Agilent SureSelect KAPA Hyper Prep Kit DNA 10 ng : FFPE DNA DNA V2.5BJ 2015 5 & : KAPA Hyper Prep Kit SureSelect XT :KAPA Hyper Prep Kit, SureSelect, Agilent SureSelect XT PCR Primer KAPA Hyper Prep Kit KAPA PCR Primer PCR 2014 10 Update PCR SureSelect XT HiSeq/MiSeq Version B.2 2015 5 KAPA BIOSYSTEMS Technical Data Sheet, KAPA Hyper Prep Kit illumina platforms KR0961 v1.14 :
pdf http://agilentgenomics.jp SureSelect SureSelect TE plus Adapter TPFD-KB KAPA,, 96 SureSelect TE plus Adapter TPFD-KB KAPA,, 96, Auto SureSelect XT // / 1 Agilent 931171 1 96 Agilent 931182 1 96 SureSelect XT Human All Exon V5, 16 Agilent 5190-6208 1 16 SureSelect XT Human All Exon V5, 96 Agilent 5190-6209 1 96 SureSelect XT Human All Exon V5+UTRs, 16 Agilent 5190-6213 1 16 SureSelect XT Human All Exon V5+UTRs, 96 Agilent 5190-6214 1 96 SureSelect XT Human All Exon V5+lncRNA, 16 Agilent 5190-6446 1 16 SureSelect XT Human All Exon V5+lncRNA, 96 Agilent 5190-6447 1 96 SureSelect XT Human All Exon V5 Plus, 16 Agilent 5190-6211 1 16 SureSelect XT Human All Exon V5 Plus, 96 Agilent 5190-6212 1 96 SureSelect XT Human All Exon V5+Regulatory, 16 Agilent 931071 1 16 SureSelect XT Human All Exon V5+Regulatory, 96 Agilent 931072 1 96 SureSelect XT Focused Exome, 16 Agilent 5190-7787 1 16 SureSelect XT Focused Exome, 96 Agilent 5190-7788 1 96 SureSelect XT Clinical Research Exome, 16 Agilent 5190-7338 1 16 SureSelect XT Clinical Research Exome, 96 Agilent 5190-7339 1 96 ClearSeq SS Comprehensive Cancer, 16 Agilent 5190-8011 1 16 ClearSeq SS Comprehensive Cancer, 96 Agilent 5190-8012 1 96 ClearSeq SS, 16 Agilent 5190-7518 1 16 ClearSeq SS, 96 Agilent 5190-7519 1 96 ClearSeq SS DNA Kinome, 16 Agilent 5190-4646 1 16 ClearSeq SS DNA Kinome, 96 Agilent 5190-4647 1 96 SureSelect XT Mouse All Exon, 16 Agilent 5190-4641 1 16 SureSelect XT Mouse All Exon, 96 Agilent 5190-4642 1 96 16 Bravo NGS 24 4 SureSelect XT 1-499 kb, 16 Agilent 5190-4806 1 16 5190-4811 SureSelect XT 1-499 kb, 96 Agilent 5190-4807 1 96 5190-4812 SureSelect XT 0.5-2.9 Mb, 16 Agilent 5190-4816 1 16 5190-4821 SureSelect XT 0.5-2.9 Mb, 96 Agilent 5190-4817 1 96 5190-4822 SureSelect XT 3-5.9 Mb, 16 Agilent 5190-4826 1 16 5190-4831 SureSelect XT 3-5.9 Mb, 96 Agilent 5190-4827 1 96 5190-4832 SureSelect XT 6-11.9 Mb, 16 Agilent 5190-4836 1 16 5190-4841 SureSelect XT 6-11.9 Mb, 96 Agilent 5190-4837 1 96 5190-4842 SureSelect XT 12-24 Mb, 16 Agilent 5190-4896 1 16 5190-4901 SureSelect XT 12-24 Mb, 96 Agilent 5190-4897 1 96 5190-4902 2
KAPA Hyper Prep Kit Herculase II Fusion DNA Polymerase Dynabeads MyOne Streptavidin T1 KAPA // / 1 KK8502 24 Agilent 600677 1 µl 200 µl Life Technologies 65601 50 µl 2 ml AMPure XP Kit SPRI beads Beckman Coulter A63880 228 µl 5 ml 1xLow TE Buffer 10 mm Tris-HCl, ph 8.0, 0.1 mm EDTA Life Technologies 12090-015 100 µl 100 ml 1 M Tris-HCl Buffer, ph 8.0 Sigma Aldrich T3038 0.2 µl 1 L KK8500 8 KK8504 96 dntp 600679 400 µl 65602 10 ml, 65603 100 ml A63881 60 ml, A63882 450 ml Nuclease-free water not DEPC-treated Life Technologies AM9930 2.4 ml 500 ml DEPC 99.5% Ethanol, molecular biology grade Wako 054-07225 500 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 5067-5582 1 7 1 16 Agilent TapeStation D1000 Agilent 5067-5583 1 112 Agilent TapeStation High Sensitivity D1000 Screen Tape Agilent TapeStation High Sensitivity D1000 Agilent TapeStation Genomic DNA ScreenTape Agilent 5067-5584 1 7 1 16 Agilent 5067-5585 1 112 Agilent 5067-5365 1 7 Agilent TapeStation Genomic DNA Agilent 5067-5366 1 Agilent 2100 Agilent DNA 1000 kit Agilent 5067-1504 1 25 Agilent High Sensitivity DNA kit Agilent 5067-4626 1 10 1 15 gdna 1 12 1 11 Ver B02.07 3
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 S2 520045 Model S2 Qubit 2.0 Fluorometer Life Technologies Q32866 Agilent SureCycler DNA DNA Expert Control Software ver B.02.07 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 123-21D 8 FGSSMAG2 96 120-27 410088 Plate 410092 Tube Agilent 401425 Plate 2 Strip Tube 4 4 Strip Tubes 120 PCR Plate 25 strip caps 120 WEB-03-12 45 50 µl DNA 1~2 TU-500 GLD-051 1.5-2 ml 16 8 8 FGSSMAG2 96 65 24h 27 µl 24 µl 65 24h 27 µl 24 µl HotTop 4
// MicroAmp Clear Adhesive Film Life Technologies 4306311 4 100 films Tube-strip capping tool Tube cap strips, domed Agilent 410096 120 DNA LoBind, 1.5 ml PCR clean, 250 pieces Eppendorf 95295-0030 108.051 28 250 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 352097 Kimberly Clark 220, 330, 440 S, M, L II 37 1.5 ml WashBuffer2 PreWarm 65 Mx3005P PCR AM Pure XP Agilent 401449 DNA 5
DNA KAPA Hyper Prep Kit SureSelect 931171 931182 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 260/280 1.7-2.0 500-1000 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 5991-0960EN, 2012 HaloPlex Target Enrichment from FFPE Tissues Agilent Application Note Publication Number 5991-0666EN, 2012 FFPE-Derived DNA Quality Assessment In Preparation for HaloPlex Target Enrichment Agilent protocol G9900-90050, 2013 6
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.2 2015 5 150-200 bp DNA S220 DNA S220 Duty Factor 10 Peak Power 175.0 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 200 60 6 Frequency sweeping 4 7 3. 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
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 24 4 20 3 2. 3. Hot Top ON Step Temperature Time End Repair & A- Tailing 20 30 min 65 30 min HOLD 4 4. 1. DNA 25 ng SureSelect Adapter OligoMix Nuclease Free SureSelect Adapter OligoMix DNA 1 50 ng 1 µg 5 µl 25 ng 50 ng 1 1 5 µl 10 ng 1 5 5 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 24 4 20 3 3. 4. Hot Top OFF Hot Top Step Temperature Time Ligation 20 15 min 5. 8
1. 30 AMPure XP 4 AMPure XP 2. 3. AMPure XP 88 µl PCR DNA 110 µl 10 4. 3 5 5. 6. 80% 200 µl 80% 7. 30 9. 30 20 µl 10. 10 10 11. 21 µl elution buffer 10 mm Tris-HCl, ph 8.0 2 12. 5 DNA 13. 20 µl PCR DNA Stopping Point 20 8. 6 7 9
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 2. 3. PCR Step Temp Duration Cycles Initial denaturation 98 45 sec 1 Denaturation 98 15 sec DNA Annealing 65 30 sec Extension 72 30 sec Final Extension 72 1 min 1 HOLD 4 1 DNA DNA 100 ng 6-7 50 ng 7-8 25 ng 8-10 10 ng 11-14 PCR Duplication PCR PCR 1. 30 AMPure XP 4 AMPure XP 2. 3. AMPure XP 53 µl PCR PCR DNA 53 µl 10 4. 3 5 5. 6. 80% 200 µl 80% 7. 30 8. 6 7 9. 30 20 µl 10. 10 10 11. 30 µl Nuclease Free 2 SureSelect Nuclease Free 12. 5 DNA 13. 30 µl PCR DNA Stopping Point 20 10
DNA DNA DNA1000 2200 TapeStation DNA 1000 1. 350 µl NOTE FFPE DNA 2. Agilent 2100 expert version B.02.02 3. 4. 5 5. assay DNA1000 6. 7. 225 bp- 275 bp 1 DNA DNA1000 225 bp-275 bp 11
2200 TapeStation D1000 Screen Tape NOTE D1000 ScreenTape Agilent p/n 5067-5582 D1000 Reagents Agilent p/n 5067-5583 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 10 2 1 ng start, 18 cycles 831 ng 2. Agilent 2200 TapeStation User Manual Step1 D1000 ScreenTape Loading tip 2200 TapeStation 3. 225 bp 275 bp DNA 3 2014 10 1 ng DNA PCR 22 18 22 18 Upper Marker Upper Marker PCR NOTE 2 DNA 2200 TapeStation D1000 ScreenTape 225 bp - 275 bp FFPE NOTE SureSelect 750 ng DNA 500 ng 750 ng 221 ng/µl 221 ng/µl 45 Low, 40 250 ng DNA 750 ng 12
DNA SureSelect SureSelect P52 SureSelect XT HiSeq/MiSeq PCR Protocol Version B.2 2015 5 HapMap DNA NA12878 25 ng 10 ng 1 ng PCR Cycle ng 1 1 1 5 1 50 9 12 19 1,170-1,260 1,890-1,910 640-860 Application Note 2014 <11> SureSelect XT FFPE DNA 50 ng 13
KAPA BIOSYSTEMS http://www.n-genetics.com TEL 03-3813-0961 FAX 03-3813-0962 E-mail info@genetics-n.co.jp SureSelect http://agilentgenomics.jp/ TEL 0120-477-111 E-mail email_japan@agilent.com http://agilentgenomics.jp Agilent Technologies Agilent Technologies, Inc., 2015 Published in Japan, May. 11, 2015 5991-5756JAJP