QTaq One-Step qrt-pcr Kit and QTaq One-Step qrt-pcr SYBR Kit

User Manual QTaq One-Step qrt-pcr Kit and QTaq One-Step qrt-pcr SYBR Kit User Manual United States/Canada 800.662.2566 Asia Pacific +1.650.919.7300 Europe +33.(0)1.3904.6880 Japan +81.(0)77.543.6116 Clontech Laboratories, Inc. 1290 Terra Bella Ave. Mountain View, CA 94043 Technical Support (US) E-mail: tech@clontech.com www.clontech.com Cat. Nos. 639518 639519 639520 639521 PT4004-1 (PR0X3728) Published October 2010

Table of Contents I. Introduction and Protocol Overview...3 II. III. IV. List of Components...5 Additional Materials Required...5 General Considerations...6 A. RNA Preparation... 6 B. Primer Design... 6 C. Amplicon Design... 6 D. Good PCR Practices... 6 V. QTaq One-Step qrt-pcr Protocols...8 A. Protocol: Preparing for QTaq One-Step qrt-pcr SYBR and TaqMan assays... 8 B. Protocol: Setting up QTaq One-Step qrt-pcr SYBR assays... 10 C. Protocol: Setting up QTaq One-Step qrt-pcr TaqMan assays... 11 VI. Troubleshooting Guide...12 VII. References...13 List of Figures Figure 1. Comparison of Two-Step vs One-Step qrt-pcr Procedures...3 List of Tables Table I: Concentrations of RNA Template for Standard Curve... 9 Table IIA: Master Mix for the QTaq One-Step qrt-pcr SYBR Kit... 10 Table IIB: Master Mix for the QTaq One-Step qrt-pcr Kit (without SYBR)... 10 Table III: Master Mix for TaqMan Assays... 11 Protocol No. PT4004-1 www.clontech.com Clontech Laboratories, Inc. Version No. PR0X3728 2

I. Introduction and Protocol Overview QTaq One-Step qrt-pcr Kit and QTaq One-Step qrt-pcr SYBR Kit User Manual Quantitative reverse transcriptase PCR (qrt-pcr) is a highly sensitive and versatile technique that is widely used for measuring gene expression in tissues and cultured cells. Traditionally, RT-PCR is performed in two reaction steps. In the initial reaction, first-strand cdna is reverse-transcribed from total or poly A + RNA using a reverse transcriptase. Then, in a separate reaction, the cdna is typically diluted and then amplified by PCR using a thermostable DNA polymerase. Such two-step procedures require multiple handling steps and either multiple tubes or the sequential addition of enzymes and other reagents to a single tube. In contrast, QTaq One-Step qrt-pcr Kits allow first-strand cdna synthesis and qpcr to be performed in one step in a single tube, with a single optimized buffer, and a single enzyme mix. No additional reagents are required after the reaction is initiated (Figure 1). This significantly reduces the risk of cross-contamination, and provides a very convenient technique for detecting and quantitating gene expression. In addition, QTaq One-Step qrt-pcr Kits are available with or without SYBR Green I dye, making them compatible with both SYBR or 5 nuclease (TaqMan ) chemistries. Overview of the qrt-pcr Procedure Two-Step RT-PCR One-Step RT-PCR RNA Oligo(dT) primers or random hexamer primers RT buffer RT enzyme RNA Gene-specific primers One-step buffer PCR enzyme RT enzyme cdna cdna dilution cdna sample PCR primers PCR buffer and enzyme PCR amplicon PCR amplicon Figure 1. Comparison of Two-Step vs One-Step qrt-pcr Procedures Clontech Laboratories, Inc. www.clontech.com Protocol No. PT4004 1 Version No. PR0X3728 3

I. Introduction and Protocol Overview continued Enzyme Features QTaq One-Step qrt-pcr Kits combine a highly purified reverse transcriptase with a robust DNA polymerase, providing the most precise and accurate analysis of gene expression is a single-tube format. The qrt Mix contains a wild type Moloney murine leukemia virus reverse transcriptase (MMLV-RT) for first-strand cdna synthesis. The QTaq DNA Polymerase Mix is an optimized qpcr enzyme formulation consisting of recombinant, full-length Thermus aquaticus (Taq) DNA Polymerase (Engelke et al., 1990; Kim et al., 1995; Clontechniques, October 2003) and a cocktail of highly efficient, hot start antibodies for automatic hot start PCR (Sharkey et al., 1994). The high sensitivity and specificity exhibited by our QTaq formulation make it the perfect choice for any qpcr application. No Buffer Optimization Required QTaq One-Step qrt-pcr Kits allow you to perform qrt-pcr without tedious buffer optimization. The qrt and QTaq enzyme formulations are provided together with our convenient 2X One-Step qpcr Buffer, which eliminates the need for tedious and lengthy preparation of the master mix during the RT-PCR setup. Many of the unique advantages and features of the QTaq One-Step Kits are listed below: Wide dynamic range High sensitivity High specificity Advanced PCR enzyme system Reliably amplify cdnas from both low-abundance and high-abundance mrnas. Amplify target transcripts from as little as 500 fg of total RNA, or 5 copies of a synthetic RNA template. Antibody-mediated hot start prevents the formation of primer-dimers and other artifacts of low level synthesis that can arise prior to thermal cycling. The kits contain QTaq DNA Polymerase, a qpcr enzyme formulation consisting of recombinant full-length Taq polymerase and a highly efficient hot start antibody. Protocol No. PT4004-1 www.clontech.com Clontech Laboratories, Inc. Version No. PR0X3728 4

II. List of Components Store all components at 20 C. QTaq One-Step qrt-pcr Kit and QTaq One-Step qrt-pcr SYBR Kit User Manual Enough reagents are supplied for 200 or 1,000 PCR reactions of 25 µl each. QTaq One-Step qrt-pcr SYBR Kit: Cat. No. Cat. No. 639518 639519 200 rxns 1,000 rxns 84 µl 420 µl 60X qrt Mix 100 µl 500 µl 50X QTaq DNA Polymerase Mix (1.5 units/µl) with hot-start antibody 2.5 ml 5 x 2.5 ml 2X One-Step qrt-pcr Buffer Plus SYBR contains salts, dntps, MgCl 2, and SYBR Green I dye QTaq One-Step qrt-pcr Kit: Cat. No. Cat. No. 639520 639521 200 rxns 1,000 rxns 84 µl 420 µl 60X qrt Mix 100 µl 500 µl 50X QTaq DNA Polymerase Mix (1.5 units/µl) with hot-start antibody 2.5 ml 5 x 2.5 ml 2X One-Step qrt-pcr Buffer contains salts, dntps, and MgCl 2 III. Additional Materials Required The following materials are required but not supplied: PCR-grade water (RNase free) Avoid using autoclaved H 2 O; the recycled steam in some autoclaves can introduce contaminants that may interfere with PCR. PCR reaction tubes or plate (reaction specific) Aerosol-free PCR pipette tips suitable for use with the above pipettors and preferably equipped with hydrophobic filters Pipettors dedicated for pre-pcr Pipettors dedicated for RNA template Quantitative PCR reagents: Primers, substrate, or probes Note: If you are performing SYBR assays with the QTaq One-Step qrt-pcr Kit you will need to purchase SYBR Green I dye (Molecular Probes, Cat. No. S-7567). This is not necessary with the QTaq One-Step qrt-pcr SYBR Kit, as it already contains SYBR Green I. Real-time thermal cycler Optional: ROX passive reference dye Optional: For standard curve generation, we recommend using Clontech s qpcr Human Reference Total RNA (Cat. No. 636690) Clontech Laboratories, Inc. www.clontech.com Protocol No. PT4004 1 Version No. PR0X3728 5

IV. General Considerations A. RNA Preparation The use of pure, non-degraded total RNA is critical for synthesizing high-quality cdna for PCR. RNA should have an A 260 /A 280 ratio of 1.8 or higher, and should be evaluated on a denaturing agarose gel (containing formaldehyde) to verify its integrity prior to cdna synthesis. 1. Total RNA can be prepared by any standard method. See Sambrook, et al. (2001) as a reference. 2. To avoid contamination and degradation of RNA, follow these precautions: Wear gloves to avoid RNase contamination from hands. Wipe all pipettes with 70% ethanol or isopropanol before RNA work. Use sterile pipette tips. 3. Store pure RNA at 70 C or as an ethanol precipitate at 20 C. Avoid multiple freeze-thaw cycles. B. Primer Design Primer design is the single largest variable in PCR applications, and the single most important factor in determining the success or failure of PCR reactions. Always check (and recheck) your primer design before synthesizing (or ordering) your primers. When designing primers, consider the following: 1. Make sure your primer sequences are gene-specific. 2. To help distinguish between products amplified from cdna and those amplified from genomic DNA, target primer pairs to exons that are separated by one or more introns. Such primers will amplify much larger products from genomic DNA than from cdna, allowing you to easily distinguish between the two products by melting curve analysis. Alternatively, if intron-separated primer sites cannot be used, a negative control reaction can be performed as described in Section IV.D.4. (below). 3. Primers should be between 18 25 bases in length, and have a GC content between 40 60%. 4. Each primer should have a melting temperature (T m ) between 58-65 o C. The T m difference between the two primers should be 4 ºC. 5 Avoid runs of identical nucleotides. The last five nucleotides at the 3 end of each primer should contain no more than two guanines (G) and/or cytosines (C). 6. Avoid complementarity within each primer to prevent hairpin structures; avoid complementarity between primer pairs to prevent primer dimers. 7. Before synthesizing your primers, perform a BLAST search to determine if the primers you designed are unique and specific (www.ncbi.nlm.nih.gov/blast/). C. Amplicon Design 1. Amplicons should be between 50 200 bp long. Shorter amplicons will give higher PCR efficiencies; longer amplicons will give a higher overall fluorescence intensity (ΔRn). 2. Select amplicons with a GC content between 40 60%. 3. Avoid sequences with extensive secondary structure. Use a computer program such as Mfold, available on the Mfold web server (www.bioinfo.rpi.edu/applications/mfold/), to predict secondary structure in your proposed amplicon (Mathews et al., 1999; Zuker, 2003). D. Good PCR Practices 1. Prepare reactions with dedicated pipettors in a dedicated work space. 2. Because of the small volumes used in experiments and the potential for tube-to-tube variation, careful pipetting technique is extremely important. Always be certain not to carry over extra solution on the outside of the pipette tip during transfer. When adding liquid to a tube, immerse the tip into the reaction mixture, deliver the contents from the pipette tip into the mixture, and then rinse the tip by pipetting up and down several times. Protocol No. PT4004-1 www.clontech.com Clontech Laboratories, Inc. Version No. PR0X3728 6

IV. General Considerations continued 3. Prepare a Master Mix containing the appropriate volumes of all of the reagents required for multiple RT- PCR reactions. This eliminates the need for repeated pipetting of individual reaction components into each reaction tube. We highly recommend using a Master Mix when multiple reactions are required because it greatly reduces tube-to-tube variation. If, for example, multiple templates are being tested with the same primer pair, include the primers in the Master Mix. If one template is being tested with multiple primer sets, include the template in the Master Mix. If you are setting up several sets of parallel samples, assemble multiple Master Mixes (e.g., each with a different set of primers). Make sure the Master Mix has enough volume to allow for additional No Reaction Controls for each template, and No Template Controls (NTC) for each Master Mix, as well as an extra 25 ml to accommodate pipetting errors. The Master Mix should be thoroughly mixed before use. 4. Always include positive and negative controls (e.g., H 2 O instead of RNA template for the negative control). Note: An optional negative control can be performed to help distinguish between fragments amplified from cdna and those derived from genomic DNA contaminants in your RNA sample. To prepare this control, simply omit the 60X qrt Mix from a reaction containing your RNA sample. Clontech Laboratories, Inc. www.clontech.com Protocol No. PT4004 1 Version No. PR0X3728 7

V. QTaq One-Step qrt-pcr Protocols PLEASE READ ENTIRE PROTOCOL BEFORE STARTING. Attention Important: If you are performing SYBR assays, follow the steps in Sections V.A. (pages 8 and 9) and V.B. (page 10). If you are performing TaqMan assays, follow the steps in Sections V.A. (pages 8 and 9) and V.C. (page 11). Note: While SYBR assays can be performed with either kit, TaqMan assays can only be performed with the QTaq One-Step qrt-pcr Kit. A. Preparing for QTaq One-Step qrt-pcr SYBR and TaqMan assays 1. General considerations: a. This section (Section V.A.) must be completed first, regardless of which QTaq One-Step qrt-pcr Kit or qpcr chemistry (SYBR or TaqMan) you are using. b. Completely thaw the 2X One-Step qrt-pcr Buffer at 37 C. Vortex vigorously, then leave the buffer at room temperature until ready to use. Note: For convenience, the buffer can be stored at 4 C for several weeks. c. Place all of the other reaction components on ice and allow them to thaw completely. Gently vortex each component and centrifuge briefly to collect the contents in the bottom of the tube. Store on ice until ready to use. Attention Note: If you are performing SYBR assays using the One-Step qrt-pcr Kit (which lacks SYBR Green I), you will need to add SYBR Green I dye ((Molecular Probes, Cat. No. S-7567) to the Master Mix in Section V.B.1.a. (see Table IIB, Page 10). Before the dye is added to the Master Mix, however, it must be thawed and diluted 1:800 with DMSO: a) Completely thaw the SYBR Green I dye (purchased as a 10,000X stock) at room temperature. b) Mix well by pipetting up and down several times. c) Dilute 1:800 with DMSO. Keep at room temperature until ready to use, then store the remaining dilution at 20 o C for future use. d. Use between 100 nm and 900 nm of each primer per reaction. For most targets, 200 nm of each primer is adequate. e. Depending on the expression level of your target gene, use between 1 pg and 500 ng of template RNA per reaction. For most genes, a total of 50 ng of template RNA is sufficient. f. This protocol assumes a final reaction volume of 25 ml. If you plan to use a different reaction volume, adjust the amount of each component accordingly. g. If you are using ROX as a reference dye, use the amount recommended by the manufacturer of your real-time PCR instrument. Protocol No. PT4004-1 www.clontech.com Clontech Laboratories, Inc. Version No. PR0X3728 8

V. QTaq One-Step qrt-pcr Protocols continued 2. Dilution of RNA template for standard curve preparation: a. Dilute all RNA samples with PCR-grade dh 2 O, on ice, in an RNase-free area. b. Prepare 5-fold serial dilutions of your RNA template. Dilute the RNA to the concentrations indicated in Table I. Notes: 1) To generate the standard curve, you will need to use 5 ml of each dilution per reaction. 2) We recommend using Clontech s qpcr Human Reference Total RNA (Stock conc. 1 mg/ml; Cat. No. 636690) 3) Use a new pair of gloves when diluting the RNA template to help prevent degradation by RNases. Table I. Concentrations of RNA template for standard curve Tube # RNA Concentration RNA Quantity/Reaction 1 100 ng/µl 500 ng 2 20 ng/µl 100 ng 3 4 ng/µl 20 ng 4 800 pg/µl 4 ng 5 160 pg/µl 0.8 ng 6 32 pg/µl 0.16 ng 7 6.4 pg/µl 0.032 ng 3. Dilute your RNA test sample(s) to 10 ng/ml. Attention If you are performing One-Step qrt-pcr using SYBR Green chemistry, please proceed to Section B (pg. 10); if you are using TaqMan chemistry, please proceed to Section C (pg. 11). Clontech Laboratories, Inc. www.clontech.com Protocol No. PT4004 1 Version No. PR0X3728 9

V. QTaq One-Step qrt-pcr Protocols Attention Important: Please be sure to complete all of the steps in Section A (pages 8 and 9) before continuing on to this section. B. Setting up QTaq One-Step RT-PCR SYBR assays 1. Preparation of the Master Mix: a. In a pre-pcr area or a template-free PCR workstation, prepare a Master Mix at room temperature by adding each component as indicated in Table IIA or IIB. Add all reagents in the order shown. Notes: 1) Use Table IIA when using the QTaq One-Step qrt-pcr SYBR Kit (containing 2X One-Step qrt-pcr Buffer Plus SYBR) and Table IIB when using the QTaq One-Step qrt-pcr Kit (containing 2X One-Step qrt-pcr Buffer without SYBR). 2) To determine the amount of each reagent to add for your particular assay, multiply the Amt./rxn of each reagent in Table IIA or IIB by the number of reactions you have, plus three additional reactions to accommodate controls and pipetting errors. For example, to determine how much 60X qrt Mix to add to a Master Mix for 12 reactions, you would multiply 0.4 ml by 15, for a total of 6 ml. Table IIA. MAster Mix for the QTaq One-Step qrt-pcr SYBR Kit REAGENT Amt./rxn DEPC-treated PCR grade water 5.6 µl 2X One-Step qrt-pcr Buffer plus SYBR 12.5 µl Forward primer (10 mm) 0.5 µl Reverse primer (10 mm) 0.5 µl 50X QTaq DNA Polymerase Mix 0.5 µl 60X qrt Mix 0.4 µl Total 20.0 µl Table IIb. MAster Mix for the QTaq One-Step qrt-pcr Kit (without sybr) REAGENT Amt./rxn DEPC-treated PCR grade water 5.1 µl 2X One-Step qrt-pcr Buffer (without SYBR) 12.5 µl Forward primer (10 mm) 0.5 µl Reverse primer (10 mm) 0.5 µl 1:800 dilution of SYBR Green I Dye 0.5 µl 50X QTaq DNA Polymerase Mix 0.5 µl 60X qrt Mix 0.4 µl Total 20.0 µl 2. Mix the Master Mix well by pulse-vortexing at least 5 times. Centrifuge briefly and transfer 20 ml into each well of a PCR plate or 8-well strip. 3. Add 5 ml of RNA template or RNA test sample per well. For the No Template Control, add 5 ml of PCRgrade water instead of RNA. 4. Seal the wells according to the procedure recommended for the real time instrument being used. 5. Program the thermal cycler with the following conditions: 1 cycle: 48 C for 20 min RT Step 1 cycle: 95 C for 3 min QTaq Activation 40 45 cycles: 95 C for 15 sec 60 C for 1 min Annealing, Extension and Data Collection Melting Curve (optional): Perform as recommended by your instrument s manufacturer Note: This protocol was developed using primers with T m s between 58 65 o C. If primers with different T m s are used, some optimization of the PCR cycling conditions may be required. 6. Place the plate into the real-time PCR instrument and begin thermal cycling. Protocol No. PT4004-1 www.clontech.com Clontech Laboratories, Inc. Version No. PR0X3728 10

V. QTaq One-Step qrt-pcr Protocols continued Attention Important: Please be sure to complete all of the steps in Section A (pages 8 and 9) before continuing on to this section. C. Setting up QTaq One-Step RT-PCR TaqMan assays. 1. Preparation of the Master Mix: a. In a pre-pcr area or a template-free PCR workstation, prepare a Master Mix at room temperature by adding each component as indicated in Table IV. Add all reagents in the order shown. Notes: 1) To help prevent cross-contaminating your samples with template, use fresh gloves when preparing the Master Mix. 2) To determine the amount of each reagent to add for your particular assay, multiply the Amt./rxn of each reagent in Table III by the number of reactions you have, plus three additional reactions to accommodate controls and pipetting errors. For example, to determine how much 60X qrt Mix to add to a Master Mix for 12 reactions, you would multiply 0.4 ml by 15, for a total of 6 ml. Table III. MAster Mix For TaqMan assays REAGENT Amt./rxn DEPC-treated PCR grade water 5.35 µl 2X One-Step qrt-pcr Buffer without SYBR 12.50 µl 20X TaqMan primers and probe 1.25 µl 50X QTaq DNA Polymerase Mix 0.50 µl 60X qrt Mix 0.40 µl Total 20.00 µl 2. Mix the Master Mix well by pulse-vortexing at least 5 times. Centrifuge briefly and transfer 20 ml into each well of a PCR plate or 8-well strip. 3. Add 5 ml of RNA template or RNA test sample per well. For the No Template Control, add 5 ml of PCRgrade water instead of RNA. 4. Seal the wells according to the procedure recommended for the real time instrument being used. 5. Centrifuge at 2000 rpm for 2 min at 4 o C. 6. Program the thermal cycler with the following conditions: 1 cycle: 48 C for 20 min RT Step 1 cycle: 95 C for 3 min QTaq Activation 40 45 cycles: 95 C for 15 sec 60 C for 1 min Annealing, Extension and Data Collection 7. Place the plate into the real-time PCR instrument and begin thermal cycling. Clontech Laboratories, Inc. www.clontech.com Protocol No. PT4004 1 Version No. PR0X3728 11

VI. Troubleshooting Guide A. No signal observed in fluorescence detector Electrophorese the post-pcr reaction on an agarose gel to visualize the PCR product. 1. If there is a PCR product of the correct size on the gel, check the following: Is the wrong fluorescence channel being read? Confirm that the instrument detection parameters were set properly according to the detection substrate used. Is the data being collected at the appropriate time? Confirm that the data is being collected during the annealing and extension steps. 2. If there is no PCR product on gel, check to see if: A reaction component is missing or degraded Use a checklist when assembling reactions. Always perform a positive control to ensure that each component is functional. The annealing temperature is too high Decrease the annealing temperature in increments of 2 4 C. The template quality is poor Check template integrity by electrophoresis or other preferred methods. The template contains secondary structure. Incubate the template for 5 min at 70 o C, then for at least 2 min at 4 o C. Set reactions up on ice to prevent these structures from re-forming. There is insufficient template Increase the amount of template; use up to 500 ng per reaction. You are amplifying a rare target When amplifying rare targets, increase the number of amplification cycles up to 50 per assay. The primer concentration is too low Increase the amount of each primer up to 900 nm. The primer design is suboptimal Redesign your primer(s) after confirming the accuracy of the sequence information. B. Nonspecific amplification; multiple products observed on agarose gel The cause is most likely one of the following: 1. Contamination Contamination most often results in extra bands or smearing. It is important to include a No Template Control (in which the RNA template is replaced with PCR-grade H 2 O) and a no RT-control (in which the reverse transcriptase is replaced with PCR-grade H 2 O) in every experiment to determine if the reagents, pipettors, or reaction tubes are contaminated. 2. Annealing temperature too low Increase the annealing/extension temperature in increments of 2 3 C. Make sure you don t use an annealing temperature that is incompatible with your chemistry, especially if the data collection is done at the annealing step. 3. Suboptimal primer design Redesign your primer(s) after confirming the accuracy of the sequence information. 4. Primer/probe design not able to distinguish between gene families or splice variants Redesign your primer(s) after confirming the accuracy of the sequence information and all of the expressed sequence tags (ESTs) described for that gene. Protocol No. PT4004-1 www.clontech.com Clontech Laboratories, Inc. Version No. PR0X3728 12

VII. References QTaq One-Step qrt-pcr Kit and QTaq One-Step qrt-pcr SYBR Kit User Manual BD QTaq DNA Polymerase Mix (October 2003) Clontechniques XVIII(4):4 5. Engelke, D. R., Krikos A., Bruck M.E., & Ginsburg, D. (1990) Purification of Thermus aquaticus DNA polymerase expressed in Escherichia coli. Anal. Biochem. 191(2):396 400. Kim, Y., Eom, S. H., Wang, J., Lee, D. S., Suh, S. W. & Steitz, T. A. (1995) Crystal structure of Thermus aquaticus DNA polymerase. Nature 376(6541):612 616. Mathews, D.H., Sabina, J., Zuker, M., & Turner, D.H. (1999) Expanded Sequence Dependence of Thermodynamic Parameters Improves Prediction of RNA Secondary Structure. J. Mol. Biol. 288(5):911-940. Sambrook, J. & D. W. Russell. (2001) Molecular Cloning: A Laboratory Manual, Third Edition (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). Sharkey, D. J., Scalice, E.R., Christy, K. G. Jr, Atwood, S. M. & Daiss, J. L. (1994) Antibodies as thermolabile switches: high temperature triggering for the polymerase chain reaction. Biotechnology 12(5):506 509. Zuker, M. (2003) Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 31(13):3406-15 Notice to Purchaser Clontech products are to be used for research purposes only. They may not be used for any other purpose, including, but not limited to, use in drugs, in vitro diagnostic purposes, therapeutics, or in humans. Clontech products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without written approval of Clontech Laboratories, Inc. This product is provided under an agreement between Molecular Probes, Inc. and Clontech Laboratories, Inc. and the manufacture, use, sale or import of this product is subject to one or more of U.S. Patent Nos. 5,436,134; 5,658,751 and corresponding international equivalents, owned by Invitrogen Corp. The purchase of this product conveys to the buyer the non-transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer, where such research does not include testing, analysis or screening services for any third party in return for compensation on a per test basis. The buyer cannot sell or otherwise transfer (a) this product (b) its components or (c) materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes. Commercial Purposes means any activity by a party for consideration and may include, but is not limited to: (1) use of the product or its components in manufacturing; (2) use of the product or its components to provide a service, information, or data; (3) use of the product or its components for therapeutic, diagnostic or prophylactic purposes; or (4) resale of the product or its components, whether or not such product or its components are resold for use in research. For information on purchasing a license to this product for purposes other than research, contact Molecular Probes, Inc., Business Development, 29851 Willow Creek Road, Eugene, OR 97402. Tel: (541) 465-8300, Fax: (541) 335-0354. Use of this product is covered 6,127,155. 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. No right under any other patent claim (such as method claims in U.S. Patents Nos. 5,994,056 and 6,171,785) 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 hereby conveyed by the purchase of this product expressly, by implication, or by estoppel. This product is for research use only. Diagnostic uses require a separate license from Roche. 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. The purchase of this product includes a limited, non-transferable license under specific claims of U.S. Patent Nos. 6,174,670, 6,569,627 and 5,871,908, owned by the University of Utah Research Foundation or Evotec Biosystems GmbH and licensed to Idaho Technology, Inc. and Roche Diagnostics GmbH, to use only the enclosed amount of product according to the specified protocols. No right is conveyed, expressly, by implication, or by estoppel, to use any instrument or system under any claim of U.S. Patent Nos. 6,174,670, 6,569,627 and 5,871,908, other than for the amount of product contained herein. TaqStart Antibody and other Hot Start Antibodies are licensed under U.S. Patent No. 5,338,671. Use of Clontech s One-Step RT-PCR kits is licensed from biomerieux, is covered by U.S. Patent No. 5,817,465 and equivalents, and is for research use only. ROX is a trademark of Applera Corporation. SYBR is a registered trademark of Molecular Probes, Inc. TaqMan is a registered trademark of Roche Molecular Systems, Inc. Clontech, the Clontech logo and all other trademarks are the property of Clontech Laboratories, Inc., unless noted otherwise. Clontech is a Takara Bio Company. 2010 Clontech Laboratories, Inc. Clontech Laboratories, Inc. www.clontech.com Protocol No. PT4004 1 Version No. PR0X3728 13