GeneAmp High Fidelity PCR System Protocol

Transcription

1 GeneAmp High Fidelity PCR System Protocol For Research Use Only. Not for use in diagnostic procedures.

2 Copyright 2001, 2010 Applied Biosystems. All rights reserved. For Research Use Only. Not for use in diagnostic procedures. Information in this document is subject to change without notice. Applied Biosystems assumes no responsibility for any errors that may appear in this document. This document is believed to be complete and accurate at the time of publication. In no event shall Applied Biosystems be liable for incidental, special, multiple, or consequential damages in connection with or arising from the use of this document. NOTICE TO PURCHASER: LIMITED LICENSE AB (Design), ABI, ABI Masterpiece, Applera, AutoAssembler, BaseSprinter, CATALYST, GeneAssist, LV40, MatchMaker, PDQ, Primer Express, and ProSorb are trademarks of Applied Biosystems or its subsidiaries in the U.S. and certain other countries. AmpErase, AmpliTaq, AmpliTaq Gold, EnviroAmp, GeneAmp, and TaqMan are registered trademarks of Roche Molecular Systems, Inc. All other trademarks are the sole property of their respective owners. Applied Biosystems is committed to providing the world s leading technology and information for life scientists. Printed in the USA, 06/2010 Part Number Rev. B

3 Contents Introduction Product Features About This Protocol About GeneAmp High Fidelity PCR System System Contents and Required Materials GeneAmp High Fidelity PCR System Storage and Stability Performance Characteristics Materials Required But Not Supplied Safety Documentation User Attention Words Chemical Hazard Warning Site Preparation and Safety Guide Ordering MSDSs Preventing Contamination Overview General PCR Practices Carryover Prevention with dutp not Recommended Protocol for Amplification of Samples Overview Preparing the Master Mixes MgCl 2 Optimization Overview Tips for Optimization Thermal Cycling Optimization Guidelines Thermal Cycling Protocol iii

4 Adjusting the Denaturation Conditions Adjusting the Annealing Conditions Adjusting the Extension Conditions Appendix A. Troubleshooting Appendix B. References Appendix C. Technical Support Contacting Technical Support To Contact Technical Support by To Contact Technical Support by Telephone or Fax (North America) To Contact Technical Support by Telephone or Fax (Outside North America) To Reach Technical Support Through the Applied Biosystems Web Site To Obtain Technical Documents To Obtain Customer Training Information iv

5 Introduction Product Features The GeneAmp High Fidelity PCR System (P/Ns , ) contains an enzyme blend of AmpliTaq DNA polymerase and a proprietary proofreading enzyme (Barnes, 1994). It also contains buffers that are designed specifically for this unique enzyme blend. The enzyme mixture produces PCR products in high yields, with high fidelity, and high specificity. GeneAmp High Fidelity PCR System enables you to: Amplify DNA fragments up to 10 kb 1 Increase the fidelity of DNA synthesis three-fold, when compared to Taq DNA polymerase About This Protocol About GeneAmp High Fidelity PCR System This protocol describes how to use the GeneAmp High Fidelity PCR System to amplify DNA samples. It also provides guidelines for optimizing thermal cycling, preventing contamination, and troubleshooting problems. The inherent 3 5 exonuclease proofreading activity in the GeneAmp High Fidelity PCR System results in a DNA synthesis fidelity 3X greater than Taq DNA polymerase. Amplified products contain a mixture of 3' single-a overhang products and blunt-ended products. 1. Product yield may decrease as PCR product size increases. May require optimization. 1

6 System Contents and Required Materials GeneAmp High Fidelity PCR System The GeneAmp High Fidelity PCR System (see table below for available sizes) contains: System Contents GeneAmp High Fidelity Enzyme Mix (5 U/µL) The enzyme mix is in the following Storage and Dilution Buffer: 20mM Tris-HCl, ph 7.5 (25 C), 100 mm KCl, 1mM dithiothreitol (DTT), 0.1 mm EDTA, 0.5% Tween 2 20 (v/v), 0.5% Nonidet 3 P40 (v/v), 50% glycerol (v/v). GeneAmp High Fidelity 10X PCR Buffer with MgCl 2 GeneAmp High Fidelity 10X PCR Buffer without MgCl 2 MgCl 2 solution, 25 mm Available Sizes Reactions per System Enzyme Units Per System Part Number X Storage and Stability Upon receipt, store the GeneAmp High Fidelity PCR System at 15 C to 25 C in a constant temperature freezer. If stored under the recommended conditions, the product will maintain performance through the control date printed on the label. Performance Characteristics Each lot of the GeneAmp High Fidelity PCR System has been tested for its ability to yield a specifically expressed visible band when amplifying a 1 kb collagen gene and a 4.8 kb tissue plasminogen activator (tpa) gene fragment from human genomic DNA. 2. Tween 20 is a trademark of ICI Americas Inc., Wilmington, USA. 3. Nonidet P40 is a trademark of Shell International Petroleum Company Limited, U.K. 2

7 Materials Required But Not Supplied In addition to the GeneAmp High Fidelity PCR System, the items listed in the following table may be required. Item GeneAmp PCR System 9700, 9600, 2700, or 2400 Thermal Cyclers MicroAmp disposable plates and tubes for the 9700, 9600, 2700, or 2400 PCR System Agarose Disposable gloves Electrophoresis apparatus Microcentrifuge Pipets, positive-displacement or air-displacement Pipet tips with filter plugs Polypropylene tubes Vortex Source Applied Biosystems (Visit our Web site at Applied Biosystems (Visit our Web site at Major laboratory supplier (MLS) MLS MLS MLS MLS MLS MLS MLS 3

8 Safety Documentation User Attention Words Five user attention words appear in the text of all Applied Biosystems user documentation. Each word implies a particular level of observation or action as described below. Note Calls attention to useful information. IMPORTANT operation. Indicates information that is necessary for proper instrument! CAUTION Indicates a potentially hazardous situation which, if not avoided, may result in minor or moderate injury. It may also be used to alert against unsafe practices.! WARNING Indicates a potentially hazardous situation which, if not avoided, could result in death or serious injury.! DANGER Indicates an imminently hazardous situation which, if not avoided, will result in death or serious injury. This signal word is to be limited to the most extreme situations. Chemical Hazard Warning! WARNING CHEMICAL HAZARD. Some of the chemicals used with Applied Biosystems instruments and protocols are potentially hazardous and can cause injury, illness, or death. Read and understand the material safety data sheets (MSDSs) provided by the chemical manufacturer before you store, handle, or work with any chemicals or hazardous materials. Minimize contact with chemicals. Wear appropriate personal protective equipment when handling chemicals (e.g., safety glasses, gloves, or protective clothing). For additional safety guidelines, consult the MSDS. Minimize the inhalation of chemicals. Do not leave chemical containers open. Use only with adequate ventilation (e.g., fume hood). For additional safety guidelines, consult the MSDS. Check regularly for chemical leaks or spills. If a leak or spill occurs, follow the manufacturer s cleanup procedures as recommended on the MSDS. Comply with all local, state/provincial, or national laws and regulations related to chemical storage, handling, and disposal. 4

9 Site Preparation and Safety Guide Ordering MSDSs A site preparation and safety guide is a separate document sent to all customers who have purchased an Applied Biosystems instrument. Refer to the guide written for your instrument for information on site preparation, instrument safety, chemical safety, and waste profiles. You can order free additional copies of MSDSs for chemicals manufactured or distributed by Applied Biosystems using the contact information below.. To order documents by automated telephone service: 1 From the U.S. or Canada, dial , or from outside the U.S. and Canada, dial Follow the voice instructions to order documents (for delivery by fax). Note There is a limit of five documents per fax request. To order documents by telephone: In the U.S. Dial , and press 1. In Canada From any other country To order in English, dial and press 1, then 2, then 1 To order in French, dial and press 2, then 2, then 1 See the specific region under To Contact Technical Support by Telephone or Fax (Outside North America) on page 20. To view, download, or order documents through the Applied Biosystems web site: Step Action 1 Go to 2 Click SERVICES & SUPPORT at the top of the page, click Documents on Demand, then click MSDS. 3 Click MSDS Index, search through the list for the chemical of interest to you, then click on the MSDS document number for that chemical to open a pdf of the MSDS. For chemicals not manufactured or distributed by Applied Biosystems, call the chemical manufacturer. 5

10 Preventing Contamination Overview Due to the enormous amplification that occurs during PCR, small levels of DNA contamination can result in product formation even in the absence of purposefully added template DNA (Kwok and Higuchi, 1989). DNA contamination can come from: Previous PCR amplifications Samples with high DNA concentration Cross contamination Positive control templates Note To prevent carry-over contamination, we recommend that you use aerosol resistant pipette tips. General PCR Practices Follow these recommendations: Maintain separate areas and dedicated equipment and supplies for: Sample preparation PCR setup PCR amplification Analysis of PCR products Wear a clean lab coat (not previously worn while handling amplified PCR products or used during sample preparation) and clean gloves when preparing samples for PCR amplification. Change gloves whenever you suspect that they are contaminated. Never bring amplified PCR products into the PCR setup area. Open and close all sample tubes carefully. Try not to splash or spray PCR samples. Keep reactions and components capped as much as possible. Clean lab benches and equipment periodically with a 10% bleach solution, and dry thoroughly before use. Use dedicated or disposable sterile vessels, solutions, and pipets (preferably positive-displacement pipets or tips with hydrophobic filters) to minimize cross-contamination during DNA preparation, reaction mixing, and sample analysis (Kwok, 1990). 6

11 Carryover Prevention with dutp not Recommended Deoxyuracil triphosphate (dutp) is a poor substrate for the GeneAmp High Fidelity enzyme mix. Therefore, we do not recommend the UNG carryover prevention method with this system. 7

12 Protocol for Amplification of Samples Overview The GeneAmp High Fidelity PCR System is designed for use with two master mixes. The preparation of two separate master mixes allows you to have a simpler manual Hot Start method. In the first master mix, master mix one, dntps are combined with template and primers. In the second master mix, master mix two, the enzymes and buffers are mixed. The combination of the two master mixes results in a final reaction volume of 50 µl. Because there is no premixing of enzymes with template or primers, the potential for partial degradation of primer or template through the 3 5 exonuclease activity of the enzyme is eliminated. Preparing the Master Mixes To prepare the two master mixes: Step Action 1 Prepare master mix one by combining the following: Note Keep on ice until ready to use. Component Volume per Reaction Final Conc. Deionized H 2 O up to 25 µl a User-provided Primer 1 TBD 300 nm User-provided Primer 2 TBD 300 nm User-provided Template b TBD ( µg) datp, 10 mm 1 µl 200 µm dctp, 10 mm 1 µl 200 µm dgtp, 10 mm 1 µl 200 µm dttp, 10 mm 1 µl 200 µm Total 25 µl a. Any combination of water and template can be used as long as the total volume of the reaction equals 50 µl (25 µl from each master mix). b. Preferably µg of human genomic DNA or pg of plasmid. 8

13 To prepare the two master mixes: (continued) Step Action 2 IMPORTANT GeneAmp High Fidelity PCR Enzyme Mix (containing Storage and Dilution Buffer). Please read the MSDS, and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves. Prepare master mix two by combining the following: Component Volume per Reaction Final Concentration Deionized H 2 O up to 25 µl a GeneAmp High Fidelity 5 µl 1X 10X PCR Buffer b,c MgCl b,c 2 TBD 1 4 mm GeneAmp High Fidelity 0.5 µl 2.5 Units Enzyme Mix d Total 25 µl a. Any combination of water and template can be used as long as the total volume of the reaction equals 50 µl (25 µl from each master mix). b. When you use GeneAmp High Fidelity 10X PCR Buffer without MgCl 2, the optimal Mg 2+ concentration must be determined and depends on the template/primer pair. c. Optimal Mg 2+ concentration is in the range of 1 4 mm. The final Mg 2+ concentration of the GeneAmp High Fidelity 10X PCR Buffer with MgCl 2 is 1.5 mm. d. Optimal enzyme concentrations range from units per assay. The standard final concentration is 2.5 units. 3 On ice, pipet 25 µl of master mix one and 25 µl of master mix two into a MicroAmp disposable plate or tube. 4 Gently mix the reagents. 5 Place the sample(s) in a thermal cycler. 6 Perform thermal cycling using the parameters stated in Thermal Cycling Optimization Guidelines on page Analyze samples by loading them onto a % agarose gel. 9

14 MgCl 2 Optimization Overview Tips for Optimization The optimal concentration of MgCl 2 depends on the template/primer pair and must be determined individually. The optimal Mg 2+ concentration for this enzyme blend is in the range of 1 4 mm. The final MgCl 2 concentration in 1X High Fidelity buffer with MgCl 2 is 1.5 mm. Use the following table to adjust the MgCl 2 concentration and optimize your results. If... you have a high background smear you have unspecific products you have low product yield Then... decrease Mg 2+ -ion concentration decrease Mg 2+ -ion concentration increase Mg 2+ -ion concentration Other optimization suggestions can be found at: 10

15 Thermal Cycling Optimization Guidelines Thermal Cycling Protocol Thermal cycling parameters for the GeneAmp PCR System 9700, 9600, 2700, or 2400 are as follows: PCR CYCLE (25 to 30 cycles) Step Initial Denature Anneal Extend Final Temp 94 C 94 C C a 68 or 72 C b 72 C Time 2 min 15 sec 30 sec 45 sec to 8 min c,d 7 min a. Adjust the temperature according to the primer melting temperature. Refer to Adjusting the Annealing Conditions on page 12 for more information. b. Extension temperature depends on the length of the amplification product: 72 C is used for amplifications up to 3 kb; 68 C is used for amplifications > 3 kb. c. Extension time depends on fragment length. We recommend extension times as stated below. PCR fragment length (kb) up to 0.75 Extension time 45 sec min 2 min 4 min 8 min d. Add an additional cycle extension of 5 sec/cycle after cycle 10 with the Autox feature (automatic extension) on GeneAmp PCR Systems 9700, 9600, 2700, or For example: Cycle number Additional extension time +5 sec +10 sec +15 sec Adjusting the Denaturation Conditions The following are guidelines for adjusting the denaturation conditions: The maximum denaturation temperature should not exceed C (Gelfand et al., 1990). A 15 second denaturation time is adequate when using the GeneAmp PCR System thermal cyclers which display a calculated sample temperature. Some models of thermal cyclers may require longer denaturation times. 11

16 Adjusting the Annealing Conditions Adjusting the Extension Conditions The following are guidelines for adjusting annealing conditions: For increased product specificity, use annealing temperatures greater than 45 C (Saiki et al., 1988; Rychlik et al., 1990). The optimum annealing temperature can be determined empirically by testing at 1 2 C increments, until the maximum specificity is reached. Computer programs designed to calculate primer melting temperatures (T m ), can assist you in narrowing the range of annealing temperatures for empirical determination. A T m calculator can be found on the Applied Biosystems Web site at The GeneAmp PCR System 9700 and 2700 Thermal Cyclers also contain a T m calculator. The following are guidelines for adjusting the extension conditions: The length of the target sequence will affect the required extension time. Longer targets require increased extension times. See page 11 for details. As the amount of DNA increases, the number of DNA polymerase molecules may become limiting. This can be compensated by increasing the extension time in later cycles. 12

17 Appendix A. Troubleshooting Observation Possible Cause Recommended Action Reduced or no product band visible Product band is smeared Template concentration too low Experimental sample DNA damaged or degraded Denaturation time too short or too long Denaturation temperature too low or too high Annealing/extension temperature too high Increase sample concentration. Use sample that has been processed to minimize shearing and nicking. Adjust time in increments of 5 seconds. Adjust temperature in increments of 1 C. Lower temperature in increments of 2 C. Annealing/extension time too short Lengthen time in increments of 15 seconds. Cycle number too low Increase cycle number in increments of three cycles. Primer design not optimal Review primer design and composition. Mg 2+ -ion concentration is too low Increase the Mg 2+ -ion concentration. Carryover contamination Enzyme concentration too high Denaturation time too short or too long Denaturation temperature too low Annealing temperature too low Annealing/extension time too long Cycle number too high Experimental sample DNA degraded Mg 2+ -ion concentration too high See Preventing Contamination on page 6. Decrease enzyme concentration. Adjust time in increments of 5 seconds. Increase temperature in increments of 1 C. Increase temperature in increments of 2 C. Shorten time in increments of 15 seconds. Shorten cycle number in increments of three cycles. Test a new aliquot of sample. Decrease Mg 2+ -ion concentration. 13

18 Observation Possible Cause Recommended Action Nonspecific amplification with or without a product band Carry-over contamination Nonspecific priming Primer design not optimal Mg 2+ -ion concentration too high See Preventing Contamination on page 6. Increase the anneal temperature in 1 2 C increments. Review primer design and composition. Decrease Mg 2+ -ion concentration. 14

19 Appendix B. References Barnes, W.M PCR Amplification of up to 35 kb DNA with High Fidelity and High Yield from λ Bacteriophage Templates. PNAS 91: Gelfand, D.H. and White, T.J Thermostable DNA polymerases. In PCR Protocols: A Guide to Methods and Applications. Innis, M.A., Gelfand, D.H., Sninsky, J.J., and White, T.J., eds. San Diego: Academic Press Kwok, S Procedures to minimize PCR-product carry-over. In PCR Protocols: A Guide to Methods and Applications. Innis, M.A., Gefland, D.H., Sninsky, J.J., and White, T.J., eds. San Diego: Academic Press Kwok, S. and Higuchi, R Avoiding false positives with PCR. Nature 339: Rychlik, W., Spencer, W.J., and Rhoads, R.E Optimization of the annealing temperature for DNA amplification in vitro [published erratum appears in Nucleic Acids Res 1991 Feb 11;19(3):698]. Nucleic Acids Res. 18: Saiki, R.K., Gelfand, D.H., Stoffel, S., et al Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239:

20 Appendix C. Technical Support Contacting Technical Support You can contact Applied Biosystems for technical support: By By telephone or fax Through the Applied Biosystems web site You can order Applied Biosystems user documents, MSDSs, certificates of analysis, and other related documents 24 hours a day. In addition, you can download documents in PDF format from the Applied Biosystems web site. (Please see the section To Obtain Technical Documents following the telephone information below) To Contact Technical Support by You can contact Applied Biosystems Technical Support by for help in the following product areas: Product/Product Area Genetic Analysis (DNA Sequencing) Sequence Detection Systems (Real-Time PCR) and PCR Protein Sequencing, Peptide, and DNA Synthesis address galab@appliedbiosystems.com pcrlab@appliedbiosystems.com corelab@appliedbiosystems.com 16

21 Product/Product Area Biochromatography (BioCAD, SPRINT, VISION, and INTEGRAL Workstations and POROS Perfusion Chromatography Products) Expedite 8900 Nucleic Acid Synthesis Systems MassGenotyping Solution 1 (MGS1) Systems PNA Custom and Synthesis Pioneer Peptide Synthesizers Proteomics Solution 1 (PS1) Systems ICAT Reagent FMAT 8100 HTS Systems Mariner ESI-TOF Mass Spectrometry Workstations Voyager MALDI-TOF Biospectrometry Workstations CytoFluor 4000 Fluorescence Plate Reader LC/MS (Applied Biosystems/MDS Sciex) Chemiluminescence (Tropix) address tsupport@appliedbiosystems.com support@sciex.com tropix@appliedbiosystems.com To Contact Technical Support by Telephone or Fax (North America) To contact Applied Biosystems Technical Support in North America, use the telephone or fax numbers in the table below. Note To schedule a service call for other support needs, or in case of an emergency, dial , then press 1. Product/Product Area Telephone Fax ABI PRISM 3700 DNA Analyzer , then press 8 a DNA Synthesis , press 2, then press 1 a

22 Product/Product Area Telephone Fax Fluorescent DNA Sequencing , press 2, then press 2 a Fluorescent Fragment Analysis (including GeneScan applications) Integrated Thermal Cyclers (ABI PRISM 877 and Catalyst 800 instruments) ABI PRISM 3100 Genetic Analyzer Peptide Synthesis (433 and 43x Systems) Protein Sequencing (Procise Protein Sequencing Systems) Sequence Detection Systems (Real-Time PCR) and PCR , press 2, then press 3 a , press 2, then press 4 a , press 2, then press 6 a , press 3, then press 1 a , press 3, then press 2 a , then press: 1 for PCR a 2 for TaqMan applications and Sequence Detection Systems including ABI Prism 7700, 7900, and 5700 a 6 for the 6700 Automated Sample Prep System a or , then press 5 a

23 Product/Product Area Telephone Fax Mariner ESI-TOF Mass Spectrometry Workstations Voyager MALDI-TOF Biospectrometry Workstations MassGenotyping Solution 1 (MGS1) Systems Proteomics Solution 1 (PS1) Systems ICAT Reagent Biochromatography (BioCAD, SPRINT, VISION, and INTEGRAL Workstations and POROS Perfusion Chromatography Products) Expedite 8900 Nucleic Acid Synthesis Systems a. 5:30 AM to 5:00 PM Pacific time. b. 8:00 AM to 6:00 PM Eastern time. c. 9:00 AM to 5:00 PM Eastern time , press 1, then press 3 b , press 1, then press 4 b , press 1, then press 5 b Pioneer Peptide Synthesizers , press 1, then press 5 b PNA Custom and Synthesis , press 1, then press 5 b FMAT 8100 HTS Systems CytoFluor 4000 Fluorescence Plate Reader , press 1, then press 6 b Chemiluminescence (Tropix) (U.S. only), or c LC/MS (Applied Biosystems/MDS Sciex)

24 To Contact Technical Support by Telephone or Fax (Outside North America) To contact Applied Biosystems Technical Support or Field Service outside North America, use the telephone or fax numbers below. Region Telephone Fax Eastern Asia, China, Oceania Australia (Scoresby, Victoria) China (Beijing) or Hong Kong India (New Delhi) / Korea (Seoul) / Malaysia (Petaling Jaya) Singapore Taiwan (Taipei Hsien) Thailand (Bangkok) Europe Austria (Wien) 43 (0) (0) Belgium 32 (0) (0) Denmark (Naerum) Finland (Espoo) 358 (0) (0) France (Paris) 33 (0) (0) Germany (Weiterstadt) 49 (0) (0) Italy (Milano) 39 (0) (0) Norway (Oslo) Portugal (Lisboa) 351.(0) (0) Spain (Tres Cantos) 34.(0) (0) Sweden (Stockholm) 46 (0) (0) Switzerland (Rotkreuz) 41 (0) (0) The Netherlands (Nieuwerkerk a/d IJssel) United Kingdom (Warrington, Cheshire) 31 (0) (0) or 31 (0) (0) (0)

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26 To Reach Technical Support Through the Applied Biosystems Web Site At the Applied Biosystems web site, you can search through frequently asked questions (FAQs) or a solution database, or you can submit a question directly to Technical Support. Search FAQs To search for FAQs: Step Action 1 Go to 2 Click SERVICES & SUPPORT at the top of the page, then click Frequently Asked Questions. 3 Click you geographic region for the product area of interest. 4 Follow the instructions under the Frequently Asked Questions section (1) to display a list of FAQs for your area of interest. Search the Solution Database To search for solutions to problems using the Solution Database: Step Action 1 Go to 2 Click SERVICES & SUPPORT at the top of the page, then click Frequently Asked Questions. 3 Follow the instructions under the Search the Solution Database section (2) to find a solution to your problem. Submit a Question To submit a question directly to Technical Support: 1 Go to 2 Click SERVICES & SUPPORT at the top of the page, then click Frequently Asked Questions. 3 In the Personal Assistance Support section (3), click Ask Us RIGHT NOW. 4 In the displayed form, enter the requested information and your question, then click Ask Us RIGHT NOW. Within 24 to 48 hours, you will receive an reply to your question from an Applied Biosystems technical expert. 22

27 To Obtain Technical Documents You can obtain technical documents, such as Applied Biosystems user documents, MSDSs, certificates of analysis, and other related documents for free, 24 hours a day. You can obtain documents: By telephone Through the Applied Biosystems web site Ordering Documents by Telephone To order documents by telephone: 1 From the U.S. or Canada, dial , or from outside the U.S. and Canada, dial Follow the voice instructions to order documents (for delivery by fax). Note There is a limit of five documents per fax request. Obtaining Documents Through the Web Site To view, download, or order documents through the Applied Biosystems web site: Step Action 1 Go to 2 Click SERVICES & SUPPORT at the top of the page, then click Documents on Demand. 3 In the search form, enter and select search criteria, then click Search at the bottom of the page. 4 In the results screen, do any of the following: Click the pdf icon to view a PDF version of the document. Right-click the pdf icon, then select Save Target As to download a copy of the PDF file. Select the Fax check box, then click Deliver Selected Documents Now to have the document faxed to you. Select the check box, then click Deliver Selected Documents Now to have the document (PDF format) ed to you. Note There is a limit of five documents per fax request, but no limit on the number of documents per request. 23

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