HiSeq® Rapid Duo cBot™ Sample Loading Kit Reagent Prep Guide FOR RESEARCH USE ONLY Reagent Kit Overview Kit Contents and Storage Requirements Prepare Reagent Plate Next Steps Technical Assistance ILLUMINA PROPRIETARY Part # 15059382 Rev. A Catalog # CT-403-9001DOC November 2014 3 4 6 8 This document and its contents are proprietary to Illumina, Inc. and its affiliates ("Illumina"), and are intended solely for the contractual use of its customer in connection with the use of the product(s) described herein and for no other purpose. This document and its contents shall not be used or distributed for any other purpose and/or otherwise communicated, disclosed, or reproduced in any way whatsoever without the prior written consent of Illumina. Illumina does not convey any license under its patent, trademark, copyright, or commonlaw rights nor similar rights of any third parties by this document. The instructions in this document must be strictly and explicitly followed by qualified and properly trained personnel in order to ensure the proper and safe use of the product(s) described herein. All of the contents of this document must be fully read and understood prior to using such product(s). FAILURE TO COMPLETELY READ AND EXPLICITLY FOLLOW ALL OF THE INSTRUCTIONS CONTAINED HEREIN MAY RESULT IN DAMAGE TO THE PRODUCT(S), INJURY TO PERSONS, INCLUDING TO USERS OR OTHERS, AND DAMAGE TO OTHER PROPERTY. ILLUMINA DOES NOT ASSUME ANY LIABILITY ARISING OUT OF THE IMPROPER USE OF THE PRODUCT(S) DESCRIBED HEREIN (INCLUDING PARTS THEREOF OR SOFTWARE) OR ANY USE OF SUCH PRODUCT(S) OUTSIDE THE SCOPE OF THE EXPRESS WRITTEN LICENSES OR PERMISSIONS GRANTED BY ILLUMINA IN CONNECTION WITH CUSTOMER'S ACQUISITION OF SUCH PRODUCT (S). © 2014 Illumina, Inc. All rights reserved. Illumina, 24sure, BaseSpace, BeadArray, BlueFish, BlueFuse, BlueGnome, cBot, CSPro, CytoChip, DesignStudio, Epicentre, GAIIx, Genetic Energy, Genome Analyzer, GenomeStudio, GoldenGate, HiScan, HiSeq, HiSeq X, Infinium, iScan, iSelect, ForenSeq, MiSeq, MiSeqDx, MiSeq FGx, NeoPrep, Nextera, NextBio, NextSeq, Powered by Illumina, SeqMonitor, SureMDA, TruGenome, TruSeq, TruSight, Understand Your Genome, UYG, VeraCode, verifi, VeriSeq, the pumpkin orange color, and the streaming bases design are trademarks of Illumina, Inc. and/or its affiliate(s) in the U.S. and/or other countries. All other names, logos, and other trademarks are the property of their respective owners. To use the cBot to load 2 libraries onto a rapid flow cell, you need 1 HiSeq Rapid Duo cBot Sample Loading Kit. Each kit contains sufficient reagents for loading 1 flow cell. Clustering on the cBot allows 2 libraries, 1 in each lane, on the 2-lane rapid flow cell. After template hybridization and first extension on the cBot, the remainder of the clustering process is performed on the HiSeq 2500 or HiSeq 1500. Kit Name HiSeq Rapid Duo cBot Sample Loading Kit HiSeq Rapid Duo cBot Sample Loading Kit Reagent Prep Guide Catalog # CT-403-2001 3 Reagent Kit Overview Reagent Kit Overview Kit Contents and Storage Requirements When you receive your kit, promptly store the kit components at the indicated temperature to ensure proper performance. Rapid Duo Kit Reagents Component cBot Reagent Plate Storage -25°C to -15°C cBot Reagent Plate The cBot reagent plate contains 3 rows of foil-sealed 8-tube strips filled with template hybridization and first extension reagents. Rows 4–12 of the reagent plate are empty. Figure 1 cBot Reagent Plate A B Row 1 Row 3 Each 8-tube strip is labeled with the reagent name followed by a number. The number indicates the row that it occupies on the reagent plate. If an 8-tube strip becomes dislodged from the reagent plate holder, use the row number on the label to return the tube strip to the appropriate position on the reagent plate. 4 Part # 15059382 Rev. A Kit Contents and Storage Requirements Table 1 cBot Reagent Plate Contents Row Reagent Description HT1 Hybridization Buffer 1 AMS Fast Amplification Mix 2 USB Universal Sequencing Buffer 3 Empty Empty 4 Empty Empty 5 Empty Empty 6 Empty Empty 7 Empty Empty 8 Empty Empty 9 Empty Empty 10 Empty Empty 11 Empty Empty 12 HiSeq Rapid Duo cBot Sample Loading Kit Reagent Prep Guide 5 Prepare Reagent Plate The reagent plate takes about 30 minutes to thaw using a room temperature water bath. Alternatively, you can thaw reagents at 2°C to 8°C overnight, or up to 16 hours. After thawing, carefully follow the steps to prepare reagents for use on the cBot. Best Practices } Wear a fresh pair of gloves when preparing clustering reagents. } Hold the 96-well cBot reagent plate by the plate base to avoid dislodging reagent tubes. } Make sure that the tubes are securely seated in the reagent plate before and after vortexing or inverting reagents. Loose reagent tubes can damage the cBot manifold. NOTE The clear plastic lid on the reagent plate protects the foil seals from being damaged or punctured during thawing. Remove the protective lid only when necessary, such as when you are checking that tubes are securely seated. } After completing the reagent preparation steps, proceed to setting up the cBot for clustering and load reagents when prompted. Thaw the cBot Reagent Plate 1 Remove the cBot reagent plate from -25°C to -15°C storage. 2 Make sure that the tube strips are securely seated in the plate. Do not to puncture the foil seals. 3 Place the reagent plate in a water bath containing enough room temperature deionized water to submerge only the reagent plate base. Allow reagents to thaw for 60 minutes. Prepare the cBot Reagent Plate 1 With a hand on top of the tubes, invert the reagent plate 5 times to mix the thawed reagents. 2 With a hand on top of the tubes, vortex the plate for approximately 10 seconds to dislodge trapped air bubbles. 6 Part # 15059382 Rev. A Tap the reagent plate on a hard surface approximately 10 times to collect reagent droplets at the bottom of the tubes. Alternatively, pulse centrifuge the reagent plate. 4 Visually inspect the reagent plate to make sure that the tubes are free of air bubbles. 5 Visually inspect the tubes to make sure that the tubes are securely seated and in the correct order as numbered on the label. 6 Promptly proceed to setting up the cBot. HiSeq Rapid Duo cBot Sample Loading Kit Reagent Prep Guide 7 Prepare Reagent Plate 3 Next Steps After you have prepared the reagent plate, you are ready to prepare your libraries and load the reagent plate when prompted by cBot software. For clustering workflow instructions, see the cBot User Guide, part # 15006165. Make sure that you use the compatible recipe version and cBot software version. Library Loading Kit Version Recipe Version Software Version HiSeq Rapid Duo cBot Sample Loading Kit Version R recipes cBot software v1.5, or later After you have completed clustering on the cBot, complete the clustering process and sequence your hybridized flow cell by performing a run in Rapid Run mode on the HiSeq. For workflow instructions, see the user guide for your sequencing instrument: } HiSeq 2500 System User Guide, part # 15035786 } HiSeq 1500 System User Guide, part # 15035788 Make sure that you use the compatible version of SBS kit, cluster kit, and HiSeq Control Software (HCS). Library Loading Kit Version HiSeq Rapid Duo cBot Sample Loading Kit 8 SBS Kit Version HiSeq Rapid SBS Kit v2 Cluster Kit Version HiSeq Rapid PE Cluster Kit v2 HiSeq Rapid SR Cluster Kit v2 Control Software Version HCS v2.2 / RTA v1.18, or later Part # 15059382 Rev. A For technical assistance, contact Illumina Technical Support. Table 2 Illumina General Contact Information Address 5200 Illumina Way San Diego, CA 92122 USA Website www.illumina.com Email [email protected] Table 3 Illumina Customer Support Telephone Numbers Region Contact Number Region North America 1.800.809.4566 Italy Austria 0800.296575 Netherlands Belgium 0800.81102 Norway Denmark 80882346 Spain Finland 0800.918363 Sweden France 0800.911850 Switzerland Germany 0800.180.8994 United Kingdom Ireland 1.800.812949 Other countries Safety Data Sheets Safety data sheets (SDSs) are available on the Illumina website at support.illumina.com/sds.html. HiSeq Rapid Duo cBot Sample Loading Kit Reagent Prep Guide Contact Number 800.874909 0800.0223859 800.16836 900.812168 020790181 0800.563118 0800.917.0041 +44.1799.534000 Technical Assistance Technical Assistance *15059382* Part # 15059382 Rev. A Illumina San Diego, California 92122 U.S.A. +1.800.809.ILMN (4566) +1.858.202.4566 (outside North America) [email protected] www.illumina.com
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