17-05 THUNDERBIRD SYBR qpcr Mix (Code No. QPS-201, QPS-201T) TOYOBO CO., LTD. Life Science Department OSAKA JAPAN A4196K

17-05 THUNDERBIRD SYBR qpcr Mix (Code No. QPS-201, QPS-201T) TOYOBO CO., LTD. Life Science Department OSAKA JAPAN A4196K - 18 -

[1] 1 [2] 2 [3] 3 [4] 5 [5] : RNA cdna 13 [6] 14 [7] 17 LightCycler TM Idaho Technology Inc. Roche Molecular Systems Inc. SYBR Molecular Probes Inc. - 0 -

[1] THUNDERBIRD SYBR qpcr Mix SYBR Green I PCR 2X ROX() SYBR Green Realtime PCR Master Mix (Code No.: QPK-201) SYBR Green Realtime PCR Master Mix -Plus- (Code No.: QPK-212) PCR PCR ( ) ( ) 1. PCR 2. ( ) PCR 3. 4. 50X ROX reference dye (Applied Biosystems Agilent Technologies ) ROX 5. Taq SYBR Green I SYBR Green I DNA SYBR Green I PCR - 1 -

[2] THUNDERBIRD SYBR qpcr Mix 50X ROX reference dye -20 C ( 2 8 C 3 ) -20 C ( 2 8 C) QPS-201X5 QPS-201 5 (QPS-201) 5ml (1.67ml x 3) 250μl (QPS-201T) 1ml (1ml x 1) 50μl THUNDERBIRD SYBR qpcr Mix SYBR Green I dntps Mg 2+ rtaq DNA DNA 2X DNA (cdna genomic DNA plasmid DNA ) 1X ROX (passive reference dye) -20 C 3 2 8 C -20 C 10 SYBR Green I THUNDERBIRD SYBR qpcr Mix -20 50X ROX reference dye Applied Biosystems [4] -20 C 2 8 C -20 C ROX reference dye 50X ROX reference dye THUNDERBIRD SYBR qpcr Mix THUNDERBIRD SYBR qpcr Mix ROX [4] ROX 1X qpcr Mix : 50X ROX = 1.67ml : 66.8μl (QPS-201) 1ml : 40μl (QPS-201T) ROX 0.1X qpcr Mix : 50X ROX = 1.67ml : 6.7μl (QPS-201) 1ml : 4μl (QPS-201T) - 2 -

[3] (1) PCR PCR (2) - 20 30mer GC 40 60% - 200bp 80 150bp - cdna DNA - (melting temperature: Tm)60 65 C HPLC(OPC) - 3 -

(3) DNA DNA cdna genomic DNA plasmid DNA virus DNA DNA (a) cdna cdna PCR 10%PCR PCR 10% PCR ReverTra Ace qpcr RT Kit (Code No.: FSQ-101) PCR 20% (b) genomic DNA virus DNA genomic DNA virus DNA SYBR Green I 2 DNA 2 DNA DNA SYBR Green I genomic DNA (50μl 200ng ) (c) plasmid DNA plasmid DNA plasmid DNA DNA DNA DNA PCR (yeast RNA ) plasmid DNA 1μg plasmid DNA = 9.1 x 10 11 / plasmid DNA (kb) - 4 -

[4] (1) 50 μl 20 μl 50μl 20μl X μl X μl THUNDERBIRD SYBR qpcr Mix 25 μl 10 μl 1x Forward Primer 15 pmol 6 pmol 0.3 μm* 1 Reverse Primer 15 pmol 6 pmol 0.3 μm* 1 50X ROX reference dye 1 / 0.1 μl 0.4 / 0.04μl 1x / 0.1x* 2 DNA Y μl Y μl 50 μl 20 μl *1: 0.2 0.6 μm *2: 50X ROX reference dye Applied Biosystems 1: ROX reference dye Applied Biosystems 7000 7300 7700 7900HT StepOne StepOnePlus Applied Biosystems 7500 7500Fast Agilent Technologies ( ) Roche Bio-Rad BioFlux Line Gene ( ) 1X (1/50 ) 0.1X (1/500 ) SYBR Green Realtime PCR Master Mix (Code No.: QPK-201) SYBR Green Realtime PCR Master Mix -Plus- (Code No.: QPK-212) ROX 1X - 5 -

(2)PCR (p.3 (2)) PCR 2 PCR Tm (60 C ) 3 PCR (2)-3. 3 PCR 95 C 20 60 * 1 PCR 95 C 1 15 * 2 (40 cycles) 60 C* 3 30 60 * 4 (Data Collection ) (Melting / Dissociation Curve Analysis)* 5 *1: DNA 60 ( ) 2: Applied Biosystems (Applied Biosystems 7500Fast ) (Roche LightCycler TM 1.x 2.0) (Applied Biosystems 7700 7500 7900HT() StepOne StepOnePlus Agilent Technologies BioFlux Line Gene ) 20 30 60-6 -

*2: PCR PCR 15 () 3: PCR PCR Applied Biosystems 3 (Applied Biosystems 7500Fast ) (Roche LightCycler TM 5 1.x 2.0) (Applied Biosystems 7700 7500 7900HT() 15 StepOne StepOnePlus Agilent Technologies BioFlux Line Gene ) *3: 56 64 C *4: 300bp 30 30 (45 60 ) 30 ( : Applied Biosystems 7000/7300 31 7500 35 ) *5: - 7 -

(2)-1 Applied Biosystems 7900HT ( 2.2.2) Applied Biosystems 7900HT 1. Instrument Thermal Profile Add Dissociation Stage Sample Volume (μl) 45-8 -

2. Data Collection 3. - 9 -

(2)-2 Roche LightCycler 1.1 ( 3.5) Roche LightCycler 1.1 1. Analysis Mode None 2. PCR Analysis Mode Quantification (60 C) Acquisition Mode SINGLE - 10 -

3. ( ) Analysis Mode Melting Curves 95 C Temperature Transition Rate 0.20 Acquisition Mode CONT 4. Analysis Mode None 5. - 11 -

(2)-3 3 PCR p.6 2 PCR 3 PCR - Tm (60 C ) - (300bp ) - PCR 95 C 20 60 * 1 PCR 95 C 1 15 * 1 (40 cycles) 55 65 C* 2 5 30 * 3 72 C 30 60 * 4 (Melting / Dissociation Curve Analysis)* 5 (Data Collection ) *1: PCR p.6 *2: Tm Tm-5 C *3: 5 15 30 *4: 300bp 30 30 (45 60 ) 30 ( : Applied Biosystems 7000/7300 31 7500 35 ) *5: - 12 -

[5] : RNA cdna cdna PCR ReverTra Ace qpcr RT Kit (Code: FSQ-101) PCR cdna ReverTra Ace qpcr RT Kit PCR cdna ( ) (1)RNA RNA 65 C 5 RNA 5x RT Buffer RNA (2) Nuclease-free Water 5x RT Buffer RT Enzyme Mix Primer Mix RNA Total volume X μl 2 μl 0.5 μl 0.5 μl 0.5pg 1μg 10 μl (3) 37 C 15 98 C 5 4 C -20 C PCR RNA - 13 -

[6] DNA SYBR Green I DNA DNA SYBR Green I DNA DNA Ct PCR PCR cdna PCR DNA DNA DNA DNA - 14 -

PCR 90% (slope < -3.6) PCR [4] (2) PCR PCR PCR 110% (slope > -3.1) Tm (60 C ) PCR Ct PCR Ct Tm (p.7 *3 ) 3 PCR(p.12 ) PCR Ct PCR Ct Ct PCR Ct PCR DNA PCR DNA DNA PCR DNA DNA DNA (Yeast RNA ) - 15 -

no-template control (NTC) no-template control [4] (2) PCR PCR HPLC 50X ROX reference dye no-template control 50X ROX reference dye SYBR Green I [4] (1) 50X ROX reference dye PCR (45 60 ) DNA - 16 -

[7] PCR Code No. 1ml x 1 QPS-101T (40 ) PCR THUNDERBIRD 1.67ml x 3 QPS-101 Probe qpcr Mix (200 ) THUNDERBIRD Probe qpcr Mix 50X ROX reference dye 50μl 1000 QPS-101X5 (QPS-101 5 ) cdna Code No. PCR cdna ReverTra Ace qpcr RT Kit PCR cdna ReverTra Ace qpcr RT Master Mix PCR cdna DNA ReverTra Ace qpcr RT Master Mix with gdna Remover 200 FSQ-101 200 FSQ-201 200 FSQ-301 1 PCR Code No. 0.5ml x 2 (40 ) QRT-101T 1 PCR RNA-direct TM 0.5ml x 5 Realtime PCR Master Mix QRT-101 (100 ) SYBR Green I 1 PCR RNA-direct TM SYBR Green Realtime PCR Master Mix 0.5ml x 2 (40 ) 0.5ml x 5 (100 ) QRT-201T QRT-201 RNA-direct TM 50mM Mn(OAc) 2 50μl 500 QRT-101X5 QRT-201X5 (QRT-101 QRT-201 5 ) http://www.toyobo.co.jp/bio - 17 -

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