One Step PrimeScript™ RT-PCR Kit (Perfect Real Time)
Contents
1. RNA RNA RT PCR RNA RNA RNA total RNA NucleoSpin RNA 740955 10 50 250 AGPC RNAiso Plus 9108 9109 http wwwr takara bio coJp 14 RR064A IX Premix Ex Tag Probe qPCR RR390A B One Step SYBR PrimeScript RT PCR Kit Perfect Real Time RR066A B One Step SYBR PrimeScript RT PCR Kit Il Perfect Real Time RR086A B One Step SYBR PrimeScript PLUS RT PCR Kit Perfect Real Time RR096A B SYBR Premix Ex Tag 11 Tli RNaseH Plus RR820S A B SYBR Premix Ex Tag Tli RNaseH Plus RR420S A B SYBR Premix DimerEraser Perfect Real Time RR02. 20 20 2 x One Step RT PCR Buffer Vortex Random Primer Oligo dT Primer http wwwr takara bio coJp 5 RR064A VI RNA VIII RNA lt Thermal Cycler Dice Real Time System Lite gt 1 1 gt 2 x One Step RT PCR Buffer Ill 12 5 ul 1 Xx TakaRa Ex Tag HS 5 U ul 0 5 ul PrimeScr
3. Fatlern 2 Step PC egrnent Pattern 1 Hold 42 5 95 10 Pattern 2 PCR Temperature deg Cycle 40 Hold Time mm s 95 5 Data Colleciion 60 30 7aXaRa Ex Tag HS g PCR PCR PCR 95 5 15 PCR 95 10 3 http www takara bio coJp 7 RR064A lt Smart Cycler II System gt 1 1 gt 2 x One Step RT PC
4. Stade 1 Stage 2 Sta e 3 Repeat 1 times Repeat 40 times ol d 2 Temperature Cycle 2 Temperature Cycle Si 42C 5 Degi Sec Temp Secs Qptics DegiSec Temp Becs optics NA 42 0 300 Of NA 95 0 5 ff 95 10 NA 95 0 10 Of NA nn 20 lon Stage 2 PCR Repeat 40 95 C 5 Advance to Next Stage _ Advance to Next Stage 60 20 7aXKaRa Ex Tag HS g PCR PCR PCR 95 5 15 PCR 95 10 3 Smart Cycler System http www takara bio coJp 8 RR064A Ligh
5. 100 Hot Start PCR 7aKaRa Ex Tag HS 2 RT PCR RNA PCR RNA cDNA PCR RNA RT PCR RNA One Step RT PCR One Step RT PCR PCR Specific Primer Reverse cDNA Specific Primer Forward Reverse PCR Random Primer Oligo dT Primer http wwwr takara bio coJp 2 RR064A Specific Primer Reverse Synthesis of 1st strand cDNA with PrimeScript RTase 1s S
6. PCR 95 10 3 http www takara bio coJp 11 RR064A lt PCR gt PCR amn PCR 95 3 5 OFF 300 bp 95 3 5 sg4c 20 30 on 56 64 31 34 Life Technologies 30 7300 31 7500 34 http wwwr takara bio coJp 12 RR064A VII Mouse Actb Thermal Cycler Dice Real Time System 1
7. TakaRa Ex 7ag9 Thermal Cycler Dice SYBR Molecular Probes Inc TaqMan Roche Diagnostics GmbH PrimeScript Premix Ex 7g9 DimerEraser http wwwr takara bio coJp 15 RR064A TafdR4 Tel 077 543 6116 Fax 077 543 1977 http www takara bio coJp v201406Da
8. PCR Cycim f Progrom Dan COyctey Taagos Stage 1 ne 42 5 20C En 95 10 20 9 1 Stage 2 PCR 95 5 20 60 20 20 40 Stage 2 PCR 7aXaRa Ex Tag HS g PCR PCR PCR 95 5 15 PCR 95C 10 3 LightCycler http www takara bio coJp 9 RR064A lt Applied Biosystems 7300 7500 Real Time PCR System
9. RNase RNA RNase RNA 1 2 1 180 60 2 0 1 DEPC 37 12 DEPC 120 30 RNA RNA 180 60 DEPC 0 1 DEPC
10. gt 1 1 gt 2 x One Step RT PCR Buffer 10 ul 25 ul 1 Xx TakaRa Ex Tag HS 5 U ul 0 4 ul 1 Al PrimeScript RT enzyme Mixll 0 4 ul 1 ul PCR Forward Primer 10 uM 0 4 ul 1 ul 0 2 uM 1 PCR Reverse Primer 10 uM 0 4 gl 1 HI 0 2 uM 1 TaqMan 0 8 wl 2 ul 2 ROX Reference Dye or Dye ll 50X 3 0 4 HI 1 ul total RNA 2 ul 4 ul 4 RNase Free dH2O 5 2 ul 14 ul Total 20 ul 5 50 pul 5 1 0 2 uM 0 1 1 0 uM 2 PCR 3 ROX Reference Dye ll 50X ROX Reference Dye 50X Applied Biosystems 7500 ReaLTime PCR System ROX Reference Dye Il 50X Applied Biosystems 7300 Rea Time PCR System ROX Reference Dye 50 Xx 4 50
11. Mouse Liver total RNA 1 pg 100 ng 1 RT PCR Mouse Actb TaqMan Gene Expression Assays Life Technologies TaqMan 2 PCR Crossinq Point CP w ta E RSq 0 999 Ef 96 4 cs gt 3 2nd Derivative Maximum SDM reeves Ord wwwrel 3 total RNA 1 pg 100 ng http www takara bio coJp 13 RR064A VII Appendix RNA RNA cDNA PCR cDNA RNA RNase
12. 91A B Thermal Cycler Dice Real Time System TP900 TP960 Thermal Cycler Dice Real Time System Lite TP700 TP760 Smart Cycler ll System SC200N SC210N NucleoSpin RNA 740955 10 50 250 RNAiso Plus 9108 9109 X 1 2005 3 4 PCR 120 126 2 2008 PCR 3 1 RT PCR 39 43 XI
13. A 2 PCR Hot Start 7aKaRa Ex 7g9 HS PCR 2 V 1 10 Master Mix RNase Free dH2O Master Mix TakKaRa Ex Tag HS PrimeScript RT enzyme Mix I 50
14. R Buffer 12 5 ul 1 TakaRa Ex Tag HS 5 U uD 0 5 ul PrimeScript RT enzyme Mixl 0 5 ul PCR Forward Primer 10 uM 0 5 wl 0 2 uM 1 PCR Reverse Primer 10 uM 0 5 ul 0 2 uM 1 TaqMan 1 ul 2 total RNA 2 ul 3 RNase Free dH20 7 5 ul Total 25 ul 1 0 2 uM 0 1 1 0 uM 2 PCR Smart Cycler ll System 0 1 0 5 uM 3 totalRNA 10 pg 100 ng template 2 Smart Cycler Smart Cycler PCR 12 PCR
15. che Diagnostics 1 TaqMan Roche Diagnostics The 5 nuclease process is covered by patents owned by Roche Diagnostics Inc and F Hoffmann La Roche Ltd Purchase of the product does not provide a license to use this patented technology 2 SYBR Green 1 RT PCR One Step SYBR PrimeScript PLUS RT PCR Kit Perfect Real Time RR096A B One Step SYBR PrimeScript RT PCR Kit Perfect Real Time RR066A B One Step SYBR PrimeScript RT PCR Kit II Perfect Real Time RR086A B One Step PrimeScript RT PCR Kit Perfect Real Time PrimeScript RTase RNA cDNA 7aKaRa Ex Tag HS PCR 1 PCR TaqMan 1 PCR PCR DNA DNA DNA 3 1
16. ipt RT enzyme Mix ll 0 5 ul PCR Forward Primer 10 uM 0 5 ul 0 2 uM 1 PCR Reverse Primer 10 uM 0 5 ul 0 2 uM 1 TaqMan 1 ul 2 total RNA 2 ul 3 RNase Free dH20 7 5 ul Total 25 ul 1 0 2 uM 0 1 1 0 uM 2 PCR Thermal Cycler Dice Real Time System 0 1 0 5 uM 3 totalRNA10 pg 100 ng template http wwwr takara bio coJp 6 RR064A 2 Thermal Cycler Dice Real Time System PCR 12 PCR
17. rimeScript RT enzyme Mix II 2 100 wl 4 RNase Free dH2O 1 25 ml X2 5 ROX Reference Dye 50Xconc 3 100 HI 6 ROX Reference Dye ll 50Xconc 3 100 HI 1 dNTP Mixture Mg 2 RNase Inhibitor 3 Life Technologies PCR ROX Reference Dye Applied Biosystems 7300 RealTime PCR System Life Technologies ROX Reference Dye ll Applied Biosystems 7500 Real Time PCR System Life Technologies Thermal Cycler Dice Real Time System hermal Cycler Dice Real Time System Lite Smart Cycler System Cepheid LightCycler Roche Diagnostics http www takara bio coJp 4 RR064A PCR authorized instruments PCR TaqMan licensed probe lI 20 IV 1 One Step RT PCR RNA RN
18. tCycler gt 1 gt 2 x One Step RT PCR Buffer Ill 10 ul 1 Xx TakaRa Ex Tag HS 5 U uD 04 HI PrimeScript RT enzyme Mix 1l 0 4 HI PCR Forward Primer 10 uM 0 4 wl 02 uM 1 PCR Reverse Primer 10 uM 0 4 HI 02 uM 1 TaqMan 0 8 ul 2 total RNA 2 ul 3 RNase Free dH2O 5 6 ul Total 20 MI 1 0 2 uM 0 1 1 0 uM 2 PCR 3 totalRNA 10 pg 100 ng template 2 PCR LightCycler PCR OR 12
19. total RNA 20 pg 200 ng template 5 http wwwr takara bio coJp 10 RR064A 2 PCR 12 PCR Stage 1 Stage 2 Stage 3 Reps 1 Reps 1 Reps Stage 1 2 Reps 1 42 5 95 10 Stage 3 PCR Reps 40 95 5 60 31 or 34 7300 31 7500 34 7g42 g Ex T7ag Hs Taq PCR PCR PCR 95 5 15
20. ws r RRO64A IdfaR4 One Step PrimeScript RT PCR Kit Perfect Real Time vV201406Da One Step PrimeScript RT PCR Kit Perfect Real Time TaqMan 1 RT PCR 2 RT PCR 1 PCR RNA RNA cDNA PrimeScript RTase PCR 7g42 Ex Tag HS 1 RT PCR 1 RT PCR Thermal Cycler Dice Real Time System TP900 TP960 Thermal Cycler Dice Real Time System Lite TP700 TP760 Smart Cycler System Smart Cycler II System Cepheid Applied Biosystems 7300 7500 Real Time PCR System Life Technologies LightCycler Ro
21. ynthesis of 2nd strand cDNA with TaKaRa Ex Tag HS Specific Primer Forward 1 RT PCR http www takara bio co jp 3 RR064A 3 TaqMan 5 FAM 3 TAMRA TaqMan DNA 7aqg DNA 5 3 exonuclease TaqMan 1 RE 2 1 3 ll 100 50 ul 1 2 Xx One Step RT PCR Bufferlll 1 840 ulXx3 2 TakaRa Ex Tag HS 5 U ul 100 wl 3 P
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