PrimeScript® RT reagent Kit with gDNA Eraser (Perfect Real Time
Contents
1. PCR PCR EASY Dilution for Real Time PCR 9160 EASY Dilutton PCR 37 42 C 85 0 2 ml 1 5 ml Il 20C http wwwr takara bio coJp 2 RR047A lI 1 2 3 4 5 DNA gDNA Eraser 2 15 PCR cDNA 22. 1 2 3 10 Master Mix RNase Free dH2O Master Mix gDNA Eraser PrimeScript RT Enzyme Mix 50 5 Xx gDNA Eraser Buffer 5XPrimeScript Buffer 2 for Real Time vortex http wwwr takara bio coJp 3
3. 6 Gene Specifc Primer 42 15 PCR 50 7 cDNA 20 PCR PCR 1096 http wwwr takara bio coJp 5 RR047A VI PCR SYBR Premix Ex Tag TIi RNaseH Plus RR820A B PCR PCR TaqMan Premix Ex Tagq Perfect Real Time RR039A B Thermal Cycler Dice Real Time System gt PCR 1 gt SYBR Premix Ex Tag ll 2X 12 5 ul 1 PCR Forward Pri
4. RNA RNA RT PCR RNA RNA GTC RNA RNAisoPlus 9108 9109 NucleoSpin RNAII 740955 20 50 250 RNA total RNA RNA TE Buffer http wwwr takara bio coJp 10 RR047A VIIl PrimeScript RT reagent Kit Perfect Real Time RR037A B PrimeScript RT Master Mix Perfect Real Time RR036A B SYBR Premix Ex Tag 11 Tli RNaseH Plus RR820A B SYBR Premix Ex Tag Tli RNaseH Plus RR420A B SYBR Premix Ex Tag GC Perfect Real Time RR071A B SYBRS Premix DimerEraser Perfect Real Time
5. PCR PCR PCR 95 5 15 PCR 95 C 30 3 Thermal Cycler Dice Real Time System http www takara bio coJp 6 RR047A lt Applied Biosystems 7300 7500 Real Time PCR System gt Life Technologies 1 PCR 1 gt SYBR Premix Ex Tag ll 2X 10 ul 25 ul 1 X PCR Forward Primer 10 uM 0 8 wl 2 ul 04 uM 1 PCR Reverse Primer 10 uM 0 8 wl 2 ul 0 4 uuM 1 ROX Reference D
6. 15 20 RNA cDNA cDNA SYBRS Green TagMan SYBR Premix Ex Tag Ill Tli RNaseH Plus Premix Ex Tag Perfect Real Time Perfect Real Time PCR 1 20 ul 100 1 gDNA Eraser 100 wl 2 5 X gDNA Eraser Buffer 1 200 ul 3 PrimeScript RT Enzyme Mixl 2 100 ul 4 5 XPrimeScript Buffer 2 for Real Time 3 400 wl 5 RTPrimer Mix 4 400 ul 6 RNase Free dH20 1ml x 2 7 EASY Dilution for Real Time PCR 5 1 ml 1 5 X gDNA Eraser Buffer DNA 2 RNase inhibitor 3 dNTP mixture 4 Oligo dT Primer Random 6mers 5 total RNA cDNA TE EASY Dilution
7. 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
8. 3 step PCR PCR ThermaiCydler Prolocol Stage 1 Thermel Protle AzAo Incremenl Ramp Rews Reps 1 Stage 1 Stage 2 Stage 3 9 95 30 Reps 1_ Reps Stage 2 PCR Reps 40 95 5 60 31 34 Dissociation Stage 7300 31 7500 34 7aKafRa Ex 7g9 HS 7ag PCR PCR PCR 95 5 15 PCR 95 30 3 PCR
9. http www takara bio coJp 7 RR047A VIl Appendix A total RNA DNA Mouse Liver total RNA 2 ug gDNA Eraser DNA RTase total RNA DNA 3 PCR SYBR Premix Ex Tag Perfect Real Time RR041A 2Al 25 ll Mouse Rsp78 Perfect Real Time 102 bp Thermal Cycler Dice Real Time System gDNA Eraser J Po gDNA Eraser Pa 2 3 4 5 0 F101 12 1314 15 10 17 1019 20 1 2 3 24 45 26 37 26 29 39 31 32 33 3 39 637 39 3949 NT gDNA Eraser gDNA Eraser http www takara bio coJp 8 RR047A B cDN
10. RR091A B Premix Ex Tag Perfect Real Time RR039A B EASY Dilution for Real Time PCR 9160 Thermal Cycler Dice Real Time System TP900 TP960 Smart Cycler Il System SC200N SC210N NucleoSpin RNAII 740955 20 50 250 RNAiso Plus 9108 9109 Perfect Real Time RefSeq RT PCR SYBR Premix Ex Tag 11 Tli RNaseH Plus SYBR Premix Ex Tag Tli RNaseH Plus SYBR Green RT PCR http www takara bio coJp realtime IX 1 2005 3 4 PCR 120 126 2 2008 PCR 3 1 RT PCR
11. 39 43 SYBR Molecular Probe Inc NOTICE TO PURCHASER LIMITED LICENSE M78 gDNA Eraser This product is the subject of the pending JP patent application http wwwr takara bio coJp 11 RR047A 4KaR4 Tel 077 543 6116 Fax 077 543 197 http wwW taKara Dio CO jp v201102Da
12. RT PCR Random 6 mers Oligo dT Primer RT Primer Mix RNA SYBR Green TaqMan PCR SYBR Green TaqMan RT Primer Mix total RNA RT PCR total RNA cDNA TE EASY Dilution for Real Time PCR IV
13. RR047A V 1 DNA DNA RNA g RNA 1 gt 5 Xx gDNA Eraser Buffer gDNA Eraser total RNA RNase Free dH2O Total 42 2 min 5min 2 4 2 0 ul 1 0 ul 1 10 0 wl 1 20 SYBR Green Assay 1 ug total RNA TaqMan Probe Assay 2 ug total RNA 2 30 2 1 a Master mix 1 10 ul 3 SYBRS Green Assay 1 5 X PrimeScript Buffer 2 for Real Time PrimeScript RT Enzyme Mix RT Primer Mix 4 RNase
14. A RR047A PrimeScript RT reagent Kit with gDNA Eraser Perfect Real Time RR037A PrimeScript RT reagent Kit Perfect Real time Mouse Liver total RNA 2 pg 2 ug 20 ul RT Primer Mix PCR SYBR Premix Ex Tag Perfect Real Time RR041A 2 25 Mouse Rsp78 Perfect Real Time ThermalCycler Dice Real Time System ct ORR037 RRO47 10 100 1000 10000 100000 100000010000000 Initial Ouantity RR037A PrimeScript RT reagentKit RR047A PrimeScript RT reagent Kit with gDNAEraser Standard Curve RR037A 1 000 Y 3425 X LOG X 43 05 RRO47A 0 999 Y 3 429 X LOG X 42 94 http www takara bio co jp 9 RR047A C RNA RNA RNase RNase
15. Free dH20 Total 37C 15 min 6 85 5 sec 4C 7 http wwwr takara bio coJp 4 10 0 4 0 ul 1 0 ul 1 0 ul 4 0 20 0 ul 5 Master mix 10 ul RR047A TaqMan Probe Assay 1 10 0 wl 5 X PrimeScript Buffer 2 for Real Time 4 0 PrimeScript RT Enzyme Mix 1 0 ul Master mix RT Primer Mix 4 4 0 ul 10 ul RNase Free dH20 1 0 ul Total 20 0 ul 5 37C 15 min 6 85 5 sec 4 7 3 Master mix 1 RNase Free dH2O 5XPrimeScripte Buffer 2 for Real Time gDNA Eraser RT Primer Mix PrimeScript RT Enzyme Mix 4 RT Primer Mix mRNA cDNA Oligo dT primer Gene Specifc Primer Oligo dT primer 50 pmol 20 ull Gene Specific Primer 5 pmol 20 5
16. mer 10 uM 1 ul 04 uM 1 PCR Reverse Primer 10 uM 1 ul 04 uM 1 RT CDNA 2 ul 2 dH2O 8 5 ul Total 25 ul 1 0 4 uM 0 2 1 0 uM 2 total RNA 10 pg 100 ng cDNA template PCR 10 2 PCR PCR PCR Tm PCR 3 step PCR PCR ms mr Hold 95 30 te eg bol Fa ins 2 Step PCR Cycle 40 95 5 60 30 60 00 50 on ts 93 i Dissociation 7aKafRa Ex 7ag Hs 7ag
17. mma RRO47A IdfaR4 PrimeScript RT reagent Kit with gDNA Eraser Perfect Real Time vV201102Da cDNA DNA DNA RNA DNase DNase RNA PrimeScript RT reagent Kit with gDNA Eraser DNA RT PCR DNA gDNA Eraser DNA 42 2 DNA
18. ye or Dyell 50X 3 0 4 ul 1 ul 1 Xx RT cDNA 2 ul 4 pul 2 dH2O 6 ul 16 ul Total 20 ul 4 50 ul 4 1 0 4 uM 0 2 1 0 uM 2 20 ul total RNA 10 pg 100 ng cDNA template PCR 10 3 ROX Reference Dye ll 50X ROX Reference Dye 50X 7500 Real Time PCR System ROX Reference Dyell 50 Xx 7300 ReaLTime PCR System ROX Reference Dye 50 Xx 4 2 PCR PCR PCR Tm PCR
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