TruSeq Amplicon - Cancer Panel Protocol Guide For Research Use Only. Not for use in diagnostic procedures. Hybridize Oligo Pool Remove Unbound Oligos Extend and Ligate Bound Oligos Amplify Libraries Clean Up Libraries Normalize Libraries Pool Libraries Acronyms Technical Assistance ILLUMINA PROPRIETARY Document # 1000000007000 v01 February 2016 3 4 5 6 7 8 9 10 11 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 common-law 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). © 2016 Illumina, Inc. All rights reserved. Illumina, 24sure, BaseSpace, BeadArray, BlueFish, BlueFuse, BlueGnome, cBot, CSPro, CytoChip, DesignStudio, Epicentre, ForenSeq, Genetic Energy, GenomeStudio, GoldenGate, HiScan, HiSeq, HiSeq X, Infinium, iScan, iSelect, MiniSeq, MiSeq, MiSeqDx, MiSeq FGx, NeoPrep, NextBio, Nextera, NextSeq, Powered by Illumina, 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. Patent pending for methods performed by components in this kit. For Research Use Only– not for any clinical or therapeutic use in humans or animals. This product includes GoTaq® Hot Start Polymerase manufactured by Promega Corporation for distribution by Illumina, Inc. Licensed to Promega Corporation under U.S. Patent Nos. 5,338,671 and 5,587,287 and their corresponding foreign patents. WARNING This set of reagents contains formamide, an aliphatic amide that is a probable reproductive toxin. Personal injury can occur through inhalation, ingestion, skin contact, and eye contact. Wear protective equipment, including eye protection, gloves, and laboratory coat. Handle used reagents as chemical waste and discard in accordance with the governmental safety standards for your region. For environmental, health, and safety information, see the SDS for this kit at support.illumina.com/sds.html. Preparation 1 Set a 96-well heat block to 95°C. 2 Preheat an incubator to 37°C to prepare for the extension-ligation step. Procedure 1 Add 5 µl ACD1 to 1 well of the HYP plate. 2 Add 5 µl gDNA to each remaining well. 3 Add 5 µl ACP1 to the well containing ACD1. 4 Add 5 µl AFP1 to each well containing gDNA. 5 Centrifuge at 1000 × g for 1 minute. 6 Add 40 µl OHS1. Pipette to mix. 7 Centrifuge at 1000 × g for 1 minute. 8 Place on the preheated heat block and incubate for 1 minute. 9 With the plate on the heat block, reset the temperature to 40°C and continue incubating for 80 minutes. TruSeq Amplicon - Cancer Panel Protocol Guide 3 Hybridize Oligo Pool Hybridize Oligo Pool Remove Unbound Oligos WARNING This set of reagents contains formamide, an aliphatic amide that is a probable reproductive toxin. Personal injury can occur through inhalation, ingestion, skin contact, and eye contact. Wear protective equipment, including eye protection, gloves, and laboratory coat. Handle used reagents as chemical waste and discard in accordance with the governmental safety standards for your region. For environmental, health, and safety information, see the SDS for this kit at support.illumina.com/sds.html. Preparation 1 Assemble the filter plate unit (FPU) from top to bottom as follows: lid, filter plate, adapter collar, and midi plate. 2 Wash the wells to be used in the assay with 45 µl SW1. Procedure 1 Make sure that the heat block has cooled to 40˚C. 2 Remove from the heat block. 3 Centrifuge at 1000 × g for 1 minute. 4 Transfer each sample to the FPU plate. 5 Cover and centrifuge at 2400 × g for 2 minutes. 6 Wash 2 times with 45 µl SW1. 7 Discard flow-through. 8 Reassemble the FPU plate. 9 Add 45 µl UB1. 10 Cover and centrifuge at 2400 × g for 2 minutes. 4 Document # 1000000007000 v01 Extend and Ligate Bound Oligos Extend and Ligate Bound Oligos Procedure 1 Add 45 µl ELM3 to the FPU plate. 2 Incubate at 37°C for 45 minutes. TruSeq Amplicon - Cancer Panel Protocol Guide 5 Amplify Libraries Preparation 1 Save the following PCR program on a thermal cycler. } 95°C for 3 minutes } 27 cycles of: } 95°C for 30 seconds } 62°C for 30 seconds } 72°C for 60 seconds } 72°C for 5 minutes } Hold at 10°C Procedure 1 Arrange the Index 1 (i7) adapters in columns 1–12 of the TruSeq Index Plate Fixture. 2 Arrange the Index 2 (i5) adapters in rows A–H of the TruSeq Index Plate Fixture. 3 Place the plate on a TruSeq Index Plate Fixture. 4 Using a multichannel pipette, add 4 µl of each Index 1 (i7) adapter down each column. 5 Using a multichannel pipette, add 4 µl of each Index 2 (i5) adapter across each row. 6 Remove the FPU plate from the incubator and do the following. a b c d Replace the aluminum foil seal with the filter plate lid. Centrifuge at 2400 × g for 2 minutes. Add 25 µl 50 mM NaOH. Pipette to mix. Incubate at room temperature for 5 minutes. 7 Add 56 µl TDP1 to a full tube (2.8 ml) of PMM2. Invert to mix. 8 Transfer 22 µl PMM2/TDP1 mixture to the IAP plate. 9 Transfer eluted samples from the FPU plate to the IAP plate. 10 Centrifuge at 1000 × g for 1 minute. 11 Transfer the IAP plate to the post-amplification area. 12 Place on the preprogrammed thermal cycler and run the PCR program. SAFE STOPPING POINT If you are stopping, seal the plate and store at 2°C to 8°C for up to 2 days. Alternatively, leave on the thermal cycler overnight. 6 Document # 1000000007000 v01 Procedure 1 Centrifuge the IAP plate at 1000 × g for 1 minute. 2 Run an aliquot of library and control on 4% agarose gel (5 µl) or Bioanalyzer (1 µl). 3 Add 45 µl AMPure XP beads to the CLP plate. 4 Transfer all the supernatant from the IAP plate to the CLP plate. 5 Shake at 1800 rpm for 2 minutes. 6 Incubate at room temperature for 10 minutes. 7 Place on a magnetic stand and wait until the liquid is clear (~2 minutes). 8 Remove and discard all supernatant from each well. 9 Wash 2 times with 200 µl 80% EtOH. 10 Use a 20 µl pipette to remove residual EtOH. 11 Remove from the magnetic stand and air-dry for 10 minutes. 12 Add 30 µl EBT. 13 Shake at 1800 rpm for 2 minutes. 14 Incubate at room temperature for 2 minutes. 15 Place on a magnetic stand and wait until the liquid is clear (~2 minutes). 16 Transfer 20 µl supernatant from the CLP plate to the LNP plate. 17 Centrifuge at 1000 × g for 1 minute. TruSeq Amplicon - Cancer Panel Protocol Guide 7 Clean Up Libraries Clean Up Libraries Normalize Libraries WARNING This set of reagents contains formamide, an aliphatic amide that is a probable reproductive toxin. Personal injury can occur through inhalation, ingestion, skin contact, and eye contact. Wear protective equipment, including eye protection, gloves, and laboratory coat. Handle used reagents as chemical waste and discard in accordance with the governmental safety standards for your region. For environmental, health, and safety information, see the SDS for this kit at support.illumina.com/sds.html. WARNING This set of reagents contains ß-mercaptoethanol. Perform the following procedure in a hood or well-ventilated area. Procedure 1 For 96 samples, add 4.4 ml LNA1 to a new 15 ml conical tube. 2 Use a P1000 pipette to resuspend LNB1. 3 Transfer 800 µl LNB1 to the tube of LNA1. 4 Add 45 µl LNA1/LNB1 to the LNP plate. 5 Shake at 1800 rpm for 30 minutes. 6 Place on a magnetic stand and wait until the liquid is clear (~2 minutes). 7 Remove and discard all supernatant. 8 Remove from the magnetic stand. 9 Wash 2 times with 45 µl LNW1. 10 Use a 20 µl pipette to remove residual LNW1. 11 Remove from the magnetic stand. 12 Add 30 µl fresh 0.1 N NaOH. 13 Shake at 1800 rpm for 5 minutes. 14 Place the LNP plate on a magnetic stand and wait until the liquid is clear (~2 minutes). 15 Add 30 µl LNS1 to the SGP plate. 16 Transfer 30 µl supernatant from the LNP plate to the SGP plate. 17 Centrifuge at 1000 × g for 1 minute. SAFE STOPPING POINT If you are stopping, seal the plate and store at -25°C to -15°C for up to 30 days. 8 Document # 1000000007000 v01 Pool Libraries Pool Libraries Procedure 1 Centrifuge at 1000 × g for 1 minute. 2 Transfer 5 µl of each library to an 8-tube strip, column by column. 3 Transfer the contents of the 8-tube strip to the PAL tube. Pipette to mix. 4 Denature and dilute pooled libraries to the loading concentration for the sequencing instrument you are using. See the denature and dilute libraries guide for your instrument. TruSeq Amplicon - Cancer Panel Protocol Guide 9 Acronyms Acronym ACD1 Amplicon Control DNA 1 ACP1 Amplicon Control Oligo Pool 1 AFP1 Amplicon Fixed Panel 1 CLP Clean-up Plate EBT Elution Buffer with Tris ELM3 Extension Ligation Mix 3 FPU Filter Plate Unit HT1 Hybridization Buffer HYP Hybridization Plate IAP Indexed Amplification Plate LNA1 Library Normalization Additives 1 LNB1 Library Normalization Beads 1 LNS1 Library Normalization Storage Buffer 1 LNW1 Library Normalization Wash 1 LNP Library Normalization Plate OHS1 Oligo Hybridization for Sequencing Reagent 1 PAL PMM2 10 Definition Pooled Amplicon Library PCR Master Mix 2 SGP Storage Plate SW1 Stringent Wash 1 TDP1 TruSeq DNA Polymerase 1 UB1 Universal Buffer 1 Document # 1000000007000 v01 For technical assistance, contact Illumina Technical Support. Table 1 Illumina General Contact Information Website Email www.illumina.com [email protected] Table 2 Illumina Customer Support Telephone Numbers Region Contact Number Region North America 1.800.809.4566 Japan Australia 1.800.775.688 Netherlands Austria 0800.296575 New Zealand Belgium 0800.81102 Norway China 400.635.9898 Singapore Denmark 80882346 Spain Finland 0800.918363 Sweden France 0800.911850 Switzerland Germany 0800.180.8994 Taiwan Hong Kong 800960230 United Kingdom Ireland 1.800.812949 Other countries Italy 800.874909 Contact Number 0800.111.5011 0800.0223859 0800.451.650 800.16836 1.800.579.2745 900.812168 020790181 0800.563118 00806651752 0800.917.0041 +44.1799.534000 Safety data sheets (SDSs)—Available on the Illumina website at support.illumina.com/sds.html. Product documentation—Available for download in PDF from the Illumina website. Go to support.illumina.com, select a product, then select Documentation & Literature. TruSeq Amplicon - Cancer Panel Protocol Guide Technical Assistance Technical Assistance Illumina 5200 Illumina Way 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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