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1. O No Template Control O Ct PCR 100 0 1 uM 1 0 uM PCR O
2. RNA total RNA NucleoSpin RNA 740955 10 50 250 AGPC RNAiso Plus 9108 9109 http wwwr takara bio coJp 12 RRO86A B PCR 80 150 bp 300 bp 17 25 mer GC 40 60 45 55 Tm Forward primer Reverse primer Tm ce Tm OLIGO 1 63 68 C Primer3 2 60 65 C GC AT
3. 2 step PCR 95 5 95 5 60 30 64 C 30 O 3 step PCR 2 step PCR 3 step PCR 95 5 95C 5 l 55 C 30 55C 30 72C 30 95 95 C 5 60 30 60 1 72 1 RT PCR SYBR Green Assay 1 RT PCR 2 RT PCR 1 RT PCR 2 RT PCR 2 RT PCR
4. Random Primer Oligo dT Primer http wwwr takara bio coJp 5 RRO86A VI RNA VIII A RNA lt Thermal Cycler Dice Real Time System gt 1 1 gt 2 x One Step SYBR RT PCR Buffer 4 12 5 ul 1x PrimeScript 1step Enzyme Mix 2 1 0 ul PCR Forward Primer 10 uM 1 0 ul 04 uM PCR Reverse Primer 10 uM 1 0 ul 0 4 uM 1 total RNA 2 0 ul 2 RNase Free dH2O 7 5 ul Total 25 ul 1 primer 0 4 uM 0 2 1 0 UM 2 total RNA 10 pg 100 ng template 2 Thermal Cycler Dice Real Time System
5. 1 RT PCR One Step SYBR PrimeScript RT PCR Kit Perfect Real Time RR066A B One Step SYBR PrimeScript PLUS RT PCR Kit Perfect Real Time RR096A B http wwwr takara bio coJp 10 RRO86A Vil Human HPRT1 hypoxanthine phosphoribosyltransferase 1 Thermal Cycler Dice Real Time System Perfect Real Time total RNA Human HPRT1 1 Human Liver total RNA 6 4 pg 100 ng no template control 1 RT PCR 2 PCR y 4 6 8 9 ODA A ne NL RX nn RU CR RRRA TR NR EG MN FE MR HU amp Qn 2nd deri
6. http wwwr takara bio coJp 2 RRO86A 1 RT PCR PCR Specific Primer Reverse cDNA Specific primer Forward Reverse PCR Random Primer Oligo dT Primer 3 5 oo Specific Primer Reverse Synthesis of 1st strand CDNA with PrimeScript RTase Synthesis of 2nd strand cDNA with TaKaRa Ex Taq HS Specific Primer Forward 1 RT PCR http www takara bio coJp 3 RRO86A 3 DNA SYBR Green DNA DNA 1 F F
7. PCR Tm PCR 3 PCR 10 Pattern 1 Hold LV 42C 54 95 10 Pattern 2 PCR Cycle 40 95 C 5 60 30 Pattern 3 Dissociation TaKaRa Ex Tag HS 7ag PCR PCR PCR DISC 5 15 PCR 95 10 3 Thermal Cycler Di
8. 2 gt g F eM F gt gt F F ow 3 ll 100 50 ul 1 2 X One Step SYBR RT PCR Buffer 1 840 ul x3 2 PrimeScript 1step Enzyme Mix 2 2 200 ul 3 RNase Free dH20 1 25 ml X2 4 ROX Reference Dye 50Xconc 3 100 ul 5 ROX Reference Dye Il 50Xconc 3 100 ul x 1 dNTP Mixture Mg tE kU SYBR Green 2 PrimeScript RTase RNase Inhibitor TaKaRa Ex Taq HS 3 Life Technologies PCR ROX Reference Dye Applied Biosystems 7300 Real Time PCR System Life Technologies ABI PRISM 7000 7700 Life Technologies ROX Reference Dye Il e Applied Biosystems 7500 7500 Fast Real Time PCR System Life Technologies Thermal Cycler Dice Real Time System TP900 TP960 LightCycler Roche Diagnostics 1 PCR authorized instruments 2
9. Takaka Ex Taq Hs 7gg PCR PCR PCR 95 15 PCR 95 10 3 PCR http www takara bio co jp 9 RRO86A PCR RT PCR
10. 3 T C polypyrimidine A G polypurine 3 3 GC AT 3 G C 3 T 3 base 3 2 base BLAST 3 1 OLIGO Primer Analysis Software Molecular Biology Insight 2 Primer3 http www genome wi mit edu ftp distribution software 3 http www ncbi nlm nih gov BLAST Perfect RealTime RT PCR amp PCR SYBR Premix Ex Tagll Tli RNaseH Plus RR820A SYBR Premix Ex Taq Tli RNaseH Plus RR420A 2
11. http wwwr takara bio coJp 14 RRO86A NOTICE TO PURCHASER LIMITED LICENSE L11 SYBR Green This product is covered by the claims of U S Patent No 5 436 134 and 5 658 751 and their foreign counterpart patent claims Takara PCR products containing SYBR Green are sold under license from Molecular Probes Inc only for the usage in Real time PCR for internal research purpose These products are not to be used for the purpose such as providing medical diagnostic or any other testing analysis or screening services or providing clinical information or clinical analysis in return for compensations L15 Hot Start PCR Licensed under U S Patent No 5 338 671 and 5 587 287 and corresponding patents in other countries L46 SYBR Melting Curve Analysis The purchase of this product includes a limited non transferable license for all fields other than hu man or veterinary in vitro diagnostics under specific claims of U S Patent Nos 6 174 670 6 569 627 and 5 871 908 owned by the University of Utah Research Foundation or Evotec Biosystems GmbH and licensed to Idaho Technology Inc and Roche Diagnostics GmbH to use only the enclosed amount of product according to the specified protocols No right is conveyed expressly by implication or by estoppel to use any instrument or system under any claim of U S Patent Nos 6 174 670 6 569 627 and 5 871 908 other than for the amount
12. 3 PCR 4 PCR VIII B http www takara bio co jp 4 RRO86A Il 20 2 X One Step SYBR RT PCR Buffer 4 4 20 IV 1 2 V 1 RT PCR RNA RNA PrimeScript RTase PCR PCR TaKaRa Ex Taq HS 1 RT PCR SYBR Green 2X
13. RT PCR 1 RT PCR 1 SYBR Premix Ex Taq Il TIiRNaseH Plus SYBR Premix Ex Taq Tli RNaseH Plus 2 RT PCR http wwwr takara bio coJp 13 RRO86A IX X XI One Step SYBR PrimeScript RT PCR Kit Perfect Real Time RRO66A B One Step SYBR PrimeScript PLUS RT PCR Kit Perfect Real Time RR096A B One Step PrimeScript RT PCR Kit Perfect Real Time RRO64A B SYBR Premix Ex Tag ll Tli RNaseH Plus RR820S A B SYBR Premix Ex Tag Tli RNaseH Plus RR420S A B SYBR Premix DimerEraser Perfect Real Time RRO91A B Premix Ex Taq Probe qPCR RR390A B Thermal Cycler Dice Real Time System TP900 TP960 Thermal Cycler Dice Real Time System Single TP850 TP870 Thermal Cycler Dice Real Time System Lite
14. RNA 1 2 1 180 60 2 0 1 DEPC 37 12 DEPC 120 30 RNA RNA 180 60 DEPC 0 1 DEPC RNA RNA RT PCR RNA RNA
15. 1 2 3 4 5 10 Master Mix RNase Free dH2O Master Mix PrimeScript 1step Enzyme Mix 2 50 20 20 2 X One Step SYBR RT PCR Buffer 4 Vortex
16. TaKaRa Ex Taq HS PCR 1 PCR SYBR Green 1 PCR PCR DNA DNA DNA 3 1 100 PCR FARES TaKaRa Ex Taq HS 2 RT PCR RNA PCR RNA cDNA PCR RNA RT PCR RNA 1 RT PCR
17. TP700 TP760 NucleoSpin RNA 740955 10 50 250 RNAiso Plus 9108 9109 1 2005 3 4 PCR 120 126 2 2008 PCR 3 1 RT PCR 39 43
18. ROX Reference Dye ll 50x ABI PRISM 7000 7700 7300 Real Time PCR System ROX Reference Dye 50 X 4 2 PCR Tm PCR 3 PCR 10 lt ABI PRISM 7000 7700 7300 7500 Real Time PCR System gt Stage1 Stage2 Stages Stage 4 EN 1 2 7 eps Reps 1 Reps 1 Reps 40 Reps 1 42 C 5 95 10 Stage 3 PCR Reps 40 95 5 60 30 34 Stage 4 Dissociation Protocol 7700 30 7000 7300 31 7500 34 http www takara bio coJp 7 RRO86A lt 7500 Fast Real Time PCR System gt Holding Stage 42 5 95 10 Cycling Stage Number of Cycles 40 95 3
19. PCR Forward Primer 10 uM 0 8 ul 0 4 uM 1 PCR Reverse Primer 10 uM 0 8 ul 04 uM 1 total RNA 2 ul 2 RNase Free dH20 5 6 ul Total 20 ul 1 primer 0 4 UM 0 2 1 0 uM 2 total RNA 10 pg 100 ng template 2 PCR LightCycler PCR Tm PCR 3 PCR 10 Stage 1 42 5 20 95 10 20 C 4 Y 1 Stage 2 PCR 95 5 20 C 60 C 20 20 C 40 Stage 3 95 0 20 65 15 20 95 0 0 1 Stage2 PCR
20. 60 30 Melt Curve Stage TaKaRa Ex Taq HS 7ag PCR PCR PCR DISC 5 15 PCR 95 10 3 PCR http wwwr takara bio coJp 8 RRO86A lt LightCycler gt X LightCycler Roche Diagnostics 1 1 gt 2 X One Step SYBR RT PCR Buffer 4 10 ul 1x PrimeScript 1step Enzyme Mix 2 0 8 ul
21. ce Real Time System Thermal Cycler Dice Real Time System VII http www takara bio co jp 6 RRO86A lt ABI PRISM 7000 7700 U Applied Biosystems 7300 7500 7500 Fast Real Time PCR System gt 1 1 gt 2 X One Step SYBR RT PCR Buffer 4 10 ul 25 ul 1x PrimeScript 1step Enzyme Mix 2 0 8 ul 2 ul PCR Forward Primer 10 uM 0 8 ul 2 ul 04 uM PCR Reverse Primer 10 uM 0 8 ul 2 ul 04 uM 1 ROX Reference Dye or Dye II 50X 3 O04 ul 1 ul total RNA 2 ul 4 ul 2 RNase Free dH20 5 2 ul 14 ul Total 20 ul 2 50 pul 4 1 RK primer 0 4 UM 0 2 1 0 uM 2 50 ul total RNA 20 pg 200 ng template 3 ROX Reference Dye ll 50X ROX Reference Dye 50X 7500 7500 Fast Real Time PCR System
22. of product contained herein L52 Rox Reference Dye Research Field Use of this product is covered by one or more of the following US patents and corresponding patent claims outside the US 5 928 907 The purchase of this product includes a limited non transferable immunity from suit under the foregoing patent claims for using only this amount of product for the purchaser s own internal research No right under any other patent claim and no right to perform com mercial services of any kind including without limitation reporting the results of purchaser s activities for a fee or other commercial consideration is conveyed expressly by implication or by estoppel This product is for research use only Further information on purchasing licenses may be obtained by con tacting the Director of Licensing Applied Biosystems 850 Lincoln Centre Drive Foster City California 94404 USA M57 LA Technology This product is covered by the claims 6 16 outside the U S corresponding to the expired U S Patent No 5 436 149 http wwwr takara bio coJp 15 RRO86A TafdR4 Tel 077 543 6116 Fax 077 543 1977 http www takara bio co jp SII AA RNS AL v201307Da
23. vative DS Ct to a R2 0 999 on 73 i an Eff 97 5 Z wn m Be 3 jm 3 ees 3 tt 6 e 13 14 14 XO HH MN 10 HD MU MN Dw 9 43 1 we 1 ee een eae ei atl en 2nd derivative 3 total RNA 6 4 pg 100 ng http www takara bio co jp 11 RRO86A VIII Appendix A RNA RNA cDNA PCR cDNA RNA RNase RNase RNA RNase
24. wm RRO86A IdfaR4 One Step SYBR PrimeScript RT PCR Kit II Perfect Real Time v201307Da One Step SYBR PrimeScript RT PCR Kit II Perfect Real Time SYBR Greenl 1 1 RT PCR 2 RT PCR 1 PCR RNA RNA cDNA PrimeScript RTase PCR TaKaRa Ex Tag HS 1 RT PCR One Step SYBR PrimeScript RT PCR Kit Perfect Real Time RR066A Enzyme Mix Thermal C
25. ycler Dice Real Time System TP900 TP960 Thermal Cycler Dice Real Time System Single TP850 TP870 Thermal Cycler Dice Real Time System Lite TP700 TP760 ABI PRISM 7000 7700 Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Life Technologies LightCycler Roche Diagnostics t G Smart Cycler System Smart Cycler Il System Cepheid One Step SYBR PrimeScript RT PCR Kit Perfect Real Time RR066A One Step SYBR PrimeScript PLUS RT PCR Kit Perfect Real Time RR096A 1 Molecular Probes Inc SYBR Green SYBR Molecular Probes Inc 2 TaqMan 1 RT PCR One Step PrimeScript RT PCR Kit Perfect Real Time TaqMan Roche Diagnostics The 5 nuclease process is covered by patents owned by Roche Molecu lar Systems Inc and F Hoffmann La Roche Ltd Purchase of the product does not provide a license to use this patented technology One Step SYBR PrimeScript RT PCR Kit II Perfect Real Time PrimeScript RTase RNA cDNA
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