Premix Ex Taq ™ （Perfect Real Time）
Contents
1. TakaRa Ex 139 Thermal Cycler Dice SYBR Molecular Probes Inc TaqMan Roche Diagnostics GmbH e Ex Tag PrimeScript http wwwr takara bio coJp 18 RR039A http www takara bio cojp 19 RR039A TafdR4 Tel 077 543 6116 Fax 077 543 1977 http www takara bio coJp v201407Da2. Premix Ex 7gg 2 Xx conc 20 2 3 TaqMan 4 http wwwr takara bio coJp 5 RR039A VI lt Thermal Cycler Dice Real Time System 7e gt Thermal Cycler Dice Real Time System 1 PCR 1 gt Premix Ex 729 2 X PCR Forward Primer 10 uM PCR Reverse Primer 10 uM TaqMan template dH20
3. http wwwr takara bio coJp 2 RR039A 2 TaqMan 5 FAM 3 TAMRA TaqMan DNA 7ag DNA 5 3 exonuclease TaqMan 1 2 fp TS 3 al nm http www takara bio coJp 3 RR039A 200 50 Il Premix Ex T
4. PCR 95 30 3 http wwwr takara bio coJp 11 RR039A lt lt LightCycler gt LightCycler Roche Diagnostics 1 PCR 1 gt Premix Ex 729 2 X 10 wl 1Xx PCR Forward Primer 10 uM 0 4 wl 0 2 uM 1 PCR Reverse Primer 10 uM 0 4 wl 0 2 uM 1 TaqMan 0 8 ul 2 template 2 ul 3 dH20 6 4 ul Total 20 ul 1 0 2 uM 0 1 1 0 uM 2 PCR
5. http www takara bio coJp 7 RR039A lt Smart Cycler II System gt Smart CyclerSystem 1 PCR 1 gt Premix Ex 729 2 X PCR Forward Primer 10 uM PCR Reverse Primer 10 uM TaqMan template dH20 Total 1x 0 2 uM 1 0 2 uM 1 2 3 1 0 2 uM 0 1 1 0 uM 2 PCR Smart Cycler System Smart Cycler II System 0 1 0 5 uM 3 template DNA template 100 ng
6. 20 20 4 6 IV PCR 2 x conc 3 PCR Hot Start 7aKaRa Ex Tag HS PCR IN http www takara bio co jp 4 RR039A V 1
7. 3 template DNA template 100 ng RT PCR cDNA RT template PCR 10 http wwwr takara bio coJp 12 RR039A PCR LightCycler PCR PCR PCR 15 PCR Stage 2 PCR Program Data Analysis Mode 40 Quantification ET erature Targets Target Temperature C Incubation Time hrs min sec PCR Temperature Transition Rate CC sec Secondary Target Temperature C Stage 1 Step Size C 9 Stap Delav GyElBa 95 30 20 Acquisition Mode 1 js oo
8. 0 1 1 0 uM 2 PCR 3 ROX Reference Dye II 50X ROX Reference Dye 50X 7500 Real Time PCR System 7500 Fast ReaHTime PCR System ROX Reference Dye II 50X 7300 Real Time PCR System ROX Refer ence Dye 50 Xx 4 template 20 ul DNA template 100 ng RT PCR cDNA RT template PCR 10 5 http wwwr takara bio coJp 10 RR039A 2 PCR
9. PCR gt 95 30 DNA 95 C 30 OFF 95 1 2 2 PCR 2 PCR 30 45 PCR 95 3 5 OFF 300 bp 95 3 5 60 20 56 64 20 30 0 4 3134 1 Life Technologies 30 Applied Biosystems 7300 31 Applied Biosystems 7500 34
10. RT PCR cDNA RT template PCR 10 http wwwr takara bio coJp 8 RR039A 2 Smart Cycler Smart Cycler PCR PCR PCR 15 PCR Stage 1 Stade 2 Hold Repeat 40 times PCR Temp gecs Optics 2 TemperatureCycle Stage 1 95 0 30 Of DegBec Temp Becs optics Hold NA 5 5 Off 95C 30 NA 600 120 Ion Stage 2 PCR Repeat 40 times 95C 5 60 20 _ Adtyance to Next Stage 7aKaRa Ex Tag HS 729 PCR PCR
11. http wwwr takara bio coJp 15 RR039A VII RT PCR 2 RT PCR PrimeScript RT reagent Kit Perfect Real Time RR037A TaqMan 1 PrimeScript RT reagent Kit 1 gt 5 X PrimeScript Buffer for Real Time 2 HI 1 PrimeScript RT Enzyme Mix 0 5 ul Oligo dT Primer 50 uM 1 0 5 ul 25 pmol Random 6 mers 100 uM 1 2 ul 200 pmol total RNA RNase Free dH20 Total 10 ul 2 1 Oligo dT Primer Random 6 mers mRNA cDNA Gene Specifc Primer Oligo dT Primer 50 uM 0 5 ul 25 pmol Random 6 mers 100 uM 2 HI 200 pmol Gene Specific Primer 2 uM 0 5 ul 1 pmol 2 10 ul
12. DNA 74 30 10 nmol 1U 25mM TAPS pH9 3 25 50mM KCI 2mM MgCl2 0 1mM DTT 200 uM dATP dGTP dCTP 100 uM BEHI dTTP 0 25mg ml DNA 1 10U 0 6 ug A Hind ll 74 1 DNA 2 10 U 0 6 ug supercoiled pBR322 DNA 74 1 DNA 3 10 U 0 6 ug ADNA 74C 1 DNA http www takara bio coJp 17 RR039A IX X Xl Premix Ex Tag Probe qPCR RR390A B PrimeScript RT reagent Kit Perfect Real Time RR037A B PrimeScript RT reagent Kit with gDNA Eraser Perfect Real Time RR047A B PrimeScript RT Master Mix Perfect Real Time
13. The 5 nuclease process is covered by patents owned by Roche Molecular Systems Inc and F Hoffmann La Roche Ltd Purchase of the product does not provide a license to use this patented technology 2 SYBR Green SYBR Green SYBR Premix Ex Tag TIi RNaseH Plus RR420A SYBR Premix Ex Tag 1 Ti RNaseH Plus RR820A 7gg g Ex 7g9 HS PCR PCR TaqMan 1 PCR PCR DNA DNA DNA 3 1 100 Hot Start PCR 7aKaRa Ex 7gg HS
14. Total 1x 0 2 uM 1 0 2 uM 1 2 3 1 0 2 uM 0 1 1 0 uM 2 PCR Thermal Cycler Dice Real Time Sys tem 7 7e 0 1 0 5 uM 3 template DNA template 100 ng RT PCR cDNA RT template PCR 10 http wwwr takara bio coJp 6 RR039A 2 PCR PCR
15. 1 ug total RNA 2 37C 15 3 85 5 4C 3 Gene Specifc Primer 42 15 PCR 50 3 PCR 6 VI PCR http wwwr takara bio coJp 16 RR039A Applied Biosystems 7500 Real Time PCR System RT PCR Mouse Gapd mRNA TaqgMan Gene Expression Assays Life Technologies TaqMan total RNA 1 pg 100 ng cDNA Negative Control dH20 VIlI Smart Cycler System ADNA PCR 300 bp 7g gg Ex Tag Hs
16. PCR 95 5 15 PCR 95 30 3 Smart Cycler System http www takara bio cojp 9 RR039A lt Applied Biosystems 7300 7500 7500 Fast Real Time PCR System gt 1 PCR 1 gt Premix Ex 729 2 X 10 wl 25 ul 1 X PCR Forward Primer 10 uM 0 4 wl 1 ul 0 2 uM 1 PCR Reverse Primer 10 uM 0 4 wl 1 ul 0 2 uM 1 TaqMan 0 8 HI 2 HI 2 ROX Reference Dye or Dyell 50 Xx 3 A 0 template 2 HI 4 HI 4 dH20 6 ul 16 ul Total 20 ul 5 50 ul 1 0 2 uM
17. oo NONE Stage 2 PCR 95 5 20 DD 40 7aKaRa Ex Tag HS 729 PCR PCR i PCR 95 C 5 15 PCR 95 30 LightCycler http www takara bio coJp 13 RR039A lt Mx3000P gt Mx3000P Agilent Technologies 1 PCR 1 gt Premix Ex 729 2 X 12 5 ul 1 PCR Forward Primer 10 uM
18. PCR PCR 15 PCR lt Applied Biosystems 7300 7500 Rea Time PCR System gt Thermal Cycler Protocol Thermal Profile Auto Increment Raml PCR Stage 1 Stage2 Stage 1 1 Reps 1 Reps 3 Reps 1 95C 30 1 Stage 2 PCR Reps 40 95 5 60 31or34 7300 31 7500 34 lt Applied Biosystems 7500 Fast Real Time PCR System gt PCR Holding Stage Reps 1 95 30 Cycling Stage Number of Cycles 40 95 3 60 30 7aKaRa Ex Tag HS Tag PCR PCR PCR 95 5 15
19. PCR PCR 15 PCR PCR Segment 1 95 30 1 Segment 2 PCR 95 5 60 20 40 7aKaRa Ex Tag HS 729 PCR PCR PCR 95 5 15 PCR 95 30 http wwwr takara bio coJp 14 RR039A 3 Mx3000P
20. RR036A B One Step PrimeScript RT PCR Kit Perfect Real Time RR064A B SYBR Premix Ex Tag TE RNaseH Plus RR820S A B SYBR Premix Ex Tag Tli RNaseH Plus RR420S A B One Step SYBR PrimeScript RT PCR Kit Il Perfect Real Time RR086A B Thermal Cycler Dice Real Time System TP900 TP960 Thermal Cycler Dice Real Time System Lite TP700 TP760 Smart Cycler ll System SC200N SC210N 1 2005 3 4 PCR 120 126 2 2008 PCR 3 1 RT PCR 39 43
21. 20 30 30 15 PCR Fattern 2 Step PCR Segment 1 2 PCR Hold Cycle 1 95 30 2 Step PCR Cycle 40 95 5 60 30 40 Temperature deg 60 0 HoldTime mm ss Data Collection 7aKaRa Ex Tag HS 7g9 PCR PCR PCR 95 5 15 PCR 95 30 3 Thermal Cycler Dice Real Time System 7e
22. 0 5 ul 0 2 uM 1 PCR Reverse Primer 10 uM 0 5 ul 0 2 uM 1 TaqMan 1 ul 2 ROX Reference Il 50 Xx 3 0 5 ul 1 X template 2 HI 4 dH20 8 0 ul Total 25 ul 1 0 2 uM 0 1 1 0 uM 2 PCR 3 ROX Reference Dye Il 50X ROX Reference Dye 50 Xx Mx3000P 4 template DNA template 100 ng RT PCR cDNA RT template PCR 109 2 PCR
23. ag Perfect Real Time 2Xconc 1 1 mlX5 ROX Reference Dye 50 X conc 2 200 wl ROX Reference Dye ll 50Xconc 2 200 ul 1 TakKaRa Ex Tag HS dNTP Mixture Mg 2 Life Technologies PCR ROX Reference Dye Applied Biosystems 7300 RealTime PCR System Life Technologies ROX Reference Dye ll Applied Biosystems 7500 7500 Fast Rea Time PCR System Life Technologies Mx3000P Agilent Technologies Thermal Cycler Dice Real Time System TP900 TP960 Thermal Cycler Dice Real Time Lite TP700 TP760 Smart Cycler System Cepheid LightCycler Roche Diagnostics 1 PCR authorized instruments 2 3 PCR 4 TaqMan licensed probe 5 6 II 4 6
24. ma RRO39A IfHa Premix Ex Tag Perfect Real Time v201407Da Premix Ex Tag Perfect Real Time TaqMan 1 PCR 2 2 X 7gg 7aKaRa Ex Tag Hs PCR PCR PCR PCR Thermal Cycler Dice Real Time System TP900 TP960 Thermal Cycler Dice Real Time 7ze TP700 TP760 Smart Cycler System Smart Cycler ll System Cepheid Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Life Technologies LightCycler Roche Diagnostics Mx3000P Agilent Technologies 1 TaqMan Roche Diagnostics
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