Premium PCR Enzymes. High Fidelity PCR Real Time PCR RT- PCR qrt- PCR High-Speed PCR Long PCR Routine PCR Multiplex PCR Hot-Start PCR GC-Rich PCR

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1 Premium PCR Enzymes Introducing SapphireAmp "Load'N'Go" fast PCR master mix... p 15 EmeraldAmp "Load'N'Go" PCR master mixes for superior performance on GC or AT rich regions...p 19 SYBR Premix Ex Taq (Tli RNAseH Plus) for improved quantification by avoiding PCR inhibition from residual mrna...p 4 Samples High Fidelity PCR Real Time PCR RT- PCR qrt- PCR High-Speed PCR Long PCR Routine PCR Multiplex PCR Hot-Start PCR GC-Rich PCR Takara Bio Europe Austria: Germany: Switzerland: United Kingdom: orders@takara-bio.eu tech@takara-bio.eu info@takara-bio.eu

2 How to select The Best PCR Enzyme for your application End-Point PCR TaKaRa Taq Routine PCR High Performance PCR TaKaRa Ex Taq TaKaRa LA Taq TaKaRa Ex Taq High GC Content or Secondary Structures High Fidelity PCR Hot Start PCR or Multiplex PCR Long PCR High Speed PCR TaKaRa LA Taq PrimeSTAR HS DNA Polymerase TaKaRa Taq Hot Start Version TaKaRa LA Taq SpeedSTAR HS DNA Polymerase LA PCR Kit Version 2.1 PrimeSTAR TaKaRa Ex Taq Hot Start Version LA PCR Kit Version 2.1 PrimeSTAR TaKaRa LA Taq Hot Start Version Premix for End-point PCR Convenient Premixes High Fidelity PCR Routine PCR High Sensitivity PCR For Longer PCR Dye Added Premix for direct electrophoresis Hot Start PCR PrimeSTAR Premix Premix Taq Premix Ex Taq One Shot LA PCR Mix SapphireAmp PCR MasterMix Premix Ex Taq HS EmeraldAmp Products Premix Taq HS *Hot start enzymes contain an anti- Taq antibody to minimize PerfectShot Ex Taq Real Time PCR Reverse Transcription PCR Probe Detection SYBR Green detection Real Time RT-PCR 1-Step RT-PCR Premix Ex Taq (Probe qpcr) Premix Ex Taq (Perfect Real Time) SYBR Premix Ex Taq I&II (Perfect Real Time) SYBR Premix Ex Taq I&II (Tli RNaseH Plus) SYBR Premix DimerEraser (Perfect Real Time) PrimeScript RT Reagent Kit for Real Time (RR037A) One Step SYBR PrimeScript RT-PCR Kit (Perfect Real Time) (RR066A) One Step SYBR PrimeScript RT-PCR Kit II (Perfect Real Time) (RR086A) PrimeScript 1st Strand cdna Synthesis Kit (6210A and 6110A) 2-Step RT-PCR PrimeScript RT-PCR Kit (RR014A) PrimeScript One Step RT-PCR Kit (RR055A) Real Time RT-PCR PrimeScript RT Reagent Kit for Real Time (RR037A) One Step PrimeScript RT-PCR Kit (Perfect Real Time) (RR064A) High Fidelity PrimeScript RT-PCR Kit (R027A)

3 Product Page Real Time PCR (qpcr) and RT-qPCR qpcr SYBR Premixes (Tli RNaseH plus) 4 SYBR Premix Ex Taq (Perfect Real Time) 5 SYBR Premix Ex Taq II (Perfect Real Time) 6 Premix Ex Taq (Perfect Real Time) 7 RT-qPCR One Step SYBR PrimeScript RT-PCR Kit (Perfect Real Time) 8 One Step SYBR PrimeScript RT-PCR Kit II (Perfect Real Time) 8 One Step PrimeScript RT-PCR Kit (Perfect Real Time) 9 PrimeScript RT Reagent Kit (Perfect Real Time) 9 Table of Contents Reverse Transcription PCR PrimeScript One Step RT-PCR Kit, Version 2 10 PrimeScript 1st Strand cdna Synthesis Kit 10 PrimeScript II 1st Strand cdna Synthesis Kit 10 PrimeScript RT-PCR Kit 11 End-Point PCR High Fidelity PCR PrimeSTAR HS DNA Polymerase 12 High Speed PCR SpeedSTAR HS DNA Polymerase 14 Load N Go PCR SapphireAmp Fast PCR Master Mix 15 EmeraldAmp Products 19 Long and Complex Amplifications TaKaRa LA Taq 16 TaKaRa LA Taq with GC Buffers 17 High Efficiency and Sensitivity PCR TaKaRa Ex Taq and Premix 18 Routine PCR Amplifications TaKaRa Taq 19 Hot Start and Multiplex PCR TaKaRa Ex Taq Hot Start DNA Polymerase 20 TaKaRa LA Taq Hot Start Version 20 TaKaRa Taq Hot Start Version 21 Guide to TaKaRa s PCR Enzymes 22 See back cover for part numbers and full contact information 3

4 REAL TIME PCR Real Time PCR (qpcr) SYBR Premix Ex Taq (Tli RNaseH Plus) SYBR Premix Ex Taq II (Tli RNaseH Plus) Improved cdna quantification by the addition of thermostable Tli RNaseH (No RNase pre-treatment needed ) Quantification of various targets with much broader dynamic range Higher specificity allowed by special buffer formulation in the Premix Ex Taq II variant Compatible with a majority of real-time thermal cyclers Excellent quantification of cdna from any RT reaction Works also perfectly for genome DNA quantification or detection since Tli RNaseH doesn t interfere with the amplification reaction. Fast, specific and accurate gene expression profiling Takara introduces two new SYBR premixes for Real- Time PCR, including the thermostable Tli RNAseH: SYBR Premix Ex Taq (Tli RNaseH Plus) and SYBR Premix Ex Taq II (Tli RNaseH Plus). These 2X premixes are based on the existing highly efficient SYBR Premix Ex Taq (Perfect Real Time) (#RR041A) and SYBR Premix Ex Taq II (Perfect Real Time) (#RR081A), respectively. The new premixes are developed to address the inhibitory effect on the PCR due to the presence of remaining RNA after cdna synthesis. This inhibition is seen particularly for GC rich templates and genes with poor expression when large amount of cdna is necessary. Tli RNaseH, added to the qpcr premix, removes the inhibition without compromising the efficiency of the reaction, as shown in the figures below. Therefore these new premixes allow excellent quantification of any cdna template and thus unparalleled accuracy in gene expression studies. #RR420A (200 x 50µL reactions) SYBR Premix Ex Taq (Tli RNaseH Plus) (2X conc.) * 5 x ROX Reference Dye (50X conc.) 200 µl ROX Reference Dye II (50X conc.) 200 µl #RR820A (200 x 50µL reactions) SYBR Premix Ex Taq II (Tli RNaseH Plus) (2X conc.) * 5 x ROX Reference Dye (50X conc.) 200 µl ROX Reference Dye II (50X conc.) 200 µl *contains Ex Taq HS DNA Polymerase, dntp mix, Mg 2+,SYBR Green I and Tli RNase H Current PCR Reagent PCR primer RT reaction PCR is inhibited by the strong binding of mrna and cdna mrna cdna Oligo dt Random primer New Products Tli RNaseH Plus PCR cycles mrnas are digested immediately in a PCR solution by the strong heat stable Tli RNaseH Note The ROX Reference Dye/Dye II is supplied for performing normalization of fluorescent signal intensities within wells when used with real time PCR instruments which have this option. For ABI PRISM 7000/7700/7900HT and Applied Biosystems 7300 Real-Time PCR Systems, the use of ROX Reference Dye (50X) is recommended. For the Applied Biosystems 7500 Real-Time PCR system, use of ROX Reference Dye II is recommended. The use of ROX Reference Dye or Dye II is optional, and not required when using Smart Cycler and LightCycler real time instruments Cycles Serial dilutions of cdna synthesized with low RNaseH RTase, were subjected to amplification with SYBR premix including Tli RNaseH (blue line) or without RNaseH treatment (red line). Target gene VGLL4 (GC content: 65%). The clear shift to the left of the amplification curves obtained when using SYBR Premix Ex Taq II (Tli RNaseH Plus) indicates better amplification efficiency (blue line). This results from the degradation of residual mrna annealed to target DNA by the thermostable Tli RNaseH, and thus removal of the observed inhibitory effect. Coming soon: Premix Ex Taq (Probe qpcr) from July 2011, Premix Ex Taq (Probe qpcr) (RR390A; 5 x 1 ml), a 2X Premix for TaqMan probe detection including Tli RNaseH: a qpcr premix suitable for fast reaction giving accurate quantification of the target over a wide dynamic range. 4 See back cover for part numbers and full contact information

5 SYBR Premix Ex Taq (Perfect Real Time) Versatility: Compatible with SmartCycler, LightCycler, ABI 7000 & 7700, RotorGene, Mx3000P and other real time PCR instruments. High Sensitivity: Detects as few as 100 target copies. Wide Dynamic Range: Possesses a dynamic range of 7-8 orders of magnitude (l DNA template). Accurate Quantification: Produces excellent standard curves using numerous real time instruments. Convenient: A separate tube of ROX reference dye is supplied. SYBR Premix Ex Taq (Perfect Real Time) Amplification Curve using a Smart Cycler High sensitivity and specificity real time PCR quantitation of DNA using SYBR Green I Low Ct value qpcr TaKaRa SYBR Premix Ex Taq (Perfect Real Time) is a convenient (2X) premix consisting of Takara s high fidelity, high performance Ex Taq Hot Start Version, SYBR Green I, and an optimized real time buffer which provides superior specificity and increased amplification efficiency for real time PCR. Antibodymediated hot start technology prevents nonspecific amplification due to mispriming and/or formation of primer dimers during the reaction assembly. The Taq antibody-polymerase complex is denatured in the first cycling step, releasing the polymerase and allowing DNA synthesis to proceed. SYBR Premix Ex Taq (Perfect Real Time) has a dynamic range of 7-8 orders of magnitude and sensitivity to 100 copies. Compatible cyclers when using SYBR Premix Ex Taq (Perfect Real Time) include the SmartCycler, Light Cycler, ABI PRISM 7000/7700/7900 HT, Applied Biosystems 7300/7500, icycler, MJ Opticon, and the Stratagene Mx3000P. Additionally, two ROX reference dyes are also supplied as separate components. These serve as convenient internal reference standards for use in normalizing signals due to non-pcr related fluorescence fluctuations that occur either between wells or over time in different instruments. SYBR Premix Ex Taq (Perfect Real Time) provides superior specificity, performance and amplification yield for real time PCR on all major real time instruments. Real Time PCR (qpcr) SYBR Premix Ex Taq (Perfect Real Time) Amplification Curve using a Applied Biosystems 7500 Real Time System #RR041A (200 reactions) SYBR Premix Ex Taq Mix (2X conc.) * 1 x 5 ml ROX Reference Dye I (50X conc.) 200 µl ROX Reference Dye II (50X conc.) 200 µl *contains Ex Taq HS DNA Polymerase, dntp mix, Mg 2+ and SYBR Green I SYBR Premix Ex Taq (Perfect Real Time) Amplification Curve using a MX3000P (Stratagene) Excellent Amplification Curves Generated using SYBR Premix Ex Taq with Several qpcr Instruments. Note The ROX Reference Dye/Dye II is supplied for performing normalization of fluorescent signal intensities within wells when used with real time PCR instruments which have this option. For ABI PRISM 7000/7700/7900HT and Applied Biosystems 7300 Real-Time PCR Systems, the use of ROX Reference Dye (50X) is recommended. For the Applied Biosystems 7500 Real-Time PCR system, use of ROX Reference Dye II is recommended. The use of ROX Reference Dye or Dye II is optional, and not required when using Smart Cycler and LightCycler real time instruments. See back cover for part numbers and full contact information 5

6 REAL TIME PCR SYBR Premix Ex Taq II (Perfect Real Time) Real Time PCR (qpcr) Higher Specificity: No more non-specific amplification from primer dimers, even when starting with a minute amount of template. Wide Range of Detection: Accurate quantification over 8 logs of magnitude. Simple Protocol: Ready to use premix type. Short reaction time due to the antibody mediated hot start technology. Fast Reaction Times: Shorter reaction times due to optimized buffer. Applicable for use with Fast PCR instruments. Versatility: Use on any qpcr instrument. High sensitivity and specificity real time PCR quantitation of DNA using SYBR Green I Low Ct value qpcr SYBR Premix Ex Taq II (Perfect Real Time) features an optimized buffer formulation which limits nonspecific amplification and primer dimer formation. It also includes a hot start PCR enzyme, TaKaRa Ex Taq HS (which contains an anti-taq-antibody) for high specificity. This combination provides extremely high specificity, sensitivity, and accurate quantification over a wide range of template concentrations. In addition, SYBR Premix Ex Taq II is compatible with a variety of real-time PCR instruments and high speed qpcr applications. #RR081A (200 reactions) SYBR Premix Ex Taq II (Perfect Real Time) (2X conc.) * 5 X ROX Reference Dye (50 X) 200 µl ROX Reference Dye II (50 X) 200 µl *Contains Takara Ex Taq HS, dntp Mixture, Mg 2+, and SYBR Green I Application Examples for SYBR Premix Ex Taq II A SYBR Premix Ex Taq SYBR Premix Ex Taq Comparison between SYBR Premix Ex Taq (Perfect Real Time) and SYBR Premix Ex Taq II (Perfect Real Time) was performed using Takara s Thermal Cycler Dice Real Time System. The reaction specificity was compared between the two SYBR products. The primer pairs that were selected for this experiment were primers that usually exhibit problems commonly encountered with real-time PCR such as primer dimerization (A) and non-specific amplifcation(b). SYBR Premix Ex Taq II overcomes primer dimerization due to a modified buffer system (see figure on right). B Reaction Set Up Instrument used: Thermal Cycler Dice Real Time System* (*Not available in Europe & US) Program: Melting Curve Analysis Reaction Conditions: Template: cdna dilution series (6.4 pg-100 ng) 95 C 10 sec total RNA from HL60 cells or dh C 5 sec 40 cycles 60 C 30 sec Target Genes: (A) Human YWHAZ (B) Human RPLP2 6 See back cover for part numbers and full contact information

7 Premix Ex Taq (Perfect Real Time) Versatility: Compatible with SmartCycler, LightCycler ABI PRISM 7000/7700/7900 HT, Applied Biosystems 7500 Real-Time PCR Systems, Mx3000P and other real time PCR instruments. High Sensitivity: Detects as few as 10 target copies. Wide Dynamic Range (Refer to graphs below) Accurate Quantitation: Produces excellent standard curves with numerous real time instruments. Convenient: Two tubes of ROX reference dye are supplied. Application(s) Real time PCR using either Probes or SYBR Green I Fast qpcr Premix Ex Taq (Perfect Real Time) is a 2X premix specially designed for high speed, high sensitivity real-time PCR using either detection probes (e.g. TaqMan ) or SYBR Green I (not included). It consists of Takara s high performance Ex Taq Hot Start DNA Polymerase, and an optimized real-time buffer which provides superior specificity and increased amplification efficiency for real-time PCR. Antibody-mediated hot start technology prevents nonspecific amplification due to mispriming and/or formation of primer dimers during the reaction assembly. The Taq antibody-polymerase complex is denatured in the first cycling step, releasing the polymerase and allowing DNA synthesis to proceed. Premix Ex Taq (Perfect Real Time) has a dynamic range of 7-8 orders of magnitude for SYBR Green I detection and 10 orders of magnitude with probe detection, as well as sensitivity to 100 traget copies for SYBR Green I and 10 target copies for probe detection. Compatible cyclers include the SmartCycler, LightCycler, ABI PRISM 7000/7700/ 7900 HT, Applied Biosystems 7300/7500, icycler, MJ Opticon, and the Stratagene Mx3000P. Additionally, two tubes of ROX reference dyes are supplied as separate components. ROX dye is a convenient internal reference standard for use in normalizing signals due to non-pcr related fluorescence fluctuations that occur either between wells or over time. Premix Ex Taq (Perfect Real Time) provides superior specificity, performance and amplification yield for real-time PCR on all major real-time instruments. Real Time PCR (qpcr) Amplification curve (top panel) and standard curve (bottom panel) for Premix Ex Taq (Perfect Real Time) using the TaqMan Gene Expression Assay on the Applied Biosystems 7500 Real-Time PCR System. #RR039A (200 reactions) Premix Ex Taq Mix (2X conc.)* 5 x ROX Reference Dye (50X conc.) 200 µl ROX Reference Dye II (50X conc.) 200 µl *Contains Ex Taq HS DNA Polymerase, dntp mix, Mg 2+ Note The ROX Reference Dye/Dye II is supplied for performing normalization of fluorescent signal intensities within wells when used with real time PCR instruments which have this option. For ABI PRISM 7000/7700/7900HT and Applied Biosystems 7300 Real-Time PCR Systems, the use of ROX Reference dye (50X) is recommended. For the Applied Biosystems 7500 Real-Time PCR system, use of ROX Reference Dye II is recommended. The use of ROX Reference Dye or Dye II is optional, and not required when using Smart Cycler and LightCycler real time instruments. See back cover for part numbers and full contact information 7

8 One Step SYBR PrimeScript RT-PCR Kit (Perfect Real Time) RT-qPCR Easy & Efficient: One Step RT-PCR lowers pipeting and contamination risks. Sensitive: Accurate quantification of any RNA. One Step SYBR PrimeScript RT-PCR Kit (Perfect Real Time) is designed for real time, one-step RT-PCR using SYBR Green I (1)(2) detection and is suitable for detection of small amounts of RNA, i.e., viral RNA. In this kit, RT-PCR can be performed in a single tube minimizing contamination. Also, amplified products are monitored in real time, so there is no need to verify results through electrophoresis after PCR. This kit uses PrimeScript RTase, a robust reverse transcriptase which can quickly and efficiently synthesize cdna, and TaKaRa s Ex Taq HS, a high efficiency hot start PCR enzyme. These enzymes have been optimized for One Step RT-qPCR. #RR066A (reaction size: 50 µl) 2X One Step SYBR RT-PCR Buffer III * μl TaKaRa Ex Taq HS 5U /μl 100 μl PrimeScript RT Enzyme Mix II ** 100 μl RNase Free dh 2 O ml ROX Reference Dye 50X conc. *** 100 μl ROX Reference Dye II 50X conc. *** 100 μl * Includes dntp Mixture, Mg 2+ and SYBR Green I ** Includes RNase Inhibitor and RTase Note *** ROX Reference Dye/Dye II is used for normalization of fluorescence intensity by background subtraction. For ABI PRISM 7000/7700/7900HT and Applied Biosystems 7300 Real-Time PCR System, the use of ROX Reference Dye (50X) is recommended. For Applied Biosystems 7500 Real-Time PCR System, the use of ROX Reference Dye II is recommended. The use of ROX Reference Dye or Dye II is optional. It isnot required for use with Thermal Cycler Dice Real Time System, Smart Cycler or LightCycler real time instruments. 1 SYBR Green I is licensed by Molecular Probes Inc. for research reagents. SYBR is a registered trademark of Molecular Probes Inc.. 2 Use One Step PrimeScript RT-PCR Kit (Perfect Real Time) for real time one-step RT-PCR using TaqMan probe for detection. TaqMan is a registered trademark of Roche Molecular Systems Inc. Not available in the United States One Step SYBR PrimeScript RT-PCR Kit II (Perfect Real Time) Easy & Efficient: One Step RT-PCR lowers pipeting and contamination risks. Specificity: Improved buffer and hot start polymerase. Sensitive: Accurate quantification of any RNA inclucing RNA viruses or small amounts of RNA. One Step SYBR PrimeScript RT-PCR Kit II (Perfect Real Time) is designed for Real-Time One Step RT-PCR including SYBR Green I (1)(2). This kit is suitable for detection of minute amounts of RNA such as RNA viruses. This kit uses PrimeScript RTase, which has excellent elongation ability and can efficiently synthesize cdna in short periods of time, and TaKaRa Ex Taq HS high efficiency/hot start PCR enzyme. It is optimized for one step RT-PCR, combining the TaKaRa Bio RT-PCR technology with these enzymes allows an increase in the yield of RT-PCR product. This kit contains an improved buffer system which results in improved reaction specificity compared to the One Step SYBR PrimeScript RT-PCR Kit (Perfect Real Time) (Cat.# RR066A). Premixed components allow simple and convenient reaction assembly. #RR086A (reaction size: 50 µl) TaKaRa Ex Taq HS 5 U/μL 2X One Step SYBR RT-PCR Buffer IV * 840 μl 3 PrimeScript 1 step enzyme Mix II ** 200 μl RNase Free dh 2 O 1.25 ml 2 ROX Reference Dye *** 50X conc. 100 μl ROX Reference Dye II *** 50X conc. 100 μl * Includes dntp Mixture, Mg 2+ and SYBR Green I ** Includes PrimeScript RTase, RNase Inhibitor, TaKaRa Ex Taq HS Note *** 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 (50X) is recommended. For Applied Biosystems 7500 Real-Time PCR System, the use of ROX Reference Dye II is recommended. The use of ROX Reference Dye or Dye II is optional. It is not required for use with Thermal Cycler Dice Real Time System, Smart Cycler or LightCycler real time instruments. 1 SYBR Green I is licensed by Molecular Probes Inc. for research reagents. SYBR is a registered trademark of Molecular Probes Inc.. 2 Use One Step PrimeScript RT-PCR Kit (Perfect Real Time) for real time one-step RT-PCR using TaqMan probe for detection. TaqMan is a registered trademark of Roche Molecular Systems Inc. Not available in the United States 8 See back cover for part numbers and full contact information

9 Application Example: Performance of Real-Time 1 Step RT-PCR with SYBR Green I Detection Detection of Rat Rplp2 (ribosomal protein, large, P2) mrna from total RNA using One Step SYBR PrimeScript RT-PCR Kit (Perfect Real Time). Template: Rat liver total RNA 6.4 pg ng (negative control is dh 2 O) Standard curve (amplification in background) Melting curve RT-qPCR Primers: Specific primers supplied through custom service (Perfect Real Time Support System, only available in Japan) One Step PrimeScript RT-PCR Kit (Perfect Real Time) Robust and Efficient: RT-qPCR all in one tube with PrimeScript RT ensuring efficient transcription from any RNA One Step RT-qPCR using the TaqMan Probes One Step PrimeScript RT-PCR Kit (Perfect Real Time) is designed for real time one-step RT-qPCR using TaqMan probe (1,2) detection. In this kit, RT-PCR can be performed in a single tube, minimizing contamination and is suitable for detection of small amounts of RNA, such as a viral RNA. In addition, amplified products are monitored in real time, so there is no need to verify results through electrophoresis after PCR. #RR064A (100 reactions) 2X One Step RT-PCR Buffer III * μl TaKaRa Ex Taq HS 5U/μL 100 μl PrimeScript RT Enzyme Mix II ** 100 μl RNase Free dh 2 O ml ROX Reference Dye *** 50X conc. 100 μl ROX Reference Dye II *** 50X conc. 100 μl * Includes dntp Mixture and Mg 2+. ** Includes RNase Inhibitor and RTase. Note *** 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 (50X) is recommended. For Applied Biosystems 7500 Real-Time PCR System, the use of ROX Reference Dye II is recommended. The use of ROX Reference Dye or Dye II is optional. It is not required for use with Thermal Cycler Dice Real Time System, Smart Cycler or LightCycler real time instruments. 1 TaqMan is a registered trademark of Roche Molecular Systems Inc. 2 Use One Step SYBR PrimeScript RT-PCR Kit (Perfect Real Time) for real time one-step RT-PCR using SYBR Green I for detection. Not available in the United States PrimeScript RT Reagent Kit (Perfect Real Time) Fast, Efficient cdna Synthesis: Ready for Real- Time PCR. This kit is best suited for two-step, real-time RT-PCR. Includes Random 6 mers and Oligo dt Primer for use as reverse transcription primers. The PrimeScript RT-PCR Kit is a 2-step RT reagent kit featuring PrimeScript RTase, a new reverse transcriptase which offers robust reverse transcription on any RNA template. PrimeScript RTase is a MMLV-based RTase which provides complete elongation through any RNA template containing higher-order structures. It produces full-length cdnas of up to 12 kb in length with good yield. The kit is simple to use and is suitable for high through-put analysis. #RR037A (200 reactions) 5X PrimeScript Buffer (for Real Time) * 400 μl PrimeScript RT Enzyme Mix I 100 μl Oligo dt Primer 50 μm 100 μl Random 6 mers 100 μm 100 μl RNase Free dh 2 O EASY Dilution (for Real Time PCR) ** * Contains dntp Mixture and Mg 2+. ** This solution can be used to prepare the dilution series of total RNA or cdna for establishing a standard curve. In contrast to dilution with water or TE, EASY Dilution facilitates accurate low concentration dilution. The Easy Dilution does not inhibit either reverse transcription or PCR enzyme activity. The diluted template solutions can be used as the templates for reverse transcription or PCR reactions. EASY Dilution is also available separately (Cat.# 9160). Note Easy Dilution has been tested with TaKaRa Bio s real-time PCR reagent. Compatibility with products from other manufacturers has not yet been verified. Not available in the United States See back cover for part numbers and full contact information 9

10 RT-PCR PrimeScript One Step RT-PCR Kit, Version 2 Robust and Efficient: PrimeScript 1 step Enzyme Mix with TaKaRa Ex Taq HS featuring specific and sensitive amplification Application Powerful One Step RT-PCR The PrimeScript One Step RT-PCR Kit, Ver. 2 is a singletube RT-PCR kit featuring PrimeScript RTase, a reverse transcriptase which offers robust reverse transcription on any RNA template. PrimeScript RTase is a MMLV-based RTase which provides complete elongation through higherorder RNA template structures. This enzyme works well on challenging templates at 50 C, making high-temperature transcription, which increases the risk of RNA degradation, unnecessary. It produces full-length cdnas of up to 12 kb in length with good yield. The kit also includes TaKaRa s Ex Taq HS for efficient, sensitive, and specific amplification. The combination of PrimeScript with TaKaRa Ex Taq HS results in reliable, efficient, reproducible RT-PCR amplification from a large variety of RNA templates. The singletube, single step format utilizing the premix components (PrimeScript One Step Enzyme Mix, 2X One Step Buffer) provides simple and efficient reaction assembly. It also eliminates the need for reagent addition during the reaction process, minimizing the risk of contamination. (50 reactions): #RR055A PrimeScript One Step Enzyme Mix 100 μl 2X One Step Buffer μl Control F-1 Primer * 20 μm 20 μl Control R-1 Primer ** 20 μm 20 μl Positive Control RNA 2 x 10 5 copies/μl 20 μl RNase Free dh 2 O μl * Upstream sense primer for Positive Control RNA ** Downstream anti-sense primer for Positive Control RNA Not available in the United States PrimeScript 1st Strand cdna Synthesis Kit Excellent Elongation: able to synthesize long cdnas (up to 12 kb) with good yield. Robust and Efficient: carries out reverse transcription at standard RT temperature (42 C), even when RNA template contains high-order structures. Application Synthesis of Full Length 1st Strand cdna From Total or poly(a)+ RNA The PrimeScript 1st Strand cdna Synthesis Kit contains all of the reagents necessary for synthesis of first-strand cdna from total or poly(a)+ RNA using PrimeScript PrimeScript II 1st Strand cdna Synthesis Kit Application Synthesis of Full Length 1st Strand cdna From Total or poly(a)+ RNA PrimeScript II 1st strand cdna Synthesis Kit contains all the reagents necessary to synthesize 1st strand cdna from total or poly(a)+ RNA using PrimeScript II RTase. A major factor that interferes with cdna synthesis is non-specific binding of reverse transcriptase to higher structure RNA (RNA with complex structure or longer fragments as templates). In addition, non-specific elongation due to mis-priming of the RT can cause problems in RT-PCR or the production of full-length cdna. PrimeScript II RTase, which contains an accessory protein similar RTase. PrimeScript RTase is a MMLV-based RTase which possesses excellent elongation ability and is capable of synthesis of cdna of up to 12 kb in length with good yield. In addition, this enzyme works well on even challenging templates at 42 C, making high-temperature transcription, which increases the risk of RNA degradation, unnecessary. First strand cdnas synthesized with this kit can be used for a variety of applications including second strand synthesis, hybridization, PCR amplification, and real-time PCR. (50 reactions): #6110A PrimeScript RTase 200 U/μL 50 μl 5X PrimeScript Buffer 200 μl RNase Inhibitor 40 U/μL 25 μl dntp Mixture 10 mm each 50 μl Oligo dt Primer 50 μm 50 μl Random 6 mers 50 μm 100 μl RNase free dh 2 O Not available in the United States to PrimeScript RTase, is a reverse transcriptase which decreases these problems in cdna synthesis reactions. This kit is useful to produce high-quality, full-length cdna. (50 reactions): Not available in the United States #6210A PrimeScript II RTase 200 U/μL 50 μl 5X PrimeScript II Buffer 200 μl RNase Inhibitor 40 U/μL 25 μl dntp Mixture 10 mm each 50 μl Oligo dt Primer 50 μm 50 μl Random 6mers 50 μm 100 μl RNase free dh 2 O 10 See back cover for part numbers and full contact information

11 PrimeScript RT-PCR Kit Complete Elongation: PrimeScript RTase works efficiently on higher structured RNA templates. High Yield of Full-Length cdna: PrimeScript RTase offers high yield transcription of full length cdnas of up to 12 kb High Specificity, Efficient Elongation: RT-PCR kits combining PrimeScript RTase and TaKaRa Ex Taq HS minimizing false priming and allowing robust amplification from fragments of varying sizes. Application Robust Reverse Transcription and Amplification of Full-Length cdnas even from Challenging RNA Templates The PrimeScript RT-PCR Kit is a 2-step RT-PCR kit featuring PrimeScript RTase, a reverse transcriptase which offers robust reverse transcription on any RNA template. PrimeScript RTase is a MMLV-based RTase which provides complete elongation through any RNA template containing higher-order structures. This enzyme works well on challenging templates at 42 C, making high-temperature transcription, which increases the risk of RNA degradation, unnecessary. It produces full-length cdnas up to 12 kb in length with good yield. The kit also includes TaKaRa s Ex Taq HS for efficient, sensitive, and specific amplification. The combination of PrimeScript with TaKaRa Ex Taq HS results in reliable, efficient, reproducible RT-PCR amplification from a large variety of RNA templates.regions or complex structures. This kit includes all reagents necessary for the reverse transcription of RNA to cdna and cdna amplification for end-point PCR. (50 reactions): #RR014A PrimeScript RTase (for 2-step) 25 μl 5X PrimeScript Buffer 200 μl RNase Inhibitor 40 U/μL 25 μl dntp Mixture 10 mm each 150 μl Oligo dt Primer 2.5 μm 50 μl Random 6 mers 20 μm 50 μl TaKaRa Ex Taq HS 5 U/μL 25 μl 10X PCR Buffer II 250 μl Control F-1 Primer ** 20 μm 10 μl Control R-1 Primer *** 20 μm 10 μl Positive Control RNA copies/μl 20 μl RNase Free dh 2 O * 50 reactions of [Reverse transcription 20 μl g PCR 50 μl] ** Upstream sense primer for Positive Control RNA *** Downstream anti-sense primer for Positive Control RNA Not available in the United States RT-PCR High Fidelity PrimeScript RT-PCR Kit Not available in the United States Accurate and Robust: Reverse transcription and amplification of full length cdnas even from challenging RNA templates Application Designed for RT-PCR with High Fidelity The High Fidelity PrimeScript RT-PCR Kit is a 2-step RT-PCR kit designed to produce and amplify cdna templates using PrimeScript RTase and PrimeSTAR HS DNA Polymerase. The kit features PrimeScript RTase, a new reverse transcriptase which offers robust reverse transcription on any RNA template. PrimeScript RTase is a MMLV-based RTase which provides complete elongation through any RNA template containing higher-order (i.e., high GC) structures. This enzyme works well on challenging templates at 42 C, making high-temperature transcription, which increases the risk of RNA degradation, unnecessary. It produces full-length cdnas of up to 12 kb in length with good yield. PrimeSTAR HS DNA Polymerase is a high-fidelity DNA Polymerase which provides marketleading accuracy, superior specificity, and excellent amplification efficiency in PCR amplification. The combination of PrimeScript with PrimeSTAR HS DNA Polymerase results in reliable, efficient, reproducible, and accurate RT-PCR amplification from a large variety of RNA templates. (50 reactions): #R027A PrimeScript RTase (for 2-step) 25 μl 5X PrimeScript RT Buffer 200 μl RNase Inhibitor 40 U/μL 25 μl dntp Mixture 10 mm each 100 μl Oligo dt Primer 2.5 μm 50 μl Random 6 mers 20 μm 50 μl PrimeSTAR HS DNA Polymerase 2.5 U/μL 25 μl 5X PrimeSTAR PCR Buffer 500 μl Control F-1 Primer ** 20 μm 10 μl Control R-1 Primer *** 20 μm 10 μl Positive Control RNA copies/μl 20 μl RNase Free dh 2 O 550 μl * 50 reactions for Reverse Transcription 20 μl g PCR reaction 50 μl ** Upstream sense primer for Positive Control RNA *** Downstream anti-sense primer for Positive Control RNA See back cover for part numbers and full contact information 11

12 HIGH FIDELITY PCR PrimeSTAR HS DNA Polymerase High Fidelity PCR Superior Accuracy: A strong exonuclease activity results in an extremely low error rate, with only 12 of 250,000 bp containing errors as determined by DNA sequence analysis. Excellent Efficiency: High efficiency amplification - even higher than Taq Polymerase. Robust Amplification: Tolerance to varying reaction conditions means a single PCR cycling protocol can be used to amplify products of varying sizes. Excellent for Targeted Demanding Cloning SNP detection High Accuracy Amplifications PrimeSTAR HS (Premix) PrimeSTAR HS DNA Polymerase is a novel high fidelity PCR enzyme which provides maximum fidelity as well as extended product length (8.5 kb for human genomic DNA; 22 kb for λ DNA). Targeted for demanding cloning (i.e. amplification of cdna libraries) and sequencing applications. It offers extremely high accuracy, and fidelity calculated by sequence analysis. It also offers excellent amplification efficiency and shortened reaction times. Finally, the antibody-mediated hot start formulation prevents false initiation events during reaction assembly due to mispriming or primer digestion, thus, lowering background. #R010A (250 U) #R010B corresponds to 4xR010A PrimeSTAR HS DNA polymerase 100 µl 5X PrimeSTAR Buffer (Mg 2+ ) 2 x dntp Mixture 800 µl PrimeSTAR HS DNA Premix is a convenient 2X formulation containing PrimeSTAR HS, PCR Buffer, MgCl 2, and dntps. The 2X premix solution of enzyme and reaction components simplifies reaction assembly, minimizes the risk of contamination, and increases reaction reproducibility. The premix, along with the added benefits of the PrimeSTAR, has excellent efficiency and fidelity from the very strong 3 5 exonuclease activity. In addition, the amplification efficiency is higher than that of standard Taq. This formulation allows quick reaction assembly for high throughput applications with lowered risk of contamination. PrimeSTAR HS DNA Polymerase with GC Buffers PrimeSTAR HS DNA Polymerase with GC Buffers was developed for high-fidelity amplification of high GC ( 75%) templates. PrimeSTAR HS is a unique high fidelity DNA polymerase, offering both maximal accuracy and higher amplification efficiency than Taq Polymerase. The new GC buffer formulation facilitates robust extension through even very high GC regions efficiently and accurately. Inclusion of a monoclonal antibody suppresses both the DNA polymerase and 3 5 exonuclease activities prior to the first denaturing step, preventing false initiation events during reaction assembly and primer digestion. PrimeSTAR HS DNA Polymerase with GC buffers provides reliable amplification, high accuracy, and high specificity in applications where amplification of high-gc DNA templates for cloning or library construction is required. Mutation Frequency Comparison PrimeSTAR HS DNA Polymerase High Fidelity PCR Made Easy Thermococcus kodakaraensis - derived DNA Polymerase Pyrococcus sp. - derived DNA Polymerase Thermus aquaticus - derived DNA Polymerase Fidelity comparison with competitors sequencing results showed only 12/249,941 mismatched bases in DNA fragments amplified using PrimeSTAR HS. 12 See back cover for part numbers and full contact information

13 : PrimeSTAR HS DNA Polymerase PrimeSTAR TM Company N Company B Company I kb Comparison of PrimeSTAR HS Amplification Efficiency with Competitors on a 2 kb Human Genomic DNA Fragment. Superior amplification efficiency was apparent using PrimeSTAR HS on a human genomic (DCLRE1A) 2 kb template. Human genomic DNA was used in the following quantities: Lane 1: 0 ng (dh 2 O), Lane 2: 100 pg, Lane 3: 1 ng, Lane 4: 10 ng, Lane 5: 100 ng. High Fidelity PCR M M2 M M M1: phy Marker Template 1: 0.5 kb DNA: kb 2: 1 kb 98 C 10 sec. 3: 2 kb 60 C 5 sec. 4: 4 kb 72 C 1 min./kb 5: 6 kb DNA: kb 6: 7.5 kb 98 C 10 sec. 7: 8.5 kb 68 C 8 min. M2: λ-hind III digest Template DNA: 100 ng human genomic DNA 30 cycles 30 cycles M: λ-hind III digest 98 C 10 sec. 1: 2 kb 60 C 5 sec. 2: 4 kb 72 C 1 min./kb 3: 6 kb 4: 8 kb 5: 10 kb Template DNA: 100 pg E. coli genomic DNA 30 cycles Amplification of Human Genomic DNA fragments (0.5 to 8.5 kbp) using PrimeSTAR HS DNA Polymerase. Amplification of E. coli Genomic DNA Targets using PrimeSTAR HS DNA Polymerase. Application: PrimeSTAR HS DNA Polymerase with GC buffers PrimeSTAR with GC Company A Company B Company C 3 M M M M M 3005 bp Amplification of a 3005 bp high-gc (73.2%) TthHB8 Genomic DNA Template. The performance of high fidelity, high-gc enzymes from Companies A, B, and C were compared with PrimeSTAR HS DNA Polymerase with GC Buffer. Lanes 1, 2, and 3: 100 pg, 1 ng, 10 ng genomic DNA template. See back cover for part numbers and full contact information 13

14 HIGH SPEED PCR SpeedSTAR HS DNA Polymerase High Speed PCR High Speed Amplification: Amplify a 2 kb fragment in as little as 30 minutes. Excellent Efficiency: Robust performance, comparable to high yield polymerases. No Special Instrument Needed: Cut reaction times by two-thirds without purchasing a specialized instrument. Long Fragments: Optimized two-buffer system allows amplification of fragments up to 20 kb with reduced optimization. Amplifications of long fragments in a reduced period of time Fast PCR on any PCR cycler High efficiency amplifications in 1/3 the time SpeedSTAR HS DNA Polymerase is a convenient, efficient DNA polymerase specially designed for fast PCR. Extension times in as short as 10 sec/kb are possible, (compared to 60 sec/kb with general enzymes), dramatically reducing total reaction times. SpeedSTAR reactions can be performed using standard PCR instruments, and the robust two-buffer system facilitates efficient amplification of varying size fragments (up to 20 kb) with less optimization than other polymerases. Additionally, the hot start formulation provides increased specificity and reduced background. #RR070A (250 U) SpeedSTAR HS DNA polymerase (5 units/µl) 50 µl 10X Fast Buffer I (Mg 2+* ) 10X Fast Buffer II (Mg 2+* ) dntp Mixture (ea. 2.5 mm) 800 µl *Mg 2+ Concentration: 10X Fast Buffer I, 30mM; 10X Fast Buffer II, 20mM. M M SpeedSTAR HS DNA Polymerase M λhin d III digest 1. 1 kb 2. 2 kb 3. 4 kb 4. 6 kb 5. 8 kb kb kb kb M λhin d III digest Target Genome: E. coli Fragment size Target genome 1 kb-2 kb E. coli 4 kb- 6 kb E. coli 8 kb- 10 kb E. coli 18 kb-20 kb E. coli Standard PCR 96 min (2-step) 226 min (2-step) 346 min (2-step) 8 hrs 16 min (2-step) SpeedSTAR HS Polymerase 33 min 53 min 83 min 3 hrs 29 min M M Standard High Efficiency Hot Start PCR Enzyme M λhin d III digest 1. 1 kb 2. 2 kb 3. 4 kb 4. 6 kb 5. 8 kb kb kb kb M λhin d III digest Table 1: Time Comparison of SpeedSTAR and Standard High Efficiency Enzyme Reaction Times on E. coli Fragments of Varying Sizes. (2-step refers to PCR cycle conditions) Target Genome: Human Fragment size Target genome 8.5 kb Human Standard PCR 4 hrs 59 min (2-step) SpeedSTAR HS Polymerase 1 hr 40 min Amplification Efficiency of SpeedSTAR and a Standard High Efficiency PCR Enzyme was Performed using 8 Different Fragment Sizes. Eight different E. coli genomic DNA targets were amplified using SpeedSTAR and a standard high efficiency enzyme using the Takara DICE* thermocycler. Fast Buffer I was used in lanes 1 and 2; Fast Buffer II was used in lanes kb Human 8 hrs 16 min (2-step) 3 hr 29 min Table 2: Time Comparison of SpeedSTAR and Standard High Efficiency Enzyme Reaction Times on Large Size Human Genomic Targets. (2-step refers to PCR cycle conditions) *Purchase of this instrument conveys a limited non-transferable immunity from suit for the purchaser s own internal research and development and applied fields other than human in vitro diagnostics under one or more of U.S. Patents No. 5,038,852, 5,656,493, 5,333,675, 5,475,610, and 6,703,236 (claims 1-6 only), or corresponding claims in their non-u.s. counterparts, owned by Applera Corporation. No right is conveyed expressly, by implication or by estoppel under any other patent claim, including under U.S. Patent No. 6,814,934, which claims thermal cycler apparatus and systems capable of real-time detection. 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. Applied Biosystems does not guarantee the performance of this instrument. 14 See back cover for part numbers and full contact information

15 HIGH SPEED LOAD N GO PCR SapphireAmp Load N Go PCR SapphireAmp Fast PCR Master Mix (#RR350A) Faster than Standard Taq: reactions completed in 1/2 the time of conventional Taq. Restriction Enzyme Digestion can be directly performed on PCR Products in the PCR buffer. PCR Reaction can be directly loaded onto a Gel without purification or addition of other reagents. Saves Time, Convenient and Eliminates Purification Steps: with no loss in yields. This high speed PCR mix includes an optimized buffer, hot start PCR enzyme, dntp mixture, gel loading dye (blue) and a density reagent in a 2X premix. Only primers and DNA template need to be added to easily complete the reaction. The mix can be used for fast PCR applications, and PCR reactions performed with this mix can be loaded directly onto a gel for electrophoresis. This product can be used for E.colibased colony PCR to check inserts up to ~5 kb. Human Genomic DNA targets up to 6 kb can be amplified using this product. Amplification of human genomic DNA of 2 kb can be completed in approximately 1 hour. Load N Go PCR #RR350A (160 x 50 µl reaction) SapphireAmp Fast PCR Master Mix x 4 dh 2 O x 4 Takara SapphireAmp Fast Company A Hot Start Master Mix Company B Hot Start Dye Mix M M M M1 Template: Human Genome 100ng/50µL Targets: 1: p kb (54%) 2: FFAR2 1.0 kb (59%) 3: DCLRE1A 2.0 kb (38%) 4: p kb (48%) 5: IGF2R 5.9 kb (50%) Sapphire Amp Cycle Count Lane: 1-3 ( kb) 94 C 1min. 98 C 5 sec. 55 C 5 sec. 30 Cycles 72 C 10 sec./kb Sapphire Amp Cycle Count Lane: 4, 5 (4.2, 5.9 kb) 94 C 1min. 98 C 5 sec. 68 C 30 sec./kb 30 Cycles Company A and B Recommended protocol 1 min/kb Watch for More Dye Premix Products from Takara! (p.19) See back cover for part numbers and full contact information 15

16 LONG PCR TaKaRa s LA Taq DNA Polymerase Long PCR Long Amplifications: Up to 30 kb for human genomic and complex template DNA or 48 kb for λdna. Improved Fidelity: 6.5X better than Taq. Minimal Optimization Required. Excellent Yield Large quantities of desired template. Long PCR (up to 48 kb on λ templates, 30 kb on genomic templates) GC-rich templates, secondary structures, difficult templates High polysaccharide (plant) or dirty samples PCR from genomic DNA of various organisms (C. elegans, Helicobacter pylori, beluga whale, zebra fish, Arabidopsis, Mycobacterium bovis, etc.) akara LA Taq A B TaKaRa LA Taq is a mixture of Taq Polymerase with a proofreading polymerase optimized for amplification of long DNA templates. Using LA Taq, routine extension to 20 kb and up to 48 kb is possible on some templates, with less optimization and greater product yield than other long PCR systems. Because of the presence of the proofreading polymerase, the fidelity is significantly better (6.5X) than that of Taq Polymerase alone. #RR002M TaKaRa LA Taq 250 U (5 U/µL) * 10X LA PCR Buffer II (contains 25 mm MgCl 2 ) dntp Mixture (2.5 mm each dntp) 800 µl #RR002A TaKaRa LA Taq 125 U (5 U/µL) * 10X LA PCR Buffer II (without Mg 2+ ) 25 mm MgCl 2 dntp Mixture (2.5 mm each dntp) 400 µl *Protocol recommends the use of 2.5 U per 50 μl reaction. LA PCR Kit, Version 2.1 Amplification of large DNA templates (30 kb genomic or 48 kb λ DNA) Longer and more accurate genomic PCR products Amplification of difficult long fragment Amplification of DNA fragments from kb in size (different primer sets) using LA Taq LA Taq DNA Polymerase was used to amplify the various fragments and generated high product yields, even with very long (28 kb) fragments. Contains All Reagents Required: For optimizing long PCR. Contains both Standard and GC Buffers: For difficult long templates and high GC-content, with strong secondary structure templates. The LA PCR Kit includes all the reagents necessary for amplification of large DNA templates, with routine extension to 30 kb (and up to 48 kb possible with some templates). The Version 2.1 Kit combines TaKaRa s LA Taq DNA Polymerase with an optimized buffer system resulting in longer and more accurate PCR products than conventional PCR Trouble-Free Long PCR Try our LA Taq Hot Start DNA Polymerase (page 18) reagents. High fidelity (6.5-fold better than conventional Taq DNA Polymerase) is facilitated by an efficient 3 5 exonuclease activity. The Version 2.1 Kit contains a control template, primers and markers to ensure premium PCR performance. #RR013A TaKaRa LA Taq 125 U (5 U/µL) 10X LA PCR Buffer II (contains 25 mm Mg 2+ ) 250 µl 10X LA PCR Buffer II (without Mg 2+ ) 250 µl MgCl 2 (25 mm) 500 µl dntp Mixture (2.5 mm each dntp) 400 µl Control Template (100 ng/µl HT29 DNA) 10 µl Control Primer LA3 (10 pmol/µl) * 10 µl Control Primer LA4 (10 pmol/µl) * 10 µl λ-hind III MW Markers (100 ng/µl) 20 µl 2X GC Buffer I (contains 5 mm MgCl 2 ) 1.25 ml 2X GC Buffer II (contains 5 mm MgCl 2 ) 1.25 ml Control Primer GC1 (10 pmol/µl) ** 10 µl Control Primer GC2 (10 pmol/µl) ** 10 µl *Amplifies a 17.5 kb region of the Control Template. **Amplifies a 1,255 bp GC-rich region of the Control Template. 16 See back cover for part numbers and full contact information

17 GC-RICH PCR TaKaRa LA Taq with GC Buffers GC Buffers I & II for High GC and secondary structures. Better Results on Difficult Templates. More accurate amplification of genomic PCR amplification Amplification of GC-rich templates High Efficiency GC-Rich PCR TaKaRa LA Taq is a mixture of Taq Polymerase with a proofreading polymerase optimized for amplification of long DNA templates. Using LA Taq, routine extension to 20 kb is possible (and up to 48 kb on some templates), with less optimization than other long PCR systems. Because of the presence of the proofreading polymerase, fidelity is significantly better (6.5X) than Taq Polymerase alone. The GC-optimized Buffers I and II are specifically designed to amplify DNA templates with high GC content or a significant amount of secondary structure. GC Buffer I is recommended for amplification of fragments 5kb, GC Buffer II is recommended for 2 3 kb fragments. #RR02AG TaKaRa LA Taq 125 U (5 U/µL) * 2X GC Buffer I (contains 5 mm MgCl 2 ) 1.25 ml 2X GC Buffer II (contains 5 mm MgCl 2 ) 1.25 ml dntp Mixture (2.5 mm each dntp) 400 µl *Protocol recommends the use of 2.5 U per 50 µl reaction. Long PCR and GC-Rich PCR M M2 M bp -262 bp Comparison of Amplification Efficiency between LA Taq with GC Buffer and LA Taq using a GC-rich Target Fragment. A 2.1 kb mouse rrna gene with 63.4% GC was amplified from 1 ng of mouse genomic DNA The results show LA Taq with GC Buffer gave optimal results in amplification of the 2.1 kb fragment with both excellent yields and high specificity. M1.: λ-hind III digest 1 & 2: LA Taq / LA PCR Buffer II 3 & 4: LA Taq with GC Buffer/ GC Buffer I 5 & 6: LA Taq with GC Buffer/ GC Buffer II M2: phy Marker Amplification of a Huntington s Disease Gene (high GC content). Purified human genomic DNA (100 ng in a 50 µl reaction) was used as a template for PCR with LA Taq DNA Polymerase and either LA PCR Buffer II (lane 1), GC Buffer I (lane 2), or GC Buffer II (lane 3), or with a competing DNA Polymerase and high GC Kit (lane 4). The primers amplified regions of the HD gene IT15 CAG repeat. The sizes of the amplified products were 262 bp (GC content 73%), and 358 bp (GC content 71.5%). Lane M contains a 100 bp ladder. PCR condition: 94 C 4 min 95 C 40 sec 70 C 3 min 40 cycles See also PrimeSTAR HS with GC Buffers (page 12) See back cover for part numbers and full contact information 17

18 Routine, High Performance and Yield PCR ROUTINE, HIGH PERFORMANCE AND YIELD PCR TaKaRa Ex Taq DNA Polymerase Sensitivity and Efficiency: Start with less DNA template and make more product than Taq. Reliability/Reproducibility: Tolerant to variations in template quality and quantity. Minimal Optimization Required. Wide Length Range: Makes both small (<100 bp) and large (up to 20 kb) products. Amplify DNA fragments of varying sizes (<100 bp, up to 30 kb λ DNA, 20 kb genomic DNA) Amplify Dirty DNA, difficult templates and high polysaccharide samples (plant) High sensitivity, low abundance, high-yield PCR (works well for microarray production of DNA) PCR from genomic DNA of various organisms (Drosophila, cow rumen, beluga whale, puffer fish, Arabidopsis) TaKaRa Ex Taq combines the proven performance of TaKaRa Taq with an efficient 3 5 exonuclease activity for unsurpassed PCR performance. In routine PCR applications, the TaKaRa Ex Taq and Ex Taq Buffer system gives higher yields and lower mutation rates (approximately 4.5X lower, as determined by the Kunkel method) than standard Taq DNA Polymerase. The system also allows amplification of longer products than Taq DNA Polymerase, with 20 kb lengths possible from genomic DNA and up to 30 kb possible from λ DNA. #RR001A TaKaRa Ex Taq 250 U (5 U/µL) * 10X Ex Taq Buffer (contains 20 mm MgCl 2 ) dntp Mixture (2.5 mm each dntp) 800 µl #RR01AM TaKaRa Ex Taq 250 U (5 U/µL) * 10X Ex Taq Buffer (without MgCl 2 ) 25 mm MgCl 2 dntp Mixture (2.5 mm each dntp) 800 µl *Protocol recommends the use of 1.25 U per 50 µl reaction. M M2 M1: phy Marker 1: 0.5 kb 2: 1 kb 3: 2 kb 4: 4 kb 5: 6 kb 6: 8 kb Amplification of Varying Fragment Sizes of λ Phage DNA using TaKaRa Ex Taq. TaKaRa Ex Taq Premix 7: 10 kb 8: 12 kb 9: 15 kb 10: 20 kb 11: 28 kb 12: 35 kb M2: λ-hindiii digest High Yield and Sensitivity High Yield and Sensitivity: Yield up to 100X greater than Taq; amplify from as little as 5 template copies. Convenient: Premix format allows simple assembly. Decreased Contamination Risk: Reduced pipetting decreases contamination. High throughput amplification Convenient, minimal-assembly amplifications High sensitivity, low abdundance, high-yield PCR (works well for microarray production of DNA) Consistent well to well results TaKaRa Ex Taq Premix contains Ex Taq DNA Polymerase, buffer, Mg 2+ and dntps in a convenient, two-fold concentrate, single-solution format. This solution provides the same high performance as standard TaKaRa Ex Taq. The convenient premixed reagents save time and minimize the possibility of contamination. Reduce Pipetting Errors! #RR003 TaKaRa Ex Taq Premix (2X) * 6 x 500 µl *Contains Ex Taq DNA Polymerase, Ex Taq Buffer, Mg 2+ and dntps. 18 See back cover for part numbers and full contact information