Table of contents. I. Description...2. Principle...2. Content...3. Storage...4. V. Features...4. Precautions for Use...4. Protocol...

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1 Table of contents I. Description...2 II. III. IV. Principle...2 Content...3 Storage...4 V. Features...4 VI. VII. Precautions for Use...4 Protocol...4 A) Protocol using Smart Cycler II System...4 B) Protocol using ABI PRISM 7000/7700/7900 HT & 7300/7500/7500 Fast Real-Time PCR System...7 C) Protocol using LightCycler...9 D) Protocol using Mx3000P VIII. Application Example IX. Related products

2 I. Description Premix Ex Taq (Perfect Real Time) is a 2 convenient premix reagent, specially designed for real time PCR by using TaqMan * 1 probe. This product combines the high performance of TaKaRa Ex Taq HS, which is an enzyme for hot start PCR utilizing Taq antibody, with a newly developed buffer which provides superior specificity, increased amplification efficiency and high aptitude for high-speed real time PCR. Accordingly, a successful real time PCR is promised with high sensitivity, wide dynamic range and accurate quantification. II. Principle Applicable real time PCR instruments; ABI PRISM 7000, 7700, 7900HT, *2 Applied Biosystems 7300, 7500 Real-Time PCR System, 7500 Fast Real-Time PCR System (Life Technologies) LightCycler (Roche Diagnostics) *3 Smart Cycler System/Smart Cycler II System (Cepheid) *4 Mx3000P (Stratagene) Thermal Cysler Dice Real Time System II (TaKaRa Bio) *5 * 1 : TaqMan is a trademark of Roche Molecular Systems Inc. * 2 : ABI PRISM is a registered trademark of Applera Corporation. * 3 : LightCycler is a registered trademark of Roche Diegnostics. * 4 : Smart Cycler is a trademark of Cepheid. * 5 : This instrument is not available in the U.S. and Europe. This product employs TaKaRa Ex Taq HS for PCR reaction. PCR products are detected with TaqMan probe in real time monitoring. 1) PCR PCR (Polymerase Chain Reaction) is a simple and powerful method to amplify only a target DNA of a tiny amount by cycling three incubation steps at different temperatures: Double-stranded target DNA is heat denatured (denaturation step), the two primers complementary to the target segment are annealed at low temperature (annealing step), and the annealed primers are then extended at an intermediate temperature (extension step) with a DNA polymerase.as the target copy number doubles upon each cycle, PCR can thereby amplify DNA fragments up to fold in a short period. As this product utilizes an enzyme for Hot Start PCR, TaKaRa Ex Taq HS, non-specific amplification due to mispriming prior to cycling or due to primer dimmer can be minimized. Accordingly the highly specific and sensitive detection is achieved. 2) Fluorescence detection : TaqMan Probe method : The TaqMan method is based on a combination of TaqMan Technology and a real time PCR instrument. Oligonucleotide modified with fluorescence substance (e.g. FAM) at 5' -end and with quencher (e.g. TAMRA) at 3' -end is added in a reaction system. Under the annealing condition, TaqMan probe specifically hybridizes to template DNA, but the fluorescence is suppressed by quencher. In extension step, the 5' 3' exonuclease activity of Taq DNA polymerase degrades the TaqMan probe hybridized to a template. Accordingly, TaqMan probe is released from the suppression with a quencher, resulting in emission of fluorescence. By measuring the fluorescence intensity the amount of amplified product can be monitored. 2

3 1) Heat denaturation Primer Fluorescence Substance F Q Probe Quencher 2) Primer annealing/probe hybridization Polymerase F Hybridizes Q 3) Extension F Q F Q III. Content (250 units) Premix Ex Taq (Perfect Real Time) (2 conc.) *1 ROX Reference Dye (50 conc.) *2 ROX Reference Dye II (50 conc.) *2 1.0 ml 5 tubes 200 μl 200 μl * 1 : contains TaKaRa Ex Taq HS, dntp Mixture,and Mg 2+. * 2 : ROX Reference Dye/Dye II is used for normalization of intensity by background subtraction. For ABI PRISM 7000/7700/7900HT and Applied Biosystems 7300 Real-Time PCR System, the use of ROX Reference Dye (50 ) is recommended. For Applied Biosystems 7500 Real-Time PCR System, 7500 Fast Real-Time PCR System, and Stratagene Mx3000P, the use of ROX Reference Dye II is recommended. It is not required for use with Smart Cycler, LightCycler or Thermal Cycler Dice Real Time System II. Reagents and Instruments Required but Not Supplied in this product 1. Thermal Cycler for real time PCR (authorized instruments) 2. Reaction tube or plate for real time PCR 3. PCR primers 4. TaqMan probe for detection (licensed probes) 5. dh2o 6. Micropippets and Micropippet tips (autoclaved prior to use) 3

4 IV. Storage Stable at 4 for 6 months. Careful attention should be made not to cause contamination. * This product is shipped at -20. After delivered, store at 4. Make the concentration uniform by gently inverting a tube before use. * This product can be stored at -20 for long term storage. Once thawed, store at 4 and use up within 6 months. V. Features 1) Quick & accurate detection and quantification of target gene through real time PCR. 2) Easy-to-use 2 premix reagent designed for reducing pipetting steps. 3) High amplification efficiency and highly sensitive detection : This product utilizes an enzyme for hot start, TaKaRa Ex Taq HS. As this enzymebuffer system is optimized for real time PCR, this product offers highly efficient amplification and high sensitive detection. VI. Precautions for Use Please ensure to read through the following precautions prior to starting the protocol. 1) Prior to use, make the concentration uniform by gently inverting a tube before use. 2) A precipiate may form in this product during storage at -20. This precipitate can be completely dissolved by briefly warming the tube with your hands or placing the tube at room temperature, followed by inversion of the tube several times to mix until dissolved. USE THE REAGENT ONLY AFTER COMPLETELY REDISSOLVING THE PRECIPITATE to ensure a uniform concentration of components.do not vortex. Even in the absence of a precipitate, gentle mixing of the product (avoiding air bubbles) is recommended to provide a uniform concentration of components prior to use. 3) TaqMan probe for detection should be prepared separately. It is not supplied in this product. 4) For preparing and dispensing the reagents, a new disposable tip should be used to minimize contamination among samples. VII. Protocol A) Protocol using Smart Cycler II System 1. Prepare the following PCR reaction mixture. <per reaction> [ Reagents ] [ Volume ] [ Final concentration ] Premix Ex Taq (2 ) 12.5 μl 1 PCR Forward Primer (10 μm) 0.5 μl 0.2 μm *1 PCR Reverse Primer (10 μm) 0.5 μl 0.2 μm *1 TaqMan probe 1 μl *2 template 2 μl *3 dh2o 8.5 μl total 25 μl 4

5 * 1 : The final concentration of primers can be 0.2 μm in most reactions. When it does not work, determine the optimalconcentrations within the range μm. * 2 : Probe concentration differs depending on kind of real time PCR instrument, and kind of fluorescence labeling material. The adding amount should be determined by referring to the operation manual of instrument and a product insert supplied with probe. Generally when performing detection with Smart Cycler System, probe concentration should be determined to have the final concentration within the range μm. * 3 : Final template concentration varies depending on the copy number of target present in the template solution. Optimal amount should be determined by preparing the dilution series. DNA template should be used in less than 100 ng. When the RT reactant (cdna) is used as a template, it should be added in less 10% volume of PCR reaction mixture. 2. Start the reaction. Gently centrifuge the reaction tubes using the centrifuge exclusive use for Smart Cycler. Load the reaction tubes on Smart Cycler II System and start the reaction. Shuttle PCR standard protocol (below) is recommended. Try this protocol firstly, and optimize the reaction condition if needed. Shuttle PCR Standard Protocol Stage 1 : Initial Denaturation Stage 2 : PCR reaction Note : This product combines the high performance of TaKaRa Ex Taq HS, which is an enzyme for hot start PCR utilizing Taq antibody. Initial denaturation step prior to PCR should be at 95 for 30 sec. No need to heat at 95 for (5 - ) 15 min. as the initial denaturation, required for chemically modified Taq polymerase. If excessive heat treatment is provided, the enzyme activity descreases and the amplification efficiency and the accuracy in quantification can also be affected. 5

6 Initial Denaturation Step Temperature Time Detection Remark Initial Denaturation sec. OFF It is recommended to perform initial denaturation at 95 for 30 seconds, which is enough even when the template is genomic DNA or circular plasmid. Denaturation condition that exceeds 2 minutes unstabilizes the reaction thus not recommended. Shuttle PCR Step Temperature Time Detection Remark Denaturation sec. OFF Annealing/ Extension sec. ON General amplified size for real time PCR is less than 300 bp, so denaturation at 95 for 3-5 seconds would be sufficient. Please try at 60 for 20 seconds at first. The temperature should be optimized within the range of if optimization is required. When reaction does not proceed efficiently, extend the time. Cycle number ; cycles 3. After the reaction is completed, perform analysis. After the reaction is completed, verify the amplification curve. Establish the standard curve when quantification is done. * Refer to the operation manual of Smart Cycler for the analysis method with Smart Cycler. 6

7 B) Protocol using ABI PRISM 7000/7700/7900HT or Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System 1. Prepare PCR reaction (below). < per reaction > [ Reagents ] [ Volume ] [ Volume ] [ Final conc. ] Premix Ex Taq (2 ) 10 μl 25 μl 1 PCR Forward Primer (10 μm) 0.4 μl 1 μl 0.2 μm * 1 PCR Reverse Primer (10 μm) 0.4 μl 1 μl 0.2 μm * 1 TaqMan probe 0.8 μl 2 μl * 2 ROX Reference Dye or Dye II (50 ) * μl 1 μl 1 template 2 μl 4 μl * 4 dh2o 6 μl 16 μl total 20 μl * 5 50 μl * 5 * 1 : The final concentration of primers can be 0.2 μm in most reactions. When it does not work, determine the optimal concentrations within the range μm. * 2 : Probe concentration differs depending on kind of real time PCR instrument, and kind of fluorescence labeling material. The adding amount should be determined by referring to the operation manual of instrument and a product insert supplied with probe. * 3 : The ROX Reference Dye/Dye II is supplied for performing normalization of fluorescent signal intensities among wells when used with real time PCR instruments that have option. For ABI PRISM 7000/7700/7900HT and Applied Biosystems 7300 Real-Time PCR Syatem, the use of ROX Reference Dye (50 ) is recommended. For Applied Biosystems 7500 Real-Time PCR System and 7500 Fast Real- Time PCR System, the use of ROX Reference Dye II is recommended. * 4 : Final template concentration varies depending on the copy number of target present in the template solution. Optimal amount should be determined by preparing the dilution series. DNA template should be used in less than 100 ng for 20 μl reaction. When the RT reactant (cdna) is used as a template, it should be added in less 10% volume of PCR reaction mixture. * 5 : The reaction of 50 μl is for 96-well plate, single tube and 8-strip tube. The reaction of 20 μl is for 384-well plate and 96-well Fast Themal Cycling Plate. 2. Start the reaction. Shuttle PCR standard protocol (below) is recommended. Try this protocol firstly, and optimize the reaction condition if needed. Note : This product combines the high performance of TaKaRa Ex Taq HS, which is an enzyme for hot start PCR utilizing Taq antibody. Initial denaturation step prior to PCR should be at 95 for 30 sec. No need to heat at 95 for (5 - ) 15 min. as the initial denaturation, required for chemically modified Taq polymerase. If excessive heat treatment is provided, the enzyme activity descreases and the amplification efficiency and the accuracy in quantification can also be affected. 7

8 [ABI PRISM 7000/7700/7900HT, 7300/7500 Real-Time PCR System] Shuttle PCR Standard Protocol Stage 1 : Initial denaturation Reps : sec. Stage 2 : PCR Reps : sec sec. (31sec, 34sec) * * : Fluorescence detection step should be 30 seconds with ABI PRISM 7700 and 7900HT, 31 seconds with ABI PRISM 7000 and 7300 Real-Time PCR System, and 34 seconds with 7500 Real-Time PCR System. [7500 Fast Real-Time PCR System] Shuttle PCR Standard Protocol Holding Stage Reps:1 95, 30 min. Cycling Stage Number of Cycles : 40 95, 3 min. 60, 30 min. Melt Curve Stage 8

9 Initial denaturation step Step Temperature Time Detection Remark Initial denaturation sec. OFF It is recommended to perform initial denaturation at 95 for 30 seconds, which is enough even when the template is genomic DNA or circular plasmid. Denaturation condition that exceeds 2 minutes unstabilizes the reaction thus not recommended. Shuttle PCR Step Temperature Time Detection Remark Denaturation sec. OFF Annealing/ Extension sec. (25, 30, 31, 34 sec.) * ON Since the target size amplified for real time PCR is generally shorter than 300 bp, the denaturation at 95 for 3-5 seconds is a sufficient condition. Please try at 60 for 30, 31, 34 or 25 seconds at first. The temperature should be optimized within the range of if optimization is required. When reaction does not proceed efficiently, extend the time. * : Fluorescence detection step should be 30 seconds with ABI PRISM 7700 and 7900HT, 31 seconds with 7000 and 7300, and 34 seconds with 7500 Real-Time PCR System. When used with 7500 Fast, set 25 sec. Cycle number;30-45 cycles 3. After the reaction is completed, verify the amplification curve. Establish the standard curve when quantification is done. * Refer to the operation manual of an used real time PCR instrument. C) Protocol using LightCycler 1. Prepare the following PCR reaction mixture. <per reaction> [ Reagents ] [ Volume ] [ Final concentration ] Premix Ex Taq (2 ) 10 μl 1 PCR Forward Primer (10 μm) 0.4 μl 0.2 μm *1 PCR Reverse Primer (10 μm) 0.4 μl 0.2 μm *1 TaqMan probe 0.8 μl *2 template 2 μl *3 dh2o 6.4 μl total 20 μl 9

10 * 1 : The final concentration of primers can be 0.2 μm in most reactions. When it does not work, determine the optimalconcentrations within the range μm. * 2 : Probe concentration differs depending on kind of real time PCR instrument, and kind of fluorescence labeling material. The adding amount should be determined by referring to the operation manual of instrument and a product insert supplied with probe. * 3 : Final template concentration varies depending on the copy number of target present in the template solution. Optimal amount should be determined by preparing the dilution series. DNA template should be used in less than 100 ng. When the RT reactant (cdna) is used as a template, it should be added in less 10% volume of PCR reaction mixture. 2. Start the reaction. After gently centrifugation of LightCycler capillaries, set capillaries in a LightCycler and start the reaction. Shuttle PCR standard protocol (below) is recommended. Try this protocol firstly, and optimize the reaction condition if needed. Note : This product combines the high performance of TaKaRa Ex Taq HS, which is an enzyme for hot start PCR utilizing Taq antibody. Initial denaturation step prior to PCR should be at 95 for 30 sec. No need to heat at 95 for (5 -) 15 min. as the initial denaturation, required for chemically modified Taq polymerase. If excessive heat treatment is provided, the enzyme activity descreases and theamplification efficiency and the accuracy in quantification can also be affected. Stage2 : PCR reaction Shuttle PCR Standard Protocol Stage 1 : Initial denaturation Reps : 1 95, 30 sec. 20 /sec. Stage 2 : PCR Reps : 40 95, 5 sec. 20 /sec. 60, 20 sec. 20 /sec. 10

11 Initial denaturation Step Temperature Time Acquisition Mode Initial denaturation sec. None Remark It is recommended to perform initial denaturation at 95 for 30 seconds, which is enough even when the template is genomic DNA or circular plasmid. Denaturation condition that exceeds 2 minutes unstabilizes the reaction thus not recommended. Shuttle PCR Step Temperature Time Acquisition Mode Denaturation sec. None Annealing /Extension sec. Single Remark Since the target size amplified for real time PCR is generally shorter than 300 bp, the denaturation at 95 for 3-5 seconds is a sufficient condition. Please try at 60 for 20 seconds at first. The temperature should be optimized within the range of if optimization is required.when reaction does not proceed efficiently, extend the time. Cycle number : cycles 3. After the reaction is completed, verify the amplification curve. Establish the standard curve when quantification is done. * Refer to the operation manual of an used real time PCR instrument. 11

12 D) Protocol using Mx3000P 1. Prepare the following PCR reaction mixture on ice. <per reaction> [ Reagents ] [ Volume ] [ Final conc. ] Premix Ex Taq (2 ) 12.5 μl 1 PCR Forward Primer (10 μm) 0.5 μl 0.2 μm *1 PCR Reverse Primer (10 μm) 0.5 μl 0.2 μm *1 TaqMan probe 1 μl *2 ROX Reference Dye II (50 ) *3 0.5 μl 1 template (<100 ng) 2 μl *4 dh2o 8.0 μl total 25 μl * 1 : The final concentration of primers can be 0.2 μm in most reactions. When it does not work, determine the optimal concentrations within the range of μm. * 2 : Probe concentration differs depending on kind of real time PCR instrument and type of fluorescence labeling material. The adding amount should be determined by referring to the operation manual of instrument and a product insert supplied with probe. * 3 : For Mx3000P, the use of ROX Reference Dye II is recomended. ROX Reference Dye is not suitable for the use with Mx3000P, since it has higher concentration than ROX Reference Dye II. * 4 : Final template concentration varies depending on the copy number of target present in the template solution. Optimal amount should be determined by preparing the dilution series. It is recomended to apply DNA template in less than 100 ng. When the RT reactant (cdna) is used as a template, it should be added in less than 10% volume of PCR reaction mixture. 2. Start the reaction. Shuttle PCR standard protocol (below) is recommended. Try this protocol firstly, and optimize the reaction condition if needed. Shuttle PCR Standard Protocol Stage 1 : Initial denaturation Reps : 1 95, 30 sec. Stage 2 : PCR Reps : 40 95, 5 sec. 60, 20 sec. Note : This product combines the high performance of TaKaRa Ex Taq HS, which is an enzyme for hot start PCR utilizing Taq antibody. Initial denaturation step prior to PCR should be at 95 for 30 sec. No need to heat at 95 for (5 -) 15 min. as the initial denaturation, required for chemically modified Taq polymerase. If excessive heat treatment is provided, the enzyme activity descreases and theamplification efficiency and the accuracy in quantification can also be affected. 12

13 Initial denaturation Step Temperature Time Detection Remark Initial denaturation sec. OFF It is recommended to perform initial denaturation at 95 for 30 seconds, which is enough even when the template is genomic DNA or circular plasmid. Denaturation condition that exceeds 2 minutes unstabilizes the reaction thus not recommended. Shuttle PCR Step Temperature Time Detection Remark Denaturation sec. OFF Annealing /Extension sec. ON Since the target size amplified for real time PCR is generally shorter than 300 bp, the denaturation at 95 for 3-5 seconds is a sufficient condition. Please try at 60 for 20 seconds at first. The temperature should be optimized within the range of if optimization is required. When reaction does not proceed efficiently, extend the time. Cycle number : cycles 3. After the reaction is completed, perform analysis. After the reaction is completed, verify the amplification curve. Establish the standard curve when quatitation is done. * Refer to the operation manual of an used real time PCR instrument. 13

14 VIII. Application example Detection example with Applied Biosystems 7500 Fast Real-Time PCR System Target : Mouse Gapd Primer/Probe : TaqMan Gene Expression Assays (Life Technologies) Template : cdna serial dilution (corresponding to total RNA 500 fg - 50 ng) Amplification curve sec sec sec. 45 cycle Standard curve R 2 = Slope = IX. Related products PrimeScript RT reagent Kit (Perfect Real Time) (Cat. #RR037A) One Step PrimeScript RT-PCR Kit (Perfect Real Time) (Cat. #RR064A) SYBR Premix Ex Taq II (Tli RNaseH Plus) (Cat. #RR820A/B) SYBR Premix Ex Taq (Tli RNaseH Plus) (Cat. #RR420A/B) One Step SYBR PrimeScript RT-PCR Kit II (Perfect Real Time) (Cat. #RR086A) One Step SYBR PrimeScript RT-PCR Kit (Perfect Real Time) (Cat. #RR066A) 14

15 NOTICE TO PURCHASER: LIMITED LICENSE [P7] PCR Notice A license to perform the patented 5' Nuclease Process for research is obtained by the purchase of (i) both Authorized 5' Nuclease Core Kit and Licensed Probe, (ii) a Licensed 5' Nuclease Kit, or (iii) license rights from Applied Biosystems. This product is an Authorized 5' Nuclease Core Kit. Use of this product is covered by one or more of the following US patents and corresponding patent claims outside the US: 5,079,352, 5,789,224, 5,618,711, 6,127,155, 5,677,152, 5,773,258, 5,407,800, 5,322,770, 5,310,652, 5,210,015, 5,487,972, and claims outside the US corresponding to US Patent No. 4,889,818. 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. Separate purchase of a Licensed Probe would convey rights under the applicable claims of US Patents Nos. 5,538,848, 5,723,591, 5,876,930, 6,030,787, 6,258,569, 5,804,375 (claims 1-12 only), and 6,214,979, and corresponding claims outside the United States. No right under any other patent claim and no right to perform commercial 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. Diagnostic uses under Roche patents require a separate license from Roche. Further information on purchasing licenses may be obtained from the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA. [L15] Hot Start PCR Licensed under U.S. Patent No ,671 and 5,587,287, and corresponding patents in other countries. [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 purchasing 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 of U.S. Patent No. 5,436,149 and its foreign counterpart patent claims. TaqMan The 5' nuclease process is covered by patents owned by Roche Molecular Systems, Inc. and F. Hoffmann-La Roche Ltd. Purchase of the product does not provide a license to use this patented technology. 15

16 NOTE : This product is for research use only. It is not intended for use in therapeutic or diagnostic procedures for humans or animals. Also, do not use this product as food, cosmetic, or household item, etc. Takara products may not be resold or transferred, modified for resale or transfer, or used to manufacture commercial products without written approval from TAKARA BIO INC. If you require licenses for other use, please contact us by phone at or from our website at 16