Realtime PCR Master Mix - TOYOBO

1 JAPAN CHINA TOYOBO CO., LTD. TOYOBO Bio-Technology, CO., LTD. Tel(81)-6-6348-3888 Tel(86) FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DIAGNOSTIC USE. 1 Instruction manual Realtime PCR Master Mix 0810 F0923K Realtime PCR Master Mix QPK-101 1 mL x 5 Store at -20 C, protected from lightContents [1] Introduction [2] Components [3] Primer/Probe design [4] Detection [5] Specimens [6] Protocol 1. TaqMan assay protocol using ABI PRISM 7700 2. Hybridization probe assay protocol using Roche LightCycler 3. One-step RT-PCR with reverse transcriptase [7] Troubleshooting [8] Related products CAUTION All reagents in this kit are intended for research purposes.

2 Do not use for diagnosis or clinical purposes. Please observe general laboratory precaution and utilize safety while using this kit. -LightCycler is a trademark of Idaho Technology, Inc. and Roche Molecular Systems, Inc. -TaqMan is a registered trademark of Roche Molecular Systems, Inc. -ABI PRISM is a registered trademark of the Perkin-Elmer Corporation. JAPAN CHINA TOYOBO CO., LTD. TOYOBO Bio-Technology, CO., LTD. Tel(81)-6-6348-3888 Tel(86) FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DIAGNOSTIC USE. 1 [ 1 ] Introduction [ 2 ] Components [ 3 ] Primer/Probe design Description This product is a Taq DNA polymerase-based 2 x Master mix for real-time PCR, which contains all components, except for the primer and probe.

3 This reagent is applicable in TaqMan assays or hybridization probe assays, in combination with each probe. Features -This reagent can be used in glass capillary systems ( LightCycler, Roche Molecular Systems, Inc.). -This reagent can be used in a passive reference system ( , ABI PRISM 7700, Applied Biosystems, Inc.). The passive reference dye does not affect any other systems. -Hot Start technology with anti-Taq DNA polymerase antibodies enables high specificity and reproducible amplification. This reagent includes the following components for 200 reactions (QPK-101), 50 l total reaction volume: <QPK-101> Realtime PCR Master Mix 1 ml x 5 Notes: This reagent can be stored at 4 C for 2 months and protected from light. For longer storage, this reagent should be kept at -20 C and protected from light.

4 1. Primer conditions Primers should be designed according to the following guidelines: -Primer length: 20-30 mer -GC content of primer: 40-60% -Target length: 200 bp (optimally, 150 bp) Notes: Longer targets (>200 bp) reduce efficiency and specificity of amplification. The ideal optimal target length range is 50-150 bp. 2. Probe conditions Probes should be designed in accordance with the manufacture s protocol for each individual assay system. JAPAN CHINA TOYOBO CO., LTD. TOYOBO Bio-Technology, CO., LTD. Tel(81)-6-6348-3888 Tel(86) FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DIAGNOSTIC USE. 2 [ 4 ] Detection [ 5 ] Specimen This reagent can be used for general detection devices, such as: -LineGene (Bioer Technology co.)

5 , ltd.) This reagent can also be used in detection equipment using glass capillaries or passive reference, such as: LightCycler (Roche Molecular Systems) ABI PRISM 7000, 7700, and 7900 (Applied Biosystems) Note: The passive reference mode of detectors should be set at ROX . 1. cDNA -Reverse transcription reactions from total or poly (A)+ RNA may be used directly, or after dilution, for real-time PCR. Purified cDNA by phenol/chloroform extraction and ethanol precipitation may also be used. Oligo dT and random primers are suitable for the reverse transcription reaction. -Up to 20% of the synthesized cDNA solution from the ReverTra Ace qPCR RT Kit (Code NO. FSQ-101) may be added to the PCR reaction solution directly, without purification.

6 2. Genomic DNA -Purified DNA, which would be used for general PCR, is also suitable for real-time PCR. In the case of mammalian genomic DNA, 1-10 ng genomic DNA is sufficient for real-time PCR. Fig. 1 Detection of -actin expression by TaqMan assay Device: ABI PRISM 7700 Target: -actin Assay method: TaqMan probe Template: cDNA from total RNA (HeLa cell) JAPAN CHINA TOYOBO CO., LTD. TOYOBO Bio-Technology, CO., LTD. Tel(81)-6-6348-3888 Tel(86) FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DIAGNOSTIC USE. 3 [ 6 ] Protocol 1. TaqMan assay protocol using ABI PRISM 7700 The following is a TaqMan assay protocol to be used with ABI PRISM 7700.

7 For other detection devices, this protocol may require modification depending on each instruction manual. (1) Preparation of reaction solution Notes -The primer and probe concentrations may require optimization. The optimal ranges for the primer and probe are M and M, respectively. In the case of commercially available primers or probes, recommended conditions from those companies should be used. (2) Cycling conditions The following condition is recommended: Notes -The extension temperature should be adjusted in accordance with the Tm of primers and probes. -The pre-denaturation condition described above is sufficient for inactivation of the anti-Taq DNA polymerase antibodies used in Hot Start PCR. To prevent unexpected and inappropriate results, do not prolong the pre-denaturation period.

8 Fifteen seconds is also sufficient for denaturation during each cycle. -Data collection step should be longer than 30 seconds. -If commercially available primers or probes are employed, the recommended conditions from each company should be used. Component Volume Final Concentration PCR grade water 14 l Realtime PCR Master Mix 25 l 1x 10 pmol/ l (10 M) Primer #1 2 l M 10 pmol/ l (10 M) Primer #2 2 l M 5 pmol/ l (5 M) TaqMan probe 2 l M Template DNA 5 l Total volume 50 l <2-step cycle> Pre-denaturation: 95 C, 1 min. Denaturation: 95 C, 15 sec. Extension: 60 C, 1 min. (data collection) 40 cycles JAPAN CHINA TOYOBO CO., LTD. TOYOBO Bio-Technology, CO.

9 , LTD. Tel(81)-6-6348-3888 Tel(86) FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DIAGNOSTIC USE. 4 2. Hybridization probe assay protocol using Roche LightCycler The hybridization probe assay is based on fluorescence resonance energy transfer (FRET) using two adjacent probes: <probe 1>, an acceptor fluorescent dye ( , LC-Red640) and <probe 2>, a donor fluorescence dye ( FITC) at the 5 and 3 ends, respectively. The following is a hybridization assay protocol using the Roche LightCycler . In the case of other detection devices, this protocol should be modified accordingly. (1) Preparation of reaction solution Notes -The primer and probe concentrations can be further optimized, if needed. In the case of commercially available primers or probes, recommended conditions from each manual should be followed.

10 (2) Cycling conditions The following condition is recommended: Notes -The annealing temperature should be set below the probe Tm value. The extension temperature should be greater than the probe Tm value. -The pre-denaturation condition described above is sufficient for inactivation of the anti-Taq DNA polymerase antibodies used in Hot Start PCR. To prevent unexpected and inappropriate results, do not prolong the pre-denaturation period. Five seconds is also sufficient for denaturation during each cycle. -Data should be collected during the annealing step. -If commercially available primers or probes are employed, the recommended conditions from each company should be used. Component Volume Final Concentration PCR grade water l Realtime PCR Master Mix 10 l 1x 10 pmol/ l (10 M) Primer #1 l M 10 pmol/ l (10 M) Primer #2 l M 10 pmol/ l (10 M) Hybridization probe 1 (5 acceptor) l M 10 pmol/ l (10 M) Hybridization probe 2 (3 donor) l M Template DNA 2 l Total volume 20 l <3-step cycle> Pre-denaturation: 95 C, 30 sec.