FastStart. Cat. No U for 25 PCR reactions Cat. No U for 50 PCR reactions Cat. No.

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1 For general laboratory use only. Not for use in diagnostic procedures. FOR IN VITRO USE ONLY. FastStart Taq DNA Polymerase Cat. No U for 25 PCR reactions Cat. No U for 50 PCR reactions Cat. No U for 250 PCR reactions Cat. No U for 500 PCR reactions Cat. No U for 1250 PCR reactions Cat. No U for 2500 PCR reactions Store this product at 15 to 25 C Instruction Manual Version Dec. 2005

2 1. Preface 1. Preface Contents Introduction Product overview Procedures and required materials Before you begin Procedures A: Standard PCR Procedure Typical results B: PCR Procedure using GC-RICH solution Typical results C: PCR Procedure for Carry-Over Prevention Trouble shooting References Ordering Information...17 P R O T O C O L 2

3 1. Preface, continued 1.1 Contents Vial Label Content and use 1 colorless cap 2 green cap 3 yellow cap 4 blue cap 5 red cap FastStart Taq DNA Polymerase 1) (5 U/µl) PCR reaction buffer, 10x conc. with 20 mm MgCl 2 PCR reaction buffer, 10x conc. without MgCl 2 MgCl 2 stock solution, 25 mm GC-RICH solution, 5x conc µl (Cat No ) 1 20 µl (Cat No ) 2 50 µl (Cat No ) 4 50 µl (Cat No ) µl (Cat No ) µl (Cat No ) enzyme storage buffer [20 mm Tris-HCl, ph 9.0/ 25 o C, 100 mm KCl, 0.1 mm EDTA, 1 mm DTT, 0.2% Tween 2) 20 (v/v), 50% glycerol (v/v)] 1 1 ml (Cat No ) 1 1 ml (Cat No ) 2 1 ml (Cat No ) 3 1 ml (Cat No ) 7 1 ml (Cat No ) 14 1 ml (Cat No ) 500 mm Tris/HCl, 100 mm KCl, 50 mm (NH 4 ) 2 SO 4, 20 mm MgCl 2, ph 8.3/ 25 o C 1 1 ml (Cat No ) 1 1 ml (Cat No ) 2 1 ml (Cat No ) 3 1 ml (Cat No ) 7 1 ml (Cat No ) 14 1 ml (Cat No ) 500 mm Tris/HCl, 100 mm KCl, 50 mm (NH 4 ) 2 SO 4, ph 8.3/ 25 o C 1 1 ml (Cat No ) 1 1 ml (Cat No ) 2 1 ml (Cat No ) 4 1 ml (Cat No ) 10 1 ml (Cat No ) 20 1 ml (Cat No ) 1 1 ml (Cat No ) 1 1 ml (Cat No ) 3 1 ml (Cat No ) 5 1 ml (Cat No ) 13 1 ml (Cat No ) 26 1 ml (Cat No ) Additional equipment required Typical equipment necessary for PCR e.g. Thermal cycler, primers, nucleotides*, PCRgrade water* and template. * available from 3

4 2. Introduction 2.1. Product overview Description Application Number of tests Storage/ stability FastStart Taq DNA Polymerase has been developed by Roche to make Polymerase Chain Reaction (PCR) more specific and sensitive in a convenient and rapid way. With FastStart Taq DNA Polymerase, "Hot Start" PCR (1,2,3,4) can be applied for genomic DNA and cdna templates, eliminating the extra handling steps or additional time required associated with up to now known "Hot Start" methods. FastStart Taq DNA Polymerase is a thermostable, modified form of recombinant Taq DNA Polymerase. It is inactive at temperatures below 75 o C, but can be activated by a 4 min 95 o C incubation step. The combination of FastStart Taq DNA Polymerase and the optimized PCR buffer minimizes non-specific amplification products and primer dimers allowing highest sensitivity. The provided GC-RICH solution, a PCR additive that facilitates amplification of difficult templates by modifying the melting behavior, will improve PCR performance on templates rich in secondary structures or GC content. FastStart Taq DNA Polymerase is an ideal tool for Hot Start PCR, because the enzyme remains inactive during PCR set-up and prior to the initial denaturation step. Since it is inactive at low temperatures, FastStart Taq DNA Polymerase cannot elongate non-specific primer-template hybrids that may form at those temperatures. Amplification of genomic DNA and cdna targets up to 3 kb long with high specificity, sensitivity, and yield. Multiplex PCR. Difficult templates e.g. secondary structures or GC-rich sequences. Carry-over prevention. Automated PCR e.g. handling at room temperatures. For a typical test 2 U of FastStart Taq DNA Polymerase are used in a 50 µl reaction volume. The number of tests depends on the pack size ordered. The unopened product is stable at 15 to 25 C through the expiration date printed on the label. The product is shipped on dry ice 4

5 2.1 Product overview, continued Quality control Unit Assay: 1 µg M13mp9ss DNA, 0.3 µg M13 sequencing primer and 0.1 µci [ - 32 P] dctp are incubated with various U of FastStart Taq DNA Polymerase in 50 µl incubation buffer with paraffin-oil overlay at 65 o C for 30 min. The amount of incorporated dntps is determined by trichloroacetic acid precipitation. Function test 1 (sensitivity) Using a serial dilution of human genomic DNA, a 365 bp fragment is amplified out of tpa gene (single copy gene) under standard conditions (2 U of FastStart Taq DNA Polymerase in a 50 µl reaction). After 44 cycles, a PCR product is detectable as a single, specific band with 50 pg of starting template. Function test 2 (GC-rich template) A PCR assay under standard conditions is performed (2 U FastStart Taq DNA Polymerase in 50 µl reaction volume) using GC-RICH solution on 200 ng human genomic DNA with primers specific for a 284 bp fragment of the ApoE gene (74% GC content). After 35 cycles a PCR product is detectable as single, specific band. Endonuclease assay 1: 1 µg lambda DNA is incubated with FastStart Taq DNA Polymerase in 50 µl test buffer with a paraffin oil overlay at 37 o C for 16 h. 25 U of enzyme show no degradation of the lambda DNA. Endonuclease assay 2: 1 µg EcoRI/HindIII-fragments of lambda DNA is incubated with FastStart Taq DNA Polymerase in 50 µl test buffer with a paraffin oil overlay at 37 o C for 16 h. 25 U of enzyme show no degradation of the EcoRI/ HindIII fragments of lambda DNA. Exonuclease assay: 5 µg of [ 3 H]-labeled calf thymus DNA is incubated with FastStart Taq DNA Polymerase in 100 µl test buffer with a paraffin overlay at 65 o C for 4 h. 15 U of enzyme show no release of radioactivity. Ribonuclease assay: 5 µg MS2 RNA is incubated with FastStart Taq DNA Polymerase in 50 µl test buffer at 37 o C for 1 h. 25 U of enzyme show no degradation of the MS2 RNA. Nicking activity 1 µg supercoiled pbr322 DNA is incubated with FastStart Taq DNA Polymerase in 50 µl test buffer with a paraffin oil overlay at 37 o C for 16 h. 25 U of enzyme show no relaxation of supercoiled DNA. 5

6 3. Procedures and required materials 3.1 Before you begin Introduction Taq DNA Polymerase Cloning Sample material Primers MgCl 2 concentration Enzyme concentration Elongation temperature GC-RICH solution dntp* concentration The optimal reaction conditions (incubation times and temperatures, concentration of FastStart Taq DNA Polymerase, template DNA, Mg 2+ -ions) depend on the template/ primer pair and must be determined individually. Three different procedures are described. Procedure A; standard PCR procedure Procedure B; PCR procedure using GC-RICH solution Procedure C: Carry-over prevention procedure using heat-labile UNG* Note: The protocols are designed for a final 50 µl reaction volume. For other volumes, the reaction and cycle conditions have to be optimized. The major differences of a typical FastStart Taq DNA procedure to standard Taq procedure are a) increased denaturation time prior to PCR of around 4 min (2-6 min) at 95 C and b) requires a minimal denaturation time in each cycle of 30 sec and c) standard Mg2+ concentration is 2 mm. All other conditions - dntp, primers, template concentrations versus cycle number are identical. FastStart Taq DNA Polymerase PCR products can be used for TA-cloning. For cloning into blunt end vectors an additional end polishing step is needed. (Refer e.g. to PCR Cloning Kit*). Every sample material suitable for PCR in terms of purity, concentration, and absence of inhibitors can be used. Typically 10 pg 500 ng human genomic DNA or 10 pg 100 ng cdna or plasmid is used. Use primers at a final concentration of µm each. A recommended starting concentration is 0.2 µm each. The optimal Mg-concentration is in the range of 1-4 mm. The recommended starting concentration is 2 mm. The optimal enzyme concentration range from U per 50 µl assay. The recommended starting concentration is 2 U per 50 µl assay. The elongation temperature is 72 o C when amplifying fragments up to 3 kb. When amplifying fragments larger than 3 kb, 68 o C might be favourable. The optimal concentration of GC-RICH solution is 10 µl per 50 µl assay. When using the GC-RICH solution the first time for a particular primer-template pair, always perform parallel reactions with and without GC-RICH solution. The optimal concentration of dntps (datp, dgtp, dctp, dttp) range from mm. The recommended concentration is 0.2 mm. For carry-over prevention 0.2 mm dttp is substituted by 0.6 mm dutp. For labeling of PCR products modified dntps (e.g. DIG-11- dutp, Biotin-16-dUTP, Fluorescein-12-dUTP) are typically used in a ratio together with dttp. For Southern blot application the ratio is 134 µm dttp + 66 µm DIG-11-dUTP, for ELISA application the ratio is 190 µm dttp + 10 µm DIG-11-dUTP. * available from 6

7 3.2 Procedures A: Standard PCR Procedure Before you begin Preparation of the master mixes Thaw the following reagents and store on ice. Vortex briefly and centrifuge all reagents before setting up the reactions. Set up Master Mix: Step Action 1 In a 1.5 ml reaction tube on ice, add the following components in the following order: Component Volume Final conc. H 2 0, sterile double distilled variable 10x PCR buffer* (vial 2, green cap) 5 l 2 mm MgCl 2 MgCl 2 solution**, 25 mm (vial 4, blue variable mm cap) 10 mm datp, PCR grade*** 1 l 200 µm 10 mm dctp, PCR grade*** 1 l 200 µm 10 mm dgtp, PCR grade*** 1 l 200 µm 10 mm dttp, PCR grade*** 1 l 200 µm downstream primer variable µm upstream primer variable µm FastStart Taq DNA Polymerase (vial 1, 0.4 l 2 U colorless cap) Read step 2 and 3 Template DNA, added at step 3 variable up to 500 ng/ reaction Total volume 50 µl Standard PCR Procedure *contains 20 mm MgCl 2 ; if Mg concentration should be titrated use 10 PCR buffer without MgCl 2, vial 3 (yellow cap) **only if Mg-titration is required ***alternatively 1 µl of 10 mm PCR Nucleotide Mix* can be used. 2 Mix the master mix thoroughly and dispense appropriate volumes into PCR tubes (preferably thin-walled PCR tubes). 3 Add template DNA to the individual tubes containing the master mix. 4 Mix each PCR tube well to produce a homogeneous sample. When using a thermal cycler without a heated lid, overlay reaction with 30 µl mineral oil. 5 Place your sample in a thermal cycler and perform PCR. An example for a cycle profile is given for the Applied Biosystems GeneAmp PCR System When using other thermal cyclers the cycle conditions have to be adjusted. continued on next page 7

8 A: Standard PCR Procedure, continued Step Action 6 PCR reaction: A typical temperature profile is given for the Applied Biosystems GeneAmp PCR System Standard PCR Procedure Denaturation/ Activation Denaturation Annealing Elongation Temperature Time No. Cycles a) This step activates the previously inactive FastStart Taq DNA Polymerase as well as denaturate DNA template. Yield of PCR product might be increased by longer activation time up to 6 min or more cycles. Activation times down to 2 min will give good results. Yield and specificity in a multiplexing-pcr (14- band multiplexing PCR with 28 primers was tested) might be increased by longer activation time up to 10 min or more cycles. Activation times down to 2 min will give good results. b) Exact annealing temperature depends on the melting temperature of the primers. c) Elongation time depends on the length of target to be amplified. Recommended time is 1 min per 1 kb of the PCR fragment. PCR product yield can be increased by using a cycle elongation feature. Usually are 15 cycles performed with a fixed elongation time than to each of the remaining cycles 5 sec are added e.g. cycle 15: 45 sec; cycle 16: 50 sec; cycle 17: 55 sec etc. d) 30 cycles are enough to produce an adequate amount of product, if there is sufficient target (preferably >10 4 copies) in the template. For low concentrations of target DNA, increase the number of cycles up to 40 cycles 7 Analyze the samples on a 1-2% agarose gel. 95 C 4 min a) 1 95 C C b) 72 C 30 sec 30 sec 45 sec - 3 min c) d) Final extension 72 C 7 min 1 8

9 Typical results Sensitivity To demonstrate the sensitivity of FastStart Taq DNA Polymerase a 365 bp fragment out of the human tpa gene (single copy gene) was amplified using various concentrations of human genomic DNA (Figure 1). PCR has been performed in a 50 µl reaction using 2 U of FastStart Taq DNA Polymerase under standard conditions [200 µm dntp (each), 200 nm primer (each), 2 mm MgCl 2 ] with 3 ng (lane 1); 1 ng (lane 2); 500 pg (lane 3); 300 pg (lane 4); 150 pg (lane 5); 60 pg (lane 6); 30 pg (lane 7); 10 pg (lane 8) human genomic DNA and no template control (lane 9). After 40 cycles with an initial 2 minutes denaturation/ activation step a specific PCR product is detectable with 10 pg of starting human genomic DNA. Figure 1: MWM MWM Amplification of 365 bp tpa fragment down to 10 pg human genomic DNA which is equivalent to 3 gene copies for a single copy gene (3 pg is equivalent to 1 copy). 9

10 Typical results, continued Specificity Specificity of FastTaq DNA Polymerase was compared to Taq DNA Polymerase by amplifying a 130 bp fragment out of human tpa gene (Figure 2). For both enzymes, standard PCR conditions were applied (2 U/ 50 µl reaction with respective buffer conditions). 100 ng (lanes 1,7); 50 ng (lanes 2,8); 10 ng (lanes 3,9); 5 ng (lanes 4,10) and without human genomic DNA (lanes 5,11) has been amplified (30 cycles with identical cycle program for both enzymes) and product visualized on agarose gel. With FastStart Taq DNA Polymerase a single specific PCR product was obtained (lanes 7-10) meanwhile with Taq DNA Polymerase unspecific PCR products as well as lower sensitivity is obtained (lanes 1 5). Figure 2: Taq DNA Polymerase FastStart Taq DNA Polymerase MWM Highly specific PCR through Hot Start capability of FastStart Taq DNA Polymerase 10

11 B: PCR Procedure using GC-RICH solution Considerations Before you begin Preparation of the master mixes When using the GC-RICH solution (vial 5) the first time for a particular primer-template pair, always perform parallel reactions with and without GC-RICH solution. Thaw the following reagents and store on ice Vortex briefly and centrifuge all reagents before setting up the reactions. Set up Master Mix: Step Action 1 In a 1.5 ml reaction tube on ice, add the following components in the following order: Component Volume Final conc. H 2 0, sterile double dist. variable 10 PCR buffer* (vial 2, green cap) 5 l 2 mm MgCl 2 MgCl 2 solution**, 25 mm (vial 4, blue variable mm cap) GC-RICH solution (5 ) (vial 5, red cap) 10 l 1 10 mm datp, PCR grade*** 1 l 200 µm 10 mmdctp, PCR grade*** 1 l 200 µm 10 mm dgtp, PCR grade*** 1 l 200 µm 10 mm dttp, PCR grade*** 1 l 200 µm downstream primer variable µm upstream primer variable µm FastStart Taq DNA Polymerase (vial 1, 0.4 l 2 U colorless cap) Read step 2 and 3 of general protocol Template DNA, added at step 3 variable up to 500 ng/ reaction Total volume 50 µl *contains 20 mm MgCl 2 ; if Mg concentration should be titrated use 10x PCR buffer without MgCl 2, vial 3 (yellow cap) **only if Mg titration is required ***alternatively 1 µl of 10 mm PCR Nucleotide Mix can be used Follow Step 2 of the standard PCR procedure (see section ) 11

12 Typical results Sensitivity Figure 3: GC-RICH solution changes the melting behavior of DNA and can be used for primertemplate pairs with high GC-content that do not work well with standard conditions. To compare the ability of the GC-RICH solution, FastStart Taq DNA Polymerase was used to amplify out of human ApoE gene a 284 bp fragment with and without the additive (Figure 3). Out of 200 ng human genomic DNA and 35 cycles a specific PCR product is visible when the GC-RICH solution is used (lane 3), without this additive PCR failes as demonstrated on FastStart Taq DNA Polymerase alone (lane 2), Taq DNA Polymerase (lane 7) or competitor "Hot Start" Taq DNA polymerases A, B (lane 4,6). Competitor s A Taq DNA polymerase with specific buffer (lane 5) permit also amplification of this target. MWM MWM Lane 2: FastStart Taq DNA Polymerase Lane 3: FastStart Taq DNA Polymerase + GC-RICH solution Lane 4: Competitor A Lane 5: Competitor A plus special buffer Lane 6: Competitor B Lane 7: Taq DNA Polymerase (Roche) Amplification of a 284 bp fragment out of human ApoE gene (GC content 74%) 12

13 C: PCR Procedure for Carry-Over Prevention Additionally reagent required Uracil-DNA Glycosylase, heat-labile*, please refer to ordering information. Before you begin Preparation of the master mixes Thaw the following reagents and store on ice. Vortex briefly and centrifuge all reagents before setting up the reactions Set up Master Mix: Step Action 1 In a 1.5 ml reaction tube on ice, add the following components in the following order: Component Volume Final conc. H 2 0, sterile double dist. variable 10x PCR buffer (vial 2, green cap) 5 l 2 mm MgCl 2 MgCl 2 solution*, 25 mm variable mm (vial 4, blue cap) PCR Nucleotide Mix Plus 1 l 200 µm (datp, dctp, dgtp) 600 µm dutp downstream primer variable µm upstream primer variable µm Heat-labile UNG (1 U/ l) 1 l 1 U FastStart Taq DNA Polymerase (vial 1, 0.4 l 2 U colorless cap) Read step 2 and 3 Template DNA, added at step 3 variable up to 500 ng/ reaction Total volume 50 µl *The optimal Mg-ions concentration depends on primer pairs and template. For best results determine optimal Mg-ions concentration empirically using 0.5 mm titration steps. 2 Mix the master mix thoroughly and dispense appropriate volumes into PCR tubes (preferably thin-walled PCR tubes). 3 Add template DNA to the individual tubes containing the master mix. 4 Mix each PCR tube well to produce a homogeneous sample. When using a thermal cycler without a heated lid, overlay reaction with 30 µl mineral oil. 5 Place your sample in a thermal cycler and perform PCR. An example for a cycle profile is given for the Applied Biosystems GeneAmp PCR System When using other thermal cyclers the cycle conditions have to be adjusted. continued on next page 13

14 C: PCR Procedure for Carry-Over Prevention, continued Step Action 6 PCR reaction: A typical temperature profile is given for the Applied Biosystems GeneAmp PCR System Temp. Time No. Cycles UNG incubation 20 C 1-10 min 1 Inactivation of UNG/ Denaturation/ Activation of polymerase 95 C 4 min a) 1 Denaturation Annealing Elongation 95 C C b) 72 C a) This step activates the previously inactive FastStart Taq DNA Polymerase, inactivates UNG and denaturates DNA template. Yield of PCR product might be increased by longer activation time up to 6 min or more cycles. Activation times down to 2 min will give good results. Yield and specificity in a multiplexing-pcr (14-band multiplexing PCR with 28 primers was tested) might be increased by longer activation time up to 10 min or more cycles. Activation times down to 2 min will give good results b) Exact annealing temperature depends on the melting temperature of the primers. c) Elongation time depends on the length of target to be amplified. Recommended time is 1 min per 1 kb of the PCR fragment. PCR product yield can be increased by using a cycle elongation feature. Usually are 15 cycles performed with a fixed elongation time than to each of the remaining cycles 5 sec are added e.g. cycle 15: 45 sec; cycle 16: 50 sec; cycle 17: 55 sec etc. d) 30 cycles are enough to produce an adequate amount of product, if there is sufficient target (preferably >10 4 copies) in the template. For low concentrations of target DNA, increase the number of cycles up to 40 cycles. e) Heat-labile UNG will not degrade du containing PCR products for at least several hrs at 2 8 C. Therefore it is necessary to freeze the PCR product immediately or to hold at 72 C until it is analyzed. If the product will not be analyzed during this time period, store it at 15 to 25 C. 7 Analyze the samples on a 1-2% agarose gel. 30 sec 30 sec 45 sec - 3 min c) d) Final extension 72 C 7 min 1 Hold 4 C up to 8 h e) 1 14

15 4. Trouble shooting Problem Possible Cause Recommendation Little or no PCR product FastStart Taq DNA Polymerase not enough activated Pipetting errors Difficult template e.g. GC-rich templates Mg 2+ concentration not optimal Primer problems due to not optimal design concentration quality or storage problems too high annealing temperature DNA template problems Check whether PCR was started with prior activation step at 95 o C for 4 min. Alternatively use 10 minutes. Check denaturation temperature during cycles. It should be 30 sec in minimum. Check cycle numbers. Increase the number of cycles in steps of 5 cycles. Repeat PCR. Check all concentrations and storage conditions of reagents. Repeat PCR under same conditions and add GC-RICH solution (see protocol B). If performance is still not satisfying titrate GC-RICH solution (4, 6, 8, µl), reduce or increase annealing temperature, titrate Mg concentration and/ or enzyme conc.. Titrate Mg concentration from 1 4 mm in 0.5 mm steps with buffer 3 (yellow cap). If you use an established primer pair, check performance on an established PCR system (control template). Titrate primer concentration ( µm). Reduce annealing temperature. Design alternative primers. Check quality/ concentration of template by Analyze an aliquot on a agarose gel. Use serial dilution of template. Make a control reaction on template with an other established primer pair/ PCR system. Check/ repeat purification of template. Enzyme concentration too low Use 2 U FastStart Taq DNA Polymerase per 50 µl reaction. If necessary, increase the amount of polymerase in 0.5 U steps. Cycle conditions are not optimal Decrease annealing temperature. Check elongation time (1 min/ 1kb PCR fragment). Denaturation time should be not below 30 sec. at 95 o C. Increase cycle number. continued on next page 15

16 4. Trouble shooting, continued Problem Possible Cause Recommendation Multiple bands or background smear PCR products in negative control experiments Problems with cloning of PCR products Specific problems in RT-PCR application Annealing temperature too low Primer design or concentration not optimal Difficult template e.g. GC- rich template Too high starting concentrations of: Mg-ions Template versus cycles Enzyme Too many cycles Increase annealing temperature. Review primer design Titrate primer concentration Performe PCR with GC-RICH solution. Reduce Mg concentration. Check template concentration by titration or on an agarose gel. Use 2 U FastStart Taq per 50 µl. Titrate enzyme units down in a 0.25 U step. Reduce cycles in steps of 3 cycles. Carry-over contamination Exchange all reagents. Use disposable pipet tips containing filters. Set up PCR reactions in an area seperate from that used for PCR product analysis. Use dutp (600 µm) instead of dttp (200 µm) in combination with thermolabile UNG (1 U/50 µl reaction). Increase Mg-ion concentration (4 mm) (see protocol C). No product, additional bands, background smear FastStart Taq DNA Polymerase does add additional A at the 3 end of PCR products similiar to Taq DNA Polymerase. Therefore, PCR products can be cloned into TA cloning vectors. Cloning in blunt end vectors need a blunt end polishing step in advance. The volume of cdna template (RTreaction) should not exceed 10% of the final concentration of the PCR reaction. Titrate cdna template. Follow trouble shooting above. 5. References 1 Chou,Q et al (1992) Nucleic Acid Res. 20: Kellogg, D.E. et al (1994) BioTechniques 16: Birch, D.E. et al (1996) Nature 381: PCR Application Manual, Roche Molecular Diagnostics, 2 nd edition (1999) 2: BIOCHEMICA (2001) 1:

17 6. Ordering Information For your further information: offers a large selection of reagents and systems for life science research. For a complete overview of related products and manuals, please visit and bookmark our Special Interest Sites including: PCR - Innovative Tools for Amplification: The LightCycler System Family for real-time, online PCR: Product Pack size Cat. No. Water, PCR Grade 25 ml ml ml GC-RICH PCR System 100 units (50 reactions Uracil-DNA Glycosylase, heat-labile 100 units units High Pure PCR Template Preparation Kit 100 purifications High Pure PCR Product Purification Kit 50 purification s purification s Transcriptor Reverse Transcriptase 250 U U U Transcriptor First Strand cdna Synthesis Kit 1 kit First Strand cdna Synthesis Kit for RT-PCR 1 kit Digoxigenin-11-dUTP (alkali-labile) 25 nmol (25 µl) nmol (125 µl) Digoxigenin-11-dUTP (alkali-stable) 25 nmol (25 µl) nmol (125 µl) x 125 nmol Biotin-16-dUTP 50 nm ol (50 µl) Fluorescein-12-dUTP 25 nmol ( 25 µl) PCR Cloning Kit (blunt end) 1 kit (35 reactions ) ) FastStart and Expand are trademarks of a member of the Roche Group. 2 ) Tween is a trademark of ICI Americas Inc., Wilmington, USA. EP Patent , US 5,773,258 and US 5,677,152 owned by Roche Molecular Systems, Inc. Equivalent patent applications are pending in other countries. Expand High Fidelity PCR System is covered by U.S. patent and

18 NOTICE TO PURCHASER: LIMITED LICENSE: A license under U.S. Patents 4,683,202, 4,683,195, and 4,965,188 or their foreign counterparts, owned by Roche Molecular Systems, Inc. and F. Hoffmann-La Roche Ltd ("Roche"), has an up-front fee component and a running-royalty component. The purchase price of this product includes limited, nontransferable rights under the running-royalty component to use only this amount of the product to practice the Polymerase Chain Reaction ("PCR") and related processes described in said patents solely for the research and development activities of the purchaser when this product is used in conjunction with a thermal cycler whose use is covered by the upfront fee component. Rights to the up-front fee component must be obtained by the end user in order to have a complete license to use this product in the PCR process. These rights under the up-front fee component may be purchased from Applied Biosystems or obtained by purchasing an Authorized Thermal Cycler. No right to perform or offer commercial services of any kind using PCR, including without limitation reporting the results of purchaser's activities for a fee or other commercial consideration, is hereby granted by implication or estoppel. Further information on purchasing licenses to practice the PCR Process may be obtained by contacting the Director of Licensing at Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California or the Licensing Department, Roche Molecular Systems, Inc., 1145 Atlantic Avenue, Alameda, California

19 19

20 Contact and Support If you have questions or experience problems with this or any Roche Applied Science (RAS) product, please contact our Technical Support staff. Our scientists commit themselves to providing rapid and effective help. We also want you to contact us if you have suggestions for enhancing RAS product performance or using our products in new or specialized ways. Such customer information has repeatedly proven invaluable to RAS and the worldwide research community. To ask questions, solve problems, suggest enhancements or report new applications, please visit our Online Technical Support Site at: To call, write, fax, or us, visit the home page, and select your home country. Country-specific contact information will be displayed. On the home page select Printed Materials to find: in-depth Technical Manuals Lab FAQS: Protocols and references for life science research our quarterly Biochemica Newsletter Material Safety Data Sheets Pack Inserts and Product Instructions or to request hard copies of printed materials ➄ Roche Diagnostics GmbH Mannheim Germany

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