/README.md)**)**

* After receiving reagent shipment, store reagent cartridge in the freezer at -20°C.
* There are several ways to thaw the reagent cartridge:
  * Thaw 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.
  * Thaw 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.

**NextSeq 1000/2000 Reagent Kits (**[**Thawing and reagent preparation guidelines for the NextSeq 1000/2000 reagents**](https://github.com/illumina-swi/illumina-knowledge/tree/master/articles/Knowledge/Thawing-Guidelines-for-the-NextSeq-2000/README.md)**)**

* After receiving reagent shipment, store reagent cartridge in the freezer at -20°C with the arrows pointing up, in their original box, to avoid tearing or puncturing the foil bag.
* There are several ways to thaw the reagent cartridge:
  * *XLEAP-SBS Sequencing Kits*\
    \- Water Bath: Thaw in temperature controlled 25°C water bath 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, 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 a minimum of 9-16 hours for 300 cycle or smaller kits; 12 hours (16 hour maximum) for 600 cycle kit.
* If using the refrigerator thaw method or removing it from fridge storage, remove the thawed cartridge from 2-8ºC storage. Set the unopened cartridge aside at room temperature for at least 15 minutes before sequencing. Do not exceed 1 hour.
* If using room temperature thaw, proceed to Load Consumables Step.

**NovaSeq 6000 Reagent Series (**[**Thawing, stability, and storage of NovaSeq 6000 reagents**](https://github.com/illumina-swi/illumina-knowledge/tree/master/articles/Knowledge/Thawing-Stability-and-Storage-of-NovaSeq-6000-reagents/README.md)**)**

* After receiving reagent shipment, store Cluster and SBS cartridges in the freezer at -20°C.
* Thaw the Cluster and SBS cartridges in a room temperature deionized water bath:
  * *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.
  * *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.
* Store thawed reagents (Cluster and SBS cartridges) 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 started right away.
* SBS cartridges are sensitive to light.

**NovaSeq X Series (**[**Reagent stability and thawing guidelines for the NovaSeq X Series**](https://github.com/illumina-swi/illumina-knowledge/tree/master/articles/Knowledge/Reagent-Stability-and-Thawing-Guidelines-for-NovaSeq-X-NovaSeq-X-Plus-Platform/README.md)**)**

* All kit components are shipped at ambient temperature, and have been qualified to remain at ambient temperature for up to 10 days during shipment. When the reagents are received, promptly store components at the indicated temperature for proper performance.

*Reagent Cartridge*

* There are two validated methods for thawing a NovaSeq X Series 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.\
    \- \[25B, 10B, 5B] To confirm reagents are thawed, inspect the position #30 well on the underside of the cartridge and make sure that the contents are free of ice. For the NovaSeq X Series 1.5B Reagent Kit, Illumina recommends strictly following the provided thaw conditions and times. Visual checks to confirm reagents are thawed might not be accurate for this kit.
  * 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.
* Invert the cartridge 10 times to mix reagents.
* Gently tap the bottom of the cartridge on the bench to reduce air bubbles.

*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.

**MiSeq i100 (**[**Reagent Stability Guidelines for the MiSeq i100 Series Consumables**](https://github.com/illumina-swi/illumina-knowledge/tree/master/articles/Knowledge/Reagent-Stability-Guidelines-for-the-MiSeq-i100-Series-Consumables/README.md)**)**

* All kit components are shipped and stored at room temperature, so there are no thawing steps required when preparing a sequencing run. However, once the packaging has been opened, the reagent kit and associated consumables should be used within the time specified below for optimal performance.

*Dry Cartridge*

* Keep dry cartridge in the foil packaging until it is ready to be loaded. The Dry Cartridge must be used within 4 hours of opening the foil packaging.

*Wet Cartridge*

* Keep wet cartridge in the foil packaging until it is ready to be loaded. The Wet Cartridge must be used within 4 hours of opening the foil packaging.

*Denatured Libraries (Libraries + KLD Buffer)*

* Store the denatured libraries on ice after mixing the diluted libraries with the KLD buffer. The denatured libraries are stable on ice or at 4° C for up to 6 hours.

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| *For any feedback or questions regarding this article (Illumina Knowledge Article #1533), contact Illumina Technical Support* [*techsupport@illumina.com*](mailto:techsupport@illumina.com?subject=Question%2FFeedback%20Regarding%20Illumina%20Knowledge%20Article%20#000009351%20-%20Instrumentation%20\&body=Dear%20Illumina%20Technical%20Support,%0D%0A%0D%0A)*.* |


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