Cycleave®PCR Library Quantification Kit (for
Contents
1. 2 ul Paired End Sample Prep Kit PE 102 1001 Mutiplexing Sample Prep Oligo Kit 96plex PE 400 1001 12 1 pM a Agilent 2100 Bioanalyzer 0 75 pM b Agilent 2100 Bioanalyzer2. GC PCR DNA PCR 3 DNA B ei C
3. 0 1 10 pM DNA PCR PCR
4. a zsooooo b ioooooo NO 2 500 000 2 000 000 1 500000 Ed 150o000 mb mb 1000 000 1 000 000 500 000 366066 0 Eb ner Ea eR CMS NRL EK OSSS AA ASS SA ST SS SOS SS SS SA SN SN SA SS SRN NN NN NN NEFF 2 Agilent 2100 Bioanalyzer a b Agilent 2100 Bioanalyzer Genome Analyzer x SCS2 8 TruSeq SR Cluster Kitv5 GD 203 5001 TruSeq SBS Kit v5 FC 104 5001 http www takara bio coJp 13 5500 VI Thermal Cycler Dice Real Time System TP900 TP960 Thermal Cycler Dice Real Time System Lite TP700 TP760 96well Hi Plate for Real Time NJ400 Sealing Film for Real Time NJ500 Plate Sealing Pads 9090
5. DNA DNA GC PCR NN RN DNA http www takara bio co jp 12 5500 Multiplexed Sequence
6. 4 PCR PCR Auto PCR Manual N 5 EA RS 122 2002 F F RS P F TruSeq DNA PCR Free LT Sample Prep Kit Set B FC 121 3002 F F RS RS 122 2002 TruSeq Nano DNA LT Sample Prep Kit Set A C 121 4001 TruSeq Nano DNA LT Sample Prep Kit Set B C 121 4002 TruSed Stranded mRNA LT Sample Prep Kit Set A 122 2101 TruSed Stranded mRNA LT Sample Prep Kit Set B 122 2102 TruSed Stranded Total RNA with Ribo Zero LT Sample Prep RS 122 2201 Kit Set A TruSed Stranded Total RNA with Ribo Zero LT Sample Prep RS 122 2202 Kit Set B TruSed Stranded Total RNA with Ribo Zero Gold LT Sample RS 122 2301 Prep Kit Set A TruSed Stranded Total RNA with Ribo Zero Gold LT Sample RS 122 2302 Prep Kit Set B SureSelectX Target Enrichment System N
7. Primer Probe Mix DNA FAM PCR Primer Probe 0 IV R2 0 99 DNA Eff 70 2 X Cycleave Reaction Mixture
8. TafdR4 Tel 077 543 6116 Fax 077 543 1977 http www takara bio coJp v201410Da
9. EO OD OD CL RNA RNase H RNA RNA RNA 1 5 RNase H 1 http www takara bio co jp 2 5500 1 25 MI 50 1 2 Xx Cycleave Reaction Mixture 625 ul 2 Primer Probe Mix FAM 5 X conc 250 ul 3 EASY Dilution 1mlX 5 4 dH2O 1ml 5 ROX Reference Dye 50 X conc 25 ul 6 ROX Reference Dye ll 50 X co
10. PCR http wwwr takara bio coJp 3 5500 20 PCR Thermal Cycler Dice Real Time System Software Ver 3 00 TP900 960 Thermal Cycler Dice Real Time System Lite TP700 TP760 1 0 2 ml 8 Thermal Cycler Dice Real Time System 0 2 ml 8 strip tube individual Flat Caps NJ600 StepOnePlus Real Time PCR System Applied Biosystems 7500 Fast Real Time PCR System Life technologies 1 000 200 20 10 4 http wwwr takara bio
11. PCR 4 V 1 DNA 0 01 10 pM 2 1 1 5 ml EASY Dilution 45 ul 3 2 Standard 10 pM 5ul 1 Standard 1 pM 0 1 pM 0 01 pM Standard DNA 3 lt http www takara bio co jp 6 5500 V 2 2 1 pM EASY Dilution DNA w v 1 bp X 660 2
12. nM ng X 106 V 3 1 1 1 DNA g 23 ul 5 DNA 4 2 Thermal Cycler Dice Real Time System gt 1 2 x Cycleave Reaction Mixture 12 5 ul 1 Primer Probe Mix 5 X conc 5 ul 1 X or DNA or 2 uD dH2O 5 5 ul Total 25 ul DNA StepOnePlus Applied Biosystems 7500 Fast Real Time PCR System gt 1
13. 00 IdfaR4 Cycleave PCR Library Quantification Kit for lllumina vV201410Da MiSeq Genome Analyzer x HiScanSQ HiSeq 1000 HiSeq 2000 DNA DNA PCR 1 DNA DNA DNA DN
14. 2 X Cycleave Reaction Mixture 12 5 ul 1 Primer Probe Mix 5 X conc 5 ul 1 X ROX Reference Dye 50 X conc or Dye ll 50 X conc 1 0 5 wl 1 or DNA or 2 wl 2 dH20 5 ul Total 25 ul 1 StepOnePlus ROX Reference Dye 50 Xx 7500 Fast Real Time PCR System ROX Reference Dye ll 50 xX 2 DNA 2 3 1 DNA 2Ml 3 PCR 1 http wwwr takara bio coJp 7 5500 V 4
15. 1 000 http wwwr takara bio coJp 9 5500 StepOnePlus ReaLTime PCR Life Technologies Applied Biosystems 7500 Fast Real Time PCR System StepOnePlus Rea Time PCR System 1 StepOne Software File New Experiment Advanced Setup 2 Experiment Properties type of experiment Quantitation Standard Curve reagents Other ramp speed Fast What ype of expernment GO you want to eet up lt tt dsa 44 uve CK 70 PIE MRL SAAD NC Use ttandar0s 0 Gatserrng Me abtOtde GU snthy Of taegat PUtier 20d 20Ueece I TDS Which reagents do you Wet to use to detect the target sequence 1 na The PCR resrtons cortam tarmers 10 Getgnod Io amgity Me Garget te and otner resoerts 0O Seto moiac sion The RatsOn Setup coen i not yaiabie or Omer reagerts iciude Me Cume Which ramgp speed do youv want to ust In he instruvmenm un7 F ot octbenl etc wn Ba Gt rarmp tpeed Appties oryitems secorrenends ving F ait teageret Cr yout PCA resctoes StepOne Software v2 1
16. PCR 3 PCR Thermal Cycler Dice Real Time System StepOnePlus Real Time PCR System Life Technologies Applied Biosystems 7500 Fast ReaLTime PCR System StepOnePlus Thermal Cycler Dice Real Time System 1 AQ Absolute Quantifcation Single Singl Ralatwe OurAfcabon ge Multglex 7 PM single mus ey PM Multolnx Y shNP SNP GenebpinO Ass ay Muktglex User ID soa Your Namez Edx wen Thermal Cycler Dice Real Time System Software Ver 4 01A 2 Plate Setup Target amp Sample Setting Dye FAM Sample list STD UNKN Update Tawue Toet Me Temole http wwwr takara bio co
17. 0 2 ml Hi 8 Tube NJ300 0 2 ml Hi 8 Flat Cap NJ302 0 2 ml 8 strip tube individual Flat Caps NJ600 EASY Dilution for Real Time PCR 9160 lt SYBR Green gt Library Quantification Kit 638324 Cycleave Thermal Cycler Dice SYBR Molecular Probes Inc
18. 3 Plate Setup Define Targets and Samples Define Target Reporter FAM Quencher TAMRA Define Samples Assign Targets and Samples Define Targets and Samples Assign Targets nd Semples 9 P 9 WD instructons Dots me rowb to goana anf he samsiwt lo tetd nn Ne re acca cie Define Targets Define Sampies fe se http www takara bio coJjp 10 5500 4 Assign Targets and Samples Define Targets and Samples n 2 rwcw 1 ye oe wowi rrowi wor eoei wowi 10 10 1 1 01 01 091 01 AMLTAMAIRA Fa TAMRaA PATAMRVA PAM TAMRA TAMA Pau ladtea FaM iatRA AM AMMA iby A Lb A Libiiy 0 ubtey 0 Libresy C bta Tr0W 1 IroW 1 rs ros ee i mai mai on moe Fa TAMRA AMLTAMRA AMLTAMRA PAM TAA PAM TAMRA 5 Run Method PCR Reaction Volume per Well 25 ul Hold Cycle 1 95 1 3 step PCR Cycle 45 95 5 55
19. 10 72 C 60 6 START RUN PCR 7 R 0 99 Eff 70 Standard Curve ss 69 n a 209 Ne 7 Guary Ta wget 1 Siope 3 494 Cini ZL107 RS 1 HPE 93277 Analysis Quality Quality pM nM X Quality 1 000 http www takara bio coJp 11 5500 NTO FAM DNA FAM PCR
20. coJp 4 5500 IV 1 PCR 2 3 3 1 2 DNA 3 DNA
21. A DNA PCR DNA DNA DNA RNA DNA RNase H
22. EBNext Singleplex Oligos for llumina E73505 E7350L E7335S E7335L E75005 E7500L 0 1 4 5 7 10 11 12 13 14 15 16 17 8 18 NEBNext Multiplex Oligos for lllumina http wwwr takara bio coJp 5 5500 V gt 1 DNA Standard 10 pM 2 DNA A DNA DNA
23. Jp 8 5500 Plate Setup Plate Image Update n 2 3 Hold Cycle 1 95 1 3 step PCR Cycle 45 95 5 55C 10 72 60 4 Start Run Run File 5 FAM Result Analysis Analysis Data Standard Curve Fluorescence 2nd Derivative Max R2 0 99 Eff 70 Ctr ee Result Analysis Analysis Data Text Report Fluorescence Data Set of Each Well Data Set of Replicate Qty SDM pM nM X Qty SDM
24. nc 25 ul 7 Standard 10 pM 50 ul 2 X Cycleave Reaction Mixture Buffer dNTP mixture PCR Primer Probe Mix PCR EASY Dilution DNA DNA EASY Dilution for Real Time PCR 9160 ROX Reference Dye ROX Reference Dyell Life Technologies PCR ROX Reference Dye StepOnePlus Real Time PCR System Life Technologies ROX Reference Dye I Applied Biosystems 7500 7500 Fast Real Time PCR System Life Technologies Thermal Cycler Dice Real Time System TP900 TP960 Standard 10 pM DNA Cycleave qPCR
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