Reference Dye N/A ROX & ROX II (as separate tubes) ROX & ROX II (as separate tubes)

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1 Comparison of TaKaRa s and qrt-pcr Kits SYBR Premix Ex Taq (Tli RNase H+) Premix Ex Taq (Probe qpcr) SYBR Premix Ex Taq II (Tli RNase H+) Cat.# RR420A RR390A RR820A Reactions/Kit 200 (50 µl reaction size) 200 (50 µl reaction size) 200 (50 µl reaction size) Premix or Separates Premix Premix Premix Reaction Volumes ABI LightCycler 0, 25, 50 µl Not Applicable 0, 25, 50 µl Enzyme TaKaRa Ex Taq Hot Start TaKaRa Ex Taq Hot Start TaKaRa Ex Taq Hot Start Buffer (Tli RNaseH) (note: buffer differs from RR390) (note: buffer differs from RR420) (note: buffer differs from RR420) Detection Method SYBR Green I (supplied in premix) SYBR Green I or TaqMan Probes (not supplied) SYBR Green I (supplied in premix) Reference Dye ROX & ROX II (as separate tubes) ROX & ROX II (as separate tubes) ROX & ROX II (as separate tubes) Instruments Supported* Applied Biosystems 7300/7700/7900 HT/7500 real time PCR system, StepOne Plus real time PCR system (Life Technologies) icycler,,, Mx3000P Note: All three enzymes include Tli Rnase H, a heat resistant RNase H Applied Biosystems 7300/7700/7900 HT/7500 real time PCR system, StepOne Plus real time PCR system (Life Technologies),, II, icycler real time PCR system, StepOne Plus real time PCR system (Life Technologies),, II One Step PrimeScript Reagent Kit One Step PrimeScript RT-PCR Kit (Perfect Real One Step SYBR PrimeScript RT-PCR Kit (Perfect Real Time) Time) (Perfect Real Time) Cat.# RR037A RR064A RR066A Reactions/Kit 200 (0 µl reverse transcription) 00 (50 µl reaction) 00 (50 µl reaction) Premix or Separates Buffer + Enzyme Mix Separate components Separate components Reaction Volumes ABI LightCycler Enzyme Buffer Detection Method 0 µl 0 µl PrimeScript RTase + RNase Inhibitor mixed (mix II) 5X PrimeScript Buffer (for Real Time) including Mg 2+ and dntp Mixture SYBR Green I or TaqMan Probes using RR04A or RR039A respectively PrimeScript Enzyme mix II +TaKaRa Ex Taq HS 2X One Step RT-PCR Buffer III, including Mg 2+ and dntp Mixture SYBR Green I or TaqMan Probes (not supplied) PrimeScript Enzyme mix II +TaKaRa Ex Taq HS 2X One Step SYBR RT-PCR Buffer III, including Mg 2+ and dntp Mixture SYBR Green I Reference Dye N/A ROX & ROX II (as separate tubes) ROX & ROX II (as separate tubes) Instruments Supported* ABI PRISM 7000/7700 icycler MJ Opticon MX3000 RotorGene ABI PRISM 7000/7700/7900 HT II ABI PRISM 7000/7700/7900 HT II EASY Dilution Solution (for real time PCR) EASY Dilution Solution (for real time PCR) Cat.# ml Dilute Template DNA or RNA used in Standard for Standard Curve This product is used to dilute template DNA or RNA when preparing serial dilutions of the standard for establishing the standard curve in real time PCR. 20 C. Store this product in aliquots to avoid contamination. 29

2 SYBR Premix Ex Taq (Tli RNase H Plus) SYBR Premix Ex Taq (Tli RNase H Plus) Cat.# RR420A 200 reactions SYBR Premix Ex Taq (Tli RNase H Plus) Cat.# RR420B 400 reactions (A size 2) SYBR Premix Ex Taq (Tli RNase H Plus) Cat.# RR420L 200 reactions SYBR Premix Ex Taq (Tli RNase H Plus) Cat.# RR420W 000 reactions (L size 5) RNase H included in Reaction Mix: Reduces inhibition from mrna/cdna hybrids Eliminates need for RNase H Digestion Step: When using low RNase H RTs Amplification of Longer Fragments: Able to amplify up to 570 bp fragments High Sensitivity: Detects as few as 00 copies Accurate Quantitation: Excellent standard curves for various real time instruments have been established Instrument Compatibility: Compatible with all qpcr instruments SYBR Premix Ex Taq (Tli RNase H Plus) provides reduced PCR inhibition caused by the presence of double stranded RNA/cDNA hybrids remaining after cdna synthesis. This inhibition is common when using low or RNase H minus RTs, and with GC rich templates and/or genes with poor expression. The presence of dsrna/cdna hybrids is a frequent cause of poor qpcr amplification and/or reaction failure. SYBR Premix Ex Taq (Tli RNase H Plus) prevents this potential problem at no additional cost and without requiring a separate RNase H digestion step prior to qpcr. Long Target qpcr Reactions RR420A SYBR Premix Ex Taq (Tli RNase H Plus) (2X conc.) * 5 ml RR420L SYBR Premix Ex Taq (Tli RNase H Plus) (2X conc.) * 5 ml * Contains TaKaRa Ex Taq HS, dntp Mixture, Mg 2+, Tli RNaseH and SYBR Green I. This component is to be used for analyses using a device that corrects fluorescent signals between wells, such as the real-time PCR device by Applied Biosystems. Please use ROX Reference Dye for Applied Biosystems 7900HT/7300 Real-Time PCR System or StepOnePlus and ROX Reference Dye II for 7500 Real-Time PCR System or 7500 Fast Real-Time PCR System. ROX Reference Dye II is compatible with Agilent Mx3000P. This component is not required with II, Smart Cycler System or. 4 C Note : Product is stable for up to 6 months when stored at 4 C. Protect this kit from light and avoid contamination. This kit is shipped in a package with dry ice. Store the kit at 4 C and in darkness. For long-term storage, store at 80 C. (Do not store at 20 C) Store a thawed product at 4 C and use within 6 months. Cq=2 SYBR Premix Ex Taq (Tli RNase H+)(Cat.# RR420) Cq=22 Company A SYBR Master Mix Target: ACTB (533 bp). Results: Superior performance on long fragments and excellent Cq values when using SYBR Premix Ex Taq (Tli RNase H+)(Cat.# RR420) Greater Results with Longer Amplicons: amplify targets up to ~533 bp 30

3 SYBR Premix Ex Taq II (Tli RNaseH Plus) SYBR Premix Ex Taq II (Tli RNase H Plus) Cat.# RR820A 200 reactions SYBR Premix Ex Taq II (Tli RNase H Plus) Cat.# RR820B 400 reactions (A size 2) SYBR Premix Ex Taq II (Tli RNase H Plus) Cat.# RR820L 200 reactions SYBR Premix Ex Taq II (Tli RNase H Plus) Cat.# RR820W 400 reactions (A size 2) Optimized for with SYBR Green I Easy Preparation Extremely High Specificity Minimization of PCR Inhibition: due to residual mrna by premixed Tli RNase H SYBR Premix Ex Taq II (Tli RNase H Plus) is a reagent specifically designed for intercalator-based real time PCR using SYBR Green I. It is supplied at a 2X concentration premixed with SYBR Green I at a concentration appropriate for real time monitoring, making it easy to prepare reaction mixtures. The 2X premixed reagent also contains Tli RNaseH, a heat-resistant RNase H, which minimizes PCR inhibition due to residual mrna when using cdna as template. This product, with a modified buffer composition, offers a higher reaction specificity than that by SYBR Premix Ex Taq (Tli RNaseH Plus) (Cat.# RR420A). With inhibition of non-specific amplifications, which interfere with quantitative determination, accurate assays over a wide range are possible. A combination of this buffer and TaKaRa Ex Taq HS, a hot start PCR enzyme that uses an anti-taq antibody, allows highly reproducible and reliable real time PCR analyses. Compatible instruments include : Real-Time PCR System, 7500 Fast Real-Time PCR System, StepOnePlus Real-Time PCR System (Life Technologies) (Roche Diagnostics) Note: We recommend using SYBR Premix Ex Taq (Tli RNaseH Plus) (Cat.# RR420A) for System/ II System (Cepheid). TAKARA BIO is under a license agreement with Molecular Probes Inc. for the use of SYBR Green I as a reagent for research purposes. SYBR is a registered trademark of Molecular Probes Inc. RR820A SYBR Premix Ex Taq II (Tli RNaseH Plus) (2X conc.) * 5 ml RR820L SYBR Premix Ex Taq II (Tli RNaseH Plus) (2X conc.) * 5 ml * Contains TaKaRa Ex Taq HS, dntp Mixture, Mg 2+, Tli RNaseH and SYBR Green I. This component is to be used for analyses using a device that corrects fluorescent signals between wells such as the real-time PCR device by Applied Biosystems. Please use ROX Reference Dye for Applied Biosystems 7300 Real-Time PCR System or StepOnePlus and ROX Reference Dye II for 7500/7500 Fast Real-Time PCR System. This component is not required with II or. 4 C Note : Product is stable for up to 6 months when stored at 4 C. Protect this kit from light and avoid contamination. This kit is shipped in a package with dry ice. Store the kit at 4 C and in darkness. For long-term storage, store at 80 C. (Do not store at 20 C) Store a thawed product at 4 C and use within 6 months. Best Performance on Standard Targets Target: ACTB (86 bp) Results: Superior Specificity, maintains signal below baseline in no template control reactions. SYBR Premix Ex Taq II (Tli RNase H+)(Cat.# RR820) Company A SYBR Master Mix Superior Specificity: limits primer dimers and off-target amplification 3

4 Premix Ex Taq (Probe qpcr) Premix Ex Taq (Probe qpcr) Cat.# RR390A 200 reactions ( 50 µl PCR) Premix Ex Taq (Probe qpcr) Cat.# RR390B 400 reactions ( 50 µl PCR) (A size x 2) Premix Ex Taq (Probe qpcr) Cat.# RR390L 200 reactions ( 50 µl PCR) Premix Ex Taq (Probe qpcr) Cat.# RR390W 000 reactions ( 50 µl PCR) (L size x 5) (qpcr) Quantitation of DNA using Probe Detection Rapid and Accurate Detection and Quantitative Gene Expression Analysis: using real-time PCR 2X Premix: makes pipetting easy Excellent Amplification Efficiency and Highly Sensitive Detection: from the optimized buffer Includes Tli RNaseH, a Heat-Resistant RNaseH: to minimize PCR inhibition by residual mrna in reactions using a cdna template Premix Ex Taq (Probe qpcr) is a 2X premix for real-time PCR (qpcr) detection with TaqMan. The 2X Premix TaKaRa Ex Taq HS, a hot start PCR enzyme with an anti-taq antibody, and a buffer optimized for real-time PCR. The resulting premix allows excellent suppression of non-specific amplification, high amplification efficiency, and high detection sensitivity in real-time PCR analyses. In addition, Tli RNaseH, a heatresistant RNaseH, is included to minimize PCR inhibition from residual mrna in reactions using cdna templates. This product is excellent for high-speed PCR and allows accurate target quantification and detection over a broad dynamic range, making it possible to conduct highly reproducible and reliable real-time PCR analyses. Store at 4 C (stable for up to 6 months). *Every precaution should be taken to avoid contamination. This product is shipped frozen at 20 C. Store the product at 4 C after receipt. Before use, gently invert tube to make sure reagent is completely dissolved and evenly mixed. This product may be frozen at 20 C for long term storage. Once thawed, it should be stored at 4 C and used within 6 months. RR390A Premix Ex Taq (Probe qpcr) (2 conc.) * 5 ml ROX Reference Dye (50 conc.) 200 µl ROX Reference Dye II (50 conc.) 200 µl RR390L Premix Ex Taq (Probe qpcr) (2 conc.) * 5 ml ROX Reference Dye (50 conc.) 200 µl ROX Reference Dye II (50 conc.) 200 µl * Contains TaKaRa Ex Taq HS, dntp Mixture, Mg 2+, and Tli RNase H. Use when performing analyses with a device such as real-time PCR instruments by Life Technologies that normalize fluorescent signals between wells. Use ROX Reference Dye for ABI PRISM 7000/7700, 7300 Real-Time PCR System, and StepOnePlus Real-Time PCR System. Use ROX Reference Dye II for 7500 Real-Time PCR System and 7500 Fast Real-Time PCR System. Use the ROX Reference Dye at a final concentration of X and the ROX Reference Dye II at a final concentration of 0.5X. No dye is required when using II, System, or. Best Performance with TaqMan Probe Data using the Premix Ex Taq (Probe qpcr) product on the Applied Biosystems StepOnePlus instrument and the Roche LightCycler 480 is shown. Template: mouse liver cdna (equivalent total RNA pg ~ 00 ng) Target: Yeast (using TaqMan Gene Expression Assays) Applied Biosystems StepOnePlus Roche 480 R 2 = PCR Efficiency= 0.0% R 2 = PCR Efficiency= 00.0% 32

5 SYBR Premix DimerEraser (Perfect Real Time) SYBR Premix DimerEraser (Perfect Real Time) Cat.# RR09A 200 reactions SYBR Premix DimerEraser (Perfect Real Time) Cat.# RR09B 400 reactions (A size x 2) For Real time PCR Quantification of DNA using SYBR Green I Quick and accurate detection and quantification of target gene through real time PCR Easy-to-use 2X premix reagent including SYBR Green I: Ready to perform real time PCR in the presence of a fluorescent intercalater. Just add PCR primers, template and dh2o to start the reaction. High amplification efficiency and highly sensitive detection: This product utilizes an enzyme for hot start, TaKaRa Ex Taq HS. As this enzyme buffer system is optimized for real time PCR, this product offers highly efficient amplification and highly sensitive detection. Moreover, the addition of the accessory protein strongly suppresses mis-priming of primers during PCR reaction and nonspecific amplification, such as primer-dimer. SYBR Premix DimerEraser (Perfect Real Time) is a real-time PCR specific reagent using the SYBR Green I intercalater method. The reagent is supplied as a 2X premix making preparation of the reaction mixture easier. This product has an improved buffer system which results in improved reaction specificity. It effectively depresses primer dimers, which is especially important in SYBR Green Assay. SYBR Premix DimerEraser (Perfect Real Time) also makes it possible to measure accurate quantitative analysis over a broad range of template concentration by suppression of non-specific amplification and more sensitive detection. We recommend a 3-step PCR method in the standard protocol of this product. Applicable Instruments ABI PRISM , Applied Biosystems 7500 Real-Time PCR System, 7500 Fast Real- Time PCR System (Applied Biosystems) LightCycler (Roche Diagnostics) 3 Thermal Cycler Dice Real Time System (TAKARA BIO) (Note: This instrument is not available in the U.S. or Europe.) Smart Cycler System/Smart Cycler II System (Cepheid) 4. SYBR Green I is provided under licensing agreement with Molecular Probes Inc. for internal research use only. SYBR is a registered trademark of Molecular Probes Inc. 2. ABI PRISM is a registered trademark of Applera Corporation. 3. LightCycler is a registered trademark of Roche Diagnostics. 4. Smart Cycler is a registered trademark of Roche Cepheid. RR09A SYBR Premix DimerEraser (Perfect Real Time) (2X conc.) 5 ml ROX Reference Dye (50X conc.) μl ROX Reference Dye II (50X conc.) μl. Contains TaKaRa Ex Taq HS, dntp Mixture, Mg 2+, and SYBR Green I. Refer to IX. Specifications for further details of TaKaRa Ex Taq HS. 2. ROX Reference Dye/Dye II is used for normalization of intensity by background subtraction. For ABI PRISM 7000, the use of ROX Reference Dye (50X) 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. It is not required for use with LightCycler, Smart Cycler System/Smart Cycler II System and Thermal Cycler Dice Real Time System. Reagents and Instruments Required but Not Supplied with this Product Thermal Cycler for real time PCR Reaction tube or plate for real time PCR PCR primer dh2o Micropippets and Micropippet tips (autoclaved prior to use) Stable at 4 C for 6 months. (Note: product should be protected from light. Avoid contamination.) This product is shipped on dry ice and on delivery should be stored at 4 C and protected from light. Make the concentration uniform by gently inverting a tube before use. For longer-term storage, 80 C is recommended. Notes TaKaRa Ex Taq HS used in this product is an enzyme for hot start PCR utilizing a Taq antibody. The initial denaturation step prior to PCR should be 95 C for 30 sec. It does not require the activation step of heating at 95 C for 0 5 min prior to the PCR. SYBR Premix DimerEraser (Perfect Real Time) does not contain Tli RNase H. Transgene Detection Primer Set for Real Time (Mouse) Transgene Detection Primer Set for Real Time (Mouse) Cat.# reactions Detection and Quantification of Transgene Inserted in Transgenic Mouse Genome This kit is a primer set for real-time PCR to detect GFP (EGFP, AcGFP) and lacz, which are gene markers in transgenic mice. It is possible to screen transgenic mice by detecting these marker genes with real-time PCR. The primer sets (Ywhaz, Raver2) for detecting genes on mouse genomic DNA are also included as a reference. Therefore, it is possible to make a comparison of the transgene contents between samples by relative quantification GFP Primer- (2 μm ea.) GFP Primer-2 (2 μm ea.) 2 lacz Primer- (2 μm ea.) 3 lacz Primer-2 (2 μm ea.) 3 Reference Primer- (2 μm ea.) 4 Reference Primer-2 (2 μm ea.) 5 GFP Primer- is designed for consensus sequence common to both EGFP and AcGFP 2 GFP Primer-2 is designed for EGFP. It is not used for AcGFP detection 3 lacz primer - and -2 are designed for lacz (β-galactosidase) gene and positioned at different sites 4 Reference Primer- is for a region of Ywhaz gene on mouse chromosome 5 5 Reference Primer-2 is for a region of Raver2 gene on mouse chromosome C 33

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