150602_AN_SureSelect と KAPA Hyper Prep.indd

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1 Agilent SureSelect KAPA Hyper Prep Kit DNA 10 ng : FFPE DNA DNA V2.5BJ & : KAPA Hyper Prep Kit SureSelect XT :KAPA Hyper Prep Kit, SureSelect, Agilent SureSelect XT PCR Primer KAPA Hyper Prep Kit KAPA PCR Primer PCR Update PCR SureSelect XT HiSeq/MiSeq Version B KAPA BIOSYSTEMS Technical Data Sheet, KAPA Hyper Prep Kit illumina platforms KR0961 v1.14 :

2 pdf SureSelect SureSelect TE plus Adapter TPFD-KB KAPA,, 96 SureSelect TE plus Adapter TPFD-KB KAPA,, 96, Auto SureSelect XT // / 1 Agilent Agilent SureSelect XT Human All Exon V5, 16 Agilent SureSelect XT Human All Exon V5, 96 Agilent SureSelect XT Human All Exon V5+UTRs, 16 Agilent SureSelect XT Human All Exon V5+UTRs, 96 Agilent SureSelect XT Human All Exon V5+lncRNA, 16 Agilent SureSelect XT Human All Exon V5+lncRNA, 96 Agilent SureSelect XT Human All Exon V5 Plus, 16 Agilent SureSelect XT Human All Exon V5 Plus, 96 Agilent SureSelect XT Human All Exon V5+Regulatory, 16 Agilent SureSelect XT Human All Exon V5+Regulatory, 96 Agilent SureSelect XT Focused Exome, 16 Agilent SureSelect XT Focused Exome, 96 Agilent SureSelect XT Clinical Research Exome, 16 Agilent SureSelect XT Clinical Research Exome, 96 Agilent ClearSeq SS Comprehensive Cancer, 16 Agilent ClearSeq SS Comprehensive Cancer, 96 Agilent ClearSeq SS, 16 Agilent ClearSeq SS, 96 Agilent ClearSeq SS DNA Kinome, 16 Agilent ClearSeq SS DNA Kinome, 96 Agilent SureSelect XT Mouse All Exon, 16 Agilent SureSelect XT Mouse All Exon, 96 Agilent Bravo NGS 24 4 SureSelect XT kb, 16 Agilent SureSelect XT kb, 96 Agilent SureSelect XT Mb, 16 Agilent SureSelect XT Mb, 96 Agilent SureSelect XT Mb, 16 Agilent SureSelect XT Mb, 96 Agilent SureSelect XT Mb, 16 Agilent SureSelect XT Mb, 96 Agilent SureSelect XT Mb, 16 Agilent SureSelect XT Mb, 96 Agilent

3 KAPA Hyper Prep Kit Herculase II Fusion DNA Polymerase Dynabeads MyOne Streptavidin T1 KAPA // / 1 KK Agilent µl 200 µl Life Technologies µl 2 ml AMPure XP Kit SPRI beads Beckman Coulter A µl 5 ml 1xLow TE Buffer 10 mm Tris-HCl, ph 8.0, 0.1 mm EDTA Life Technologies µl 100 ml 1 M Tris-HCl Buffer, ph 8.0 Sigma Aldrich T µl 1 L KK KK dntp µl ml, ml A ml, A ml Nuclease-free water not DEPC-treated Life Technologies AM ml 500 ml DEPC 99.5% Ethanol, molecular biology grade Wako ml 80% 70% Ethanol for SPRI clean-up molecular biology grade 1.8 ml Qubit dsdna BR Assay Kit Life Technologies Q32850 Qubit dsdna HS Assay Kit Life Technologies Q32851 Agilent QPCR NGS GA Agilent G4880A 1 5 TapeStation Agilent 2200 TapeStation 98% gdna BR gdna 1 21 Agilent TapeStation D1000 Screen Tape Agilent Agilent TapeStation D1000 Agilent Agilent TapeStation High Sensitivity D1000 Screen Tape Agilent TapeStation High Sensitivity D1000 Agilent TapeStation Genomic DNA ScreenTape Agilent Agilent Agilent Agilent TapeStation Genomic DNA Agilent Agilent 2100 Agilent DNA 1000 kit Agilent Agilent High Sensitivity DNA kit Agilent gdna Ver B

4 Agilent 2200 TapeStation System Agilent G2964AA/ G2965AA // Agilent 2100 Bioanalyzer Agilent G2943CA Covaris S-series Single Tube Sample Preparation System, Model S2 Covaris microtube with AFA fiber and snap cap Covaris Covaris Model S Model S2 Qubit 2.0 Fluorometer Life Technologies Q32866 Agilent SureCycler DNA DNA Expert Control Software ver B Model E210, C-2000 gdna gdna 65 24h 27 µl 24 µl HotTop 100 µl Eppendorf 5417C WEV-03 CC-105 Dynal DynaMag-2 NGS MagnaStand 8 Dynal DynaMag-96 Side skirted 96 Mx3000P/Mx3005P 96-well tube plates optical strip tubes Mx3000P/Mx3005P optical strip caps flat type Life Technologies LifeTechnologies Agilent D 8 FGSSMAG Plate Tube Agilent Plate 2 Strip Tube 4 4 Strip Tubes 120 PCR Plate 25 strip caps 120 WEB µl DNA 1~2 TU-500 GLD ml FGSSMAG h 27 µl 24 µl 65 24h 27 µl 24 µl HotTop 4

5 // MicroAmp Clear Adhesive Film Life Technologies films Tube-strip capping tool Tube cap strips, domed Agilent DNA LoBind, 1.5 ml PCR clean, 250 pieces Eppendorf Qubit assay tubes Life Technologies Q32856 Pipetman P10, P20, P200, P1000 PCR Strip 65 24h 27 µl 24 µl HotTop PCR LoBind Qubit gdna Rainin L12-20 Nuclease-Free 15 ml SAFE SKIN PRE S, M, L SureSelect Kimberly Clark 220, 330, 440 S, M, L II ml WashBuffer2 PreWarm 65 Mx3005P PCR AM Pure XP Agilent DNA 5

6 DNA KAPA Hyper Prep Kit SureSelect SureSelect Adapter Primer SureSelect 750 ng SureSelect XT SureSelect XT Blocker SureSelect XT SureSelect XT PCR Herculase II KAPA HiFi HotStart PCR KAPA HiFi HotStart PCR KAPA Hyper Prep Kit 1 ng DNA SureSelect XT DNA 10 ng FFPE FFPE FFPE DNA 50 ng DNA FFPE DNA 200 ng DNA PCR Duplication % FFPE DNA FFPE FFPE DNA UV-VIS 260/ bp Qubit/Nanodrop 0.35 dsdna qpcr FFPE DNA Optimizing FFPE DNA preparation for SureSelectXT2 NOTE: guidelines in this application note for FFPE gdna QC can also be used when working with SureSelectXT products Agilent Application Note Publication Number EN, 2012 HaloPlex Target Enrichment from FFPE Tissues Agilent Application Note Publication Number EN, 2012 FFPE-Derived DNA Quality Assessment In Preparation for HaloPlex Target Enrichment Agilent protocol G ,

7 DNA gdna Agilent TapeStation Genomic DNA Assay FFPE DNA Intact gdna FFPE DNA 1. DNA LoBind Qubit 10 ng DNA 1x LOW TE Buffer 52 µl 2. SureSelect XT Version B bp DNA S220 DNA S220 Duty Factor 10 Peak Power Cycles / Burst 200 Treatment 360 sec 4 8 Genomic DNA Assay DNA Agilent TapeStation D1000 Assay DNA 1000 FFPE qpcr DNA S2 Duty Factor 10% Intensity 5 Cycles per Burst Frequency sweeping PCR 8 Strip PCR 4. Qubit PicoGreen DNA DNA 50 µl DNA DNA DNA FFPE DNA FFPE DNA 10 ng 10 ng 50 ng 200 ng DNA 20-40% 7

8 DNA A 1. End Repair & A-Tailing KAPA Hyper Prep Kit Component Volume Fragmented, double-stranded DNA 50 µl End Repair & A-Tailing Buffer * 7 µl End Repair & A-Tailing Enzyme Mix * 3 µl Total Volume 60 µl * Buffer Enzyme Hot Top ON Step Temperature Time End Repair & A- Tailing min min HOLD DNA 25 ng SureSelect Adapter OligoMix Nuclease Free SureSelect Adapter OligoMix DNA 1 50 ng 1 µg 5 µl 25 ng 50 ng µl 10 ng µl Note DNA 100 ngsureselect Adapter Oligo Mix 10 µl SureSelect Adapter Oligo Mix 10 µl Adapter Ligation Nuclease Free 110 µl 2. Adapter Ligation KAPA Hyper Prep Kit SureSelect Target Enrichment Component Volume End Repair & A-Tailing reaction product 60 µl Nuclease Free 5 µl Ligation Buffer 30 µl DNA Ligase 10 µl diluted SureSelect Adapter Oligo Mix 5 µl Total Volume 110 µl Nuclease Free Buffer Enzyme Hot Top OFF Hot Top Step Temperature Time Ligation min 5. 8

9 1. 30 AMPure XP 4 AMPure XP AMPure XP 88 µl PCR DNA 110 µl % 200 µl 80% µl µl elution buffer 10 mm Tris-HCl, ph DNA µl PCR DNA Stopping Point

10 DNA 1. PCR Component Volume 2x KAPA HiFi HotStart ReadyMix 25 µl SureSelect Primer 4.0 µl SureSelect ILM Indexing Pre Capture PCR Reverse Primer 4.0 µl Adapter Ligated Library 20 µl Total Volume 53 µl PCR Step Temp Duration Cycles Initial denaturation sec 1 Denaturation sec DNA Annealing sec Extension sec Final Extension 72 1 min 1 HOLD 4 1 DNA DNA 100 ng ng ng ng PCR Duplication PCR PCR AMPure XP 4 AMPure XP AMPure XP 53 µl PCR PCR DNA 53 µl % 200 µl 80% µl µl Nuclease Free 2 SureSelect Nuclease Free DNA µl PCR DNA Stopping Point 20 10

DNA DNA DNA1000 2200 TapeStation DNA 1000 1.

Agilent 2100 expert version B.02.02 3. 4. 5 5.

11 DNA DNA DNA TapeStation DNA µl NOTE FFPE DNA 2. Agilent 2100 expert version B assay DNA bp- 275 bp 1 DNA DNA bp-275 bp 11

12 2200 TapeStation D1000 Screen Tape NOTE D1000 ScreenTape Agilent p/n D1000 Reagents Agilent p/n Agilent 2200 TapeStation User Manual 1 ng start, 22 cycles 711 ng 1. Agilent 2200 TapeStation User Manual DNA 1 µl D1000 sample buffer 3 µl CAUTION DNA D1000 sample buffer TapeStation 2000 rpm 1 Vortex Max ng start, 18 cycles 831 ng 2. Agilent 2200 TapeStation User Manual Step1 D1000 ScreenTape Loading tip 2200 TapeStation bp 275 bp DNA ng DNA PCR Upper Marker Upper Marker PCR NOTE 2 DNA 2200 TapeStation D1000 ScreenTape 225 bp bp FFPE NOTE SureSelect 750 ng DNA 500 ng 750 ng 221 ng/µl 221 ng/µl 45 Low, ng DNA 750 ng 12

13 DNA SureSelect SureSelect P52 SureSelect XT HiSeq/MiSeq PCR Protocol Version B HapMap DNA NA ng 10 ng 1 ng PCR Cycle ng ,170-1,260 1,890-1, Application Note 2014 <11> SureSelect XT FFPE DNA 50 ng 13

14 KAPA BIOSYSTEMS TEL FAX SureSelect TEL Agilent Technologies Agilent Technologies, Inc., 2015 Published in Japan, May. 11, JAJP

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3 1 2 Agilent 4200 TapeStation Agilent 4200 TapeStation 3 1 2 2 3 ScreenTape 3 Agilent 4200 TapeStation QC Agilent 4200 TapeStation Ready-to-use ScreenTape 8 PCR 96-well plate 1 96 1 qrt-pcr DNA RNA DNA / RNA