SYBR® Premix Ex Taq ™ GC （Perfect Real Time）
Contents
1. SYBR Molecular Probe Inc 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 forthe usage in Realtime PCR for internal research purpose These products are not to be used forthe Purpose such as providing medical diagnostic or any othertesting analysis or screening services or providind clinical information or clinical analysis in return for compensations L15 Hot Start PCR Licensed2. RT PCR amp Perfect Real Time http wwwr takara bio coJp realtime RefSeq RT PCR SYBRS Green RT PCR RT PCR PCR P 5 10 17 25 mer 40 60 45 55 1 Tm Forward primer Reverse primer Tm Tm OLIGO 2 63
3. http wwwr takara bio coJp 4 RR071A VI lt Thermal Cycler Dice Real Time System gt Thermal Cycler Dice Real Time System 1 PCR 1 gt SYBR Premix Ex Tag GC 2X 12 5 ul 1 PCR Forward Primer 10 uM 0 5 ul 0 2 KUM 1 PCR Reverse Primer 10 uM 0 5 ul 0 2 uM 1 template lt 100 ng 2 ul 2 dH2O 9 5 ul Total 25 ul 3 1 primer 0 2 uM 0 1 1 0 uM 2 template DNA template 100 ng RT PCR cDNA RT template PCR 10 3
4. TEL 03 3271 8553 TEL 077 565 6969 http wwwr takara bio coJp 12 RR071A 2 RT PCR RT PrimeScript RT reagent Kit Perfect Real Time RR037A B PrimeScript RT Master Mix Perfect Real Time RR036A B PrimeScript RT reagent Kit with gDNA Eraser Perfect Real Time RR047A B PrimeScript RT reagent Kit Perfect Real Time RR037A 1 RNA RNA 1 gt 5 X PrimeScript Buffer for Real Time 2 HI 1 PrimeScript RT Enzyme Mix 0 5 ul Oligo dT Primer 50 1 0 5 ul 25 pmol Random 6 mers 100 uM 0 5 ul 50 pmol total RNA RNase Free dH20 Total 10 ul 2 1 Oligo dT Primer Random
5. ROX Reference Dye StepOnePlus Real Time PCR System Life Technologies ROX Reference Dye Applied Biosystems 7500 7500 Fast Real Time PCR System Life Technologies Thermal Cycler Dice Real Time System TP900 TP960 Smart Cycler System Smart Cycler System Cepheid LightCycler LightCycler 480 System Roche Diagnostics PCR authorized instruments PCR CO PCR VII 1 RT PCR amp Perfect Real Time
6. 3 step PCR PCR step PCR gt 95 5 _ gt 95 C 5 10 10 55 C 30 55C 30 95C 5 95 5 10 10 60C 303 60 1 72 30 72 1 95 30 DNA 95 1 2 2 http wwwr takara bio coJp 11 RR071A Appendix 1 PCR
7. 2 DNA SYBR Green DNA DNA 1 F F 3 fn SL SE SD BOO OO FL STAAL http www takara bio coJp 2 RR071A 200 50 ul IV SYBR Premix Ex Tag GC Perfect Real Time 2Xconc 7 1mlX5 ROX Reference Dye 50 X conc 2 200 ul ROX Reference Dyell 50Xconc lt 200 ul 1 TakKaRa Ex Tag HS dNTP Mixture Mg lt SYBR 8 14 2 Life Technologies PCR
8. 20 ul DNA template 100 ng RT PCR cDNA RT template PCR 10 4 http wwwr takara bio coJp 6 RR071A 2 PCR PCR PCR PCR 11 PCR lt Applied Biosystems 7500 RealTime PCR System StepOnePlus gt Thermal Cydier Protocol Thermal Profile aut Increment Ramp Rale Stage 1 Stage 2 Stage 3 Reps 1 Reps 40 Reps 1 lt 7500 Fast RealTime PCR System gt PCR Stage 1 Reps 1 95 30 Stage 2 PCR Reps 40 95 5 60 30or34 Stage 3 Melt Curve StepOnePlus 30 7500 34 toe te 2
9. http www takara bio coJp 5 RR071A lt Applied Biosystems 7500 7500 Fast Real Time PCR System StepOnePlus Real Time PCR System gt 1 PCR 1 gt SYBR Premix Ex Tag GC 2X 10 ul 25 ul 1 PCR Forward Primer 10 uM 0 4 ul 1 ul 0 2 uM 1 PCR Reverse Primer 10 uM 0 4 ul 1 ul 0 2 uM 1 ROX Reference Dye 50 X or Dyell 50524 2 0 4 gl 1 template 2 ul 4 ul 83 dH2O 6 8 ul 18 wl Total 20 ul 4 50 ul 4 1 primer 0 2 uM 0 1 1 0 uM 2 ROX Reference Dye ll 50X ROX Reference Dye 502 7500 7500 Fast Real Time PCR System ROX Reference Dyell 50X StepOnePlus ROX Reference Dye 5054 3 template
10. 74 30 10 nmol 1U 25 mM TAPS pH9 3 25 50 mM KCI 2mM MgCl2 0 1 mM 200 uM dATP dGTP dTTP 100 uM BEH dTTP 0 25 mg ml DNA 1 10 U 0 6 ug A HMnd 1 74 1 DNA 2 10 U 0 6 ug supercoiled pBR322 DNA 74 1 DNA 3 10 U 0 6 DNA 74 1 DNA IX X SYBR Premix Ex Tag Tli RNaseH Plus RR4205S A B SYBR Premix Ex Tag Ti RNaseH Plus RR820S A B SYBRS Premix DimerEraser Perfect Real Time RR091A B PrimeScript RT reagent Kit Perfect Real Time RR037A B PrimeScript RT Master Mix Perfect Real Time RR036A B PrimeScript RT reagent Kit with gDNA Eraser Perfect Real Time RR047A B
11. 2 template DNA template 100 ng RT PCR cDNA RT template PCR 1096 2 Smart Cyclers Smart Cycler PCR PCR PCR PCR 11 PCR PCR Stage 2 Repeat 40 times Optics Of Stage 3 End opticd Degrsec Stage 1 Hold 95 30 Degiecl Temp 665 optics BOO 380 02 Stade 2 PCR IT Repeat 40 times 95C 10 60 30 Stage 3 Melt Curve 7aKafa
12. http wwwr takara bio coJp realtime 4 6 6 80 20 4 6 1 PCR 2 SYBR Green 2xconc PCR 3 PCR Hot Start PCR 7aKaRa Ex HS 60 7096
13. 200 bp http wwwr takara bio coJp 3 RR071A V 1 SYBR Premix Ex Tag GC 2Xconc 80 2 3 SYBR Green 4
14. 11 PCR LightCycler gt PCR Yes Stage 1 95 30 20 Seccedoyy Tergat Tomperstare 1 Sre Dolev orcies ce Stage 2 PCR 95 10 20 60 C 30 20 ww 40 Stage 3 95 0 20 65 15 20 95 0 0 1 Stage 2 PCR http www takara bio coJp 8 RR071A lt LightCycler 480 gt PCR Denature 95 C 30 Ramp Rate 4 4 sec 1 PCR Analysis Mode Quantifcation 95 C 5 Ramp Rate 4 4 sec 60 30 Ramp Rate 2 2 sec Acquisition Mode Single 40 Melting Analysis Mode Melting Curves 95 C 5 Ramp Rate 4 4 sec 60 1 Ramp Rate 2 2 sec 95 Ramp Rate 0 11 sec Acquisition Mode Continuous Acquisitions 5 Der 1 Cooling 50 30 Ramp Rate 2 2 sec 1 Ex 7ag 7ag
15. PCR PCR PCR 95 5 15 PCR 95 30 3 PCR http wwwr takara bio coJp 9 RR071A lt Smart Cyclere System gt 1 PCR 1 gt SYBR Premix Ex Tag GC 2X 12 5 ul 1 PCR Forward Primer 10 uM 0 5 ul 0 2 uM 1 PCR Reverse Primer 10 uM 0 5 ul 0 2 uM 1 template lt 100 ng 2 HI 2 dH2O 9 5 ul Total 25 ul 1 primer 0 2 uM 0 1 1 0 uM
16. AmDsfa Tn Coe Hokaro 23 LU 4 25 a Cu Ccfmrunot ef ed 1 10 HC n 3 00 0211 i 5 5 SWo 4 Holding Stage Reps 1 95 30 Cycling Stage Reps 40 95 10 60 30 Melt Curve Stage 7aKaRa Ex Tag HS 7ag PCR PCR PCR 95 5 15 PCR 95 C 30 3 http www takara bio coJp 7 PCR RR071A lt LightCycler LightCycler 480 gt Roche Diagno
17. TafdR4 Tel 077 543 6116 Fax 077 543 1977 http www takara bio coJp v201308Da
18. 25 2 PCR PCR PCR PCR 11 PCR Hold Cycle 1 95 30 60 30 Dissociation Ex 799 HS Taq PCR PCR PCR 95 5 15 PCR 95 30 3 Thermal Cycler Dice Real Time System
19. 68 Primer3 3 60 65 C GC AT 3 T C polypyrimidine A G polypurine 3 3 GC AT 3 G C 3 T 3 base 3 2 base BLAST 4 1 GC 45 659 50 60 2 OLIGO Primer Analysis Software Molecular Biology Insights 3 Primer3 http www genome wimit edu ftp distribution software 4 http www ncbi nlIm nih gov BLAST Perfect Real Time
20. 2 Smart Cyclere System 2 Cepheid Applied Biosystems 7500 7500 Fast Rea Time PCR System StepOnePlus Real Time PCR System Life Technologies LightCycler LightCycler 480 System Roche Diagnostics 1 Molecular Probes Inc SYBR Green 1 SYBR Molecular Probes Inc 2 Smart Cyclere Cepheid 7aKaRa Ex 1 Hs PCR PCR SYBR Greenl 1 PCR PCR DNA DNA DNA 3 1 100 Hot Start PCR 7aKaRa Ex 7gg HS
21. 6 mers mRNA cDNA Gene Specific Primer Oligo dT Primer 50 uM 0 5 ul 25 pmol Random6 mers 100 uM 0 5 ul 50 pmol Gene Specifc Primer 2 uM 0 5 ul 1 pmol 2 10 ul 500 ng total RNA 2 37C 15 3 85 5 4 3 Gene Specific Primer 42 15 PCR 50 http wwwr takara bio coJp 13 RR071A 3 PCR Thermal Cycler Dice Real Time System o 22 5 24 1 gt SYB
22. 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 felds other than human 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 accordind to the specifed 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 of product contained herein L52 Rox Reference Dye Research Field Purchase of this product includes an immunity from suit under patents specified in the product insert to use only the amount purchased for the purchaser s own internal research No other patent rights are conveyed expressly by implication or by estoppel Further information on purchasind licenses may be obtained by contacting 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
23. Ex 799 HS 7ag PCR PCR PCR 95 5 15 PCR 95 30 3 Smart Cycler System http wwwr takara bio coJp 10 RR071A PCR PCR PCR 95C 5 10 95C 5 10 60 30 64 30
24. R Premix Ex Tag GC 2X 12 5 ul 1 X PCR Forward Primer 10 uM 0 5 ul 0 2 uM PCR Reverse Primer 10 uM 0 5 ul 0 2 uM dH2O x MI Total 22 5 24 ul 4 1 2 5 ul PCR 2 5 5 fuoneicwece Pnmay Cu w 3 3 4 56 7 0 91011 1313 141516 17 10 10 30 31 22 23 24 25 26 27 20233203 22317247 INH IEA Cycle 11755 0 00929294 Temoe me 45 2 R2 0 999 Eff 97 9 RT PCR Human SYNGR3 mRNA GC 63 5 total RNA5 pg 50 ng cDNA Negative Control dH20 http www takara bio coJp 14 RR071A Thermal Cycler Dice Real Time System cDNA HL60 cDNA PCR 98 bp 87 bp SYBRe Green 7akKaRa Ex Tag HS DNA
25. Thermal Cycler Dice Real Time System TP900 TP960 Thermal Cycler Dice Real Time System Single TP850 TP870 Thermal Cycler Dice Real Time System Lite TP700 TP760 Smart Cycler System SC200N SC210N Perfect Real Time http www takara bio coJp realtime RefSeg RT PCR SYBR Green RT PCR 1 2005 3 4 PCR 120 126 2 2008 PCR 3 1 RT PCR 39 43 http wwwr takara bio coJp 15 RR071A XI
26. ma RRO 1A IfHa SYBR Premix Ex Tag GC Perfect Real Time v201308Da SYBR Premix Ex 7g9 Perfect Real Time SYBR Green 1 PCR 2 SYBR Green GC 60 70 GC 200 bp GC GC 60 PCR Thermal Cycler Dice Real Time System TP900 TP960 Thermal Cycler Dice Real Time System Single TP850 TP870 Thermal Cycler Dice Real Time System Lite TP700 TP760 Smart Cycler System
27. stics 1 PCR 1 gt SYBR Premix Ex Tag GC 2X 10 ul 1 X PCR Forward Primer 10 uM 0 4 ul 0 2 MM 1 PCR Reverse Primer 10 uM 0 4 ul 0 2 uM 1 template lt 100 ng 2 ul 2 dH2O 7 2 ul Total 20 ul 1 primer 0 2 uM 0 1 1 0 uM 2 template DNA template 100 ng RT PCR cDNA RT template PCR 1096 2 PCR LightCycler PCR PCR PCR PCR
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