AmpliTaq® 360 DNA Polymerase Protocol
Contents
1. 28 Chemical alerts oce Rp EE E RUD REG C ee deg 29 AmpliTaq 360 DNA Polymerase Protocol 23 Appendix C Safety Chemical hazard warnings 24 WARNING CHEMICAL HAZARD Before handling any chemicals refer to the Material Safety Data Sheet MSDS provided by the manufacturer and observe all relevant precautions WARNING CHEMICAL HAZARD All chemicals in the instrument including liquid in the lines are potentially hazardous Always determine what chemicals have been used in the instrument before changing reagents or instrument components Wear appropriate eyewear protective clothing and gloves when working on the instrument WARNING CHEMICAL HAZARD Four liter reagent and waste bottles can crack and leak Each 4 liter bottle should be secured in a low density polyethylene safety container with the cover fastened and the handles locked in the upright position Wear appropriate eyewear clothing and gloves when handling reagent and waste bottles WARNING CHEMICAL STORAGE HAZARD Never collect or store waste in a glass container because of the risk of breaking or shattering Reagent and waste bottles can crack and leak Each waste bottle should be secured in a low density polyethylene safety container with the cover fastened and the handles locked in the upright position Wear appropriate eyewear clothing and gloves when handling reagent and waste bottles AmpliTaq 360 DNA Polymerase Proto2. Test the primer pairs then select the primer pair that produces the largest amount of specific product and the least amount of non specific product To determine specific product compare migration through an agarose gel of amplicons to that of the DNA bands of known length in a DNA ladder Adjust thermal cycling Adjusting n the early cycles make sure that your DNA template is completely denatured denaturation Do not exceed a denaturation temperature of 95 to 96 C Gelfand and White conditions 1990 A denaturation period of 30 seconds is adequate when using Veriti and GeneAmp PCR System thermal cyclers with a calculated in tube temperature Some models of thermal cyclers may require longer denaturation times Adjusting For increased product specificity use annealing temperatures higher than 45 C annealing Saiki Gelfand and Stoffel 1988 Rychlik Spencer and Rhoads 1990 conditions Determine the optimum annealing temperature by testing at increments of 5 or fewer degrees Celsius until the maximum specificity is reached Computer programs that calculate primer melting temperatures T can help you narrow the range of annealing temperatures to test For such a T calculator go to http www appliedbiosystems com then select Services amp Support Technical Tools T Calculator The GeneAmp PCR System 9700 Thermal Cycler also contains a T calculator Thirty seconds is adequate annealin
3. 1996 Simplified Hot Start PCR Product review in Nature 381 445 446 Bost D A Zalloua P and Abramson R D 1997 AmpliTaq Gold Biochemical Characterization and PCR Optimization The FASEB Journal 11 A1370 Chou Q Russell M Birch D E Raymond J and Bloch W 1992 Prevention of pre PCR mis priming and primer dimerization improves low copy number amplifications Nucleic Acids Res 20 1717 1723 Eckert K A and Kunkel T A 1992 The fidelity of DNA polymerases used in the polymerase chain reactions In PCR A Practical Approach McPherson M J Quirke P and Taylor G R eds New York Oxford University Press 225 244 Faloona F Weiss S Ferre F and Mullis K 1990 Direct detection of HIV sequences in blood high gain polymerase chain reaction abstract In 6th International Conference on AIDS University of California San Francisco San Francisco CA Abstract 1019 Gelfand D H and White T J 1990 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 129 141 Holland P M Abramson R D Watson R and Gelfand D H 1991 Detection of specific polymerase chain reaction product by utilizing the 5 9 exonuclease activity of Thermus aquaticus DNA polymerase Proc Natl Acad Sci USA 88 7276 7280 Innis M A Myambo K B Gelfand D H and Brow M A 1988 DNA
4. Appendix B Guidelines for Designing PCR Assays 17 PCR good laboratory practices AA KUIA ee 18 Select the amplicon site UA IAE Aa nee 19 Adjust thermal cycling AA do be ae ian eee Rd 19 Optimize the PCR conditions 20 Appendix C Safety 23 Chemical hazard warnings 4 kk kk kk kk kk kK kK KK KI KK KI KK KK n 24 Chemical safety guidelines os sk haa na kok A WE k Ak k jk w j kar de dok a l 25 MSDSS xa S n kuya seus e pU dan eate t Aten ra ak eo swe a y d eve ay Kalk din ide i dida Auca 26 Chemical waste hazards 26 Chemical waste safety guidelines 27 Waste disposal 27 Biological hazard Safety ix ase kass kwla bh aa ak Alk rn 28 Chemical alerts sitas Aa IS Et ated tng r ads J 29 AmpliTaq 360 DNA Polymerase Protocol iii Contents Bibliography scort aa 31 elec MP PEEL Pr PPP AR 33 Documentation and Support 35 Related documentation Tros a a paya ea eres 35 How to obtain support kk kk kk kk kk kK kk KK KK KK KK KK KK KK KK kk kk KK kK kk kk kk lk kk 36 AmpliTaq 360 DNA Polymerase Protocol Preface This preface covers Safety information How to USE THIS gde r ienei A A N eee eh LA 2 Kwa AmpliTaq 360 DNA Polymerase P
5. and the handles locked in the upright position Wear appropriate eyewear clothing and gloves when handling reagent and waste bottles 26 AmpliTaq 360 DNA Polymerase Protocol Chemical waste safety guidelines Chemical waste safety guidelines To minimize the hazards of chemical waste Waste disposal Read and understand the Material Safety Data Sheets MSDSs provided by the manufacturers of the chemicals in the waste container before you store handle or dispose of chemical waste Provide primary and secondary waste containers A primary waste container holds the immediate waste A secondary container contains spills or leaks from the primary container Both containers must be compatible with the waste material and meet federal state and local requirements for container storage Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals for example 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 for example fume hood For additional safety guidelines consult the MSDS Handle chemical wastes in a fume hood After emptying a waste container seal it with the cap provided Dispose of the contents of the waste tray and waste bottle in accordance with good laboratory practices and local state provincial or nation
6. 2 degrees Celsius for up to six different temperatures Annealing extension time is too short Increase the time in increments of 15 seconds Cycle number is too low Increase the cycle number in increments of three cycles Primer design is not optimal Review primer design and composition AmpliTaq 360 DNA Polymerase Protocol Troubleshooting 11 AmpliTaq 360 DNA Polymerase Protocol Observation Possible cause Recommended action Product band Carryover contamination Use the GeneAmp PCR Carry Over is smeared Prevention Kit PN N8080068 Dispose of reagents make fresh reagents then repeat the PCR Denaturation time is too short or too Adjust the time in increments of long 5 seconds Denaturation temperature is too low Increase the temperature in increments of 1 degree Celsius Annealing extension time is too long Shorten the time in increments of 15 seconds Cycle number is too high Shorten the cycle number in increments of 3 cycles Experimental sample DNA is degraded Test a new aliquot of sample 12 AmpliTaq 360 DNA Polymerase Protocol Appendix A Ordering Information This appendix covers Materials and equipment not included Thermalcyclers mita ida a a Other equipment and consumables AmpliTaq 360 DNA Polymerase Protocol Appendix A Ordering Information Materials and
7. Protocol 3 AmpliTag 360 DNA Polymerase Protocol Part Quantity Sumber AmpliTag 360 Buffer Kit 4398848 e AmpliTaq 360 Buffer 10X 1 x 1 5 mL 360 GC Enhancer 1 x 1 5 mL e 25 mM Magnesium Chloride 1 x 1 5 mL AmpliTag 360 Buffer Kit 4398858 e AmpliTaq 360 Buffer 10X 6 x 1 5 mL 360 GC Enhancer 6 x 1 5 mL e 25 mM Magnesium Chloride 6 x 1 5 mL AmpliTaq 360 Buffer Kit 4398868 e AmpliTaq 360 Buffer 10X 1 x 150 mL e 360 GC Enhancer 1 x 120 mL e 25 mM Magnesium Chloride 1 x 150 mL Storage Store all components at 15 to 25 C 4 AmpliTaq 360 DNA Polymerase Protocol Workflow Workflow The workflow for PCR using the AmpliTaq 360 DNA Polymerase Prepare the reaction mix Prepare the reaction plate or tubes 384 well plate 96 well plate Set up the run method 5 Load and run the plate or tubes Analyze the results AmpliTaq 360 DNA Polymerase Protocol 5 AmpliTaq 360 DNA Polymerase Protocol Before you perform PCR Prevent contamination Select an instrument and reaction plate Calculate the number of required reactions Review PCR good laboratory practices on page 18 You can perform PCR amplification with any of the instruments listed under Thermal cyclers on page 14 Use MicroAmp Optical 96 Well Reaction Plates PN N8010560 Other recommended equipment and consumables are listed beginning on page 14 Calc
8. equipment not included Thermal cyclers Other equipment and consumables 14 In addition to the reagents supplied the items listed in the following table are required Item Applied Biosystems PN VeritiTM 60 Well Thermal Cycler 4384638 Veriti 96 Well Fast Thermal 4375305 Cycler Veriti 96 Well Thermal Cycler 4375786 2720 Thermal Cycler 4359659 Aluminum 96 Well GeneAmp 4314445 PCR System 9700 Sample Block Module Gold plated 96 Well GeneAmp 4314878 PCR System 9700 t Only one thermal cycler or one PCR system is required Item Source MicroAmp Optical 96 Well Reaction Plates Applied Biosystems PN N8010560 MicroAmp Splash Free 96 Well Base Applied Biosystems 4312063 MicroAmp 8 Tube Strip 0 2 mL Applied Biosystems PN N8010580 MicroAmp 8 Cap Strip Applied Biosystems PN N8010535 MicroAmp 96 well Base Applied Biosystems N8010531 dNTP Mix Applied Biosystems PN N8080260 MicroAmp 96 Well Tray Retainer Set 10 sets Applied Biosystems PN 403081 Nuclease free water not DEPC treated 500 mL Applied Biosystems PN AM9930 MicroAmp Clear Adhesive Film Applied Biosystems PN 4306311 MicroAmp Optical Film Compression Padt Applied Biosystems PN 4312639 Centrifuge with plate adapter Major laboratory supplier MLS AmpliTaq 360 DNA Polymerase Protocol Materials and equipment
9. new MSDS packaged with a hazardous chemical be sure to replace the appropriate MSDS in your files Obtaining The MSDS for any chemical supplied by Applied Biosystems is available to you free MSDSs 24 hours a day To obtain MSDSs 1 Goto www appliedbiosystems com click Support then select MSDS 2 In the Keyword Search field enter the chemical name product name MSDS part number or other information that appears in the MSDS of interest Select the language of your choice then click Search 3 Find the document of interest right click the document title then select any of the following Open To view the document Print Target To print the document Save Target As To download a PDF version of the document to a destination that you choose Note For the MSDSs of chemicals not distributed by Applied Biosystems contact the chemical manufacturer Chemical waste hazards CAUTION HAZARDOUS WASTE Refer to Material Safety Data Sheets and local regulations for handling and disposal WARNING CHEMICAL WASTE HAZARD Wastes produced by Applied Biosystems instruments are potentially hazardous and can cause injury ilIness or death WARNING CHEMICAL STORAGE HAZARD Never collect or store waste in a glass container because of the risk of breaking or shattering Reagent and waste bottles can crack and leak Each waste bottle should be secured in a low density polyethylene safety container with the cover fastened
10. not included Source Pre cast agarose gels 1 up to 3 with MLS ethidium bromide stain Agarose MLS Disposable gloves MLS Electrophoresis apparatus MLS Microcentrifuge MLS Pipettes positive displacement or air MLS displacement Pipette tips with filter plugs MLS Polypropylene tubes MLS TBE buffer MLS TE buffer MLS Vortex MLS Disposable gloves MLS Microcentrifuge MLS 1 5 mL microcentrifuge tubes MLS Tris EDTA TE buffer pH 8 0 MLS Vortexer MLS t See instrument manual for compatibility For the MSDS of any chemical not distributed by Applied Biosystems contact the chemical manufacturer Before handling any chemicals refer to the MSDS provided by the manufacturer and observe all relevant precautions For more product recommendations visit the PCR technology page at www3 appliedbiosystems com applicationstechnologies PCR index htm newGl obalNav true AmpliTaq 360 DNA Polymerase Protocol 15 Appendix A Ordering Information 16 AmpliTaq 360 DNA Polymerase Protocol Appendix B Guidelines for Designing PCR Assays This appendix covers PCR good laboratory practices Select the amplicon site Adjust thermal cycling Optimize the PCR conditions AmpliTaq 360 DNA Polymerase Protocol Appendix B Guidelines for Designing PCR Assays PCR good laboratory
11. practices General PCR When preparing samples for PCR amplification practices When using a non hot start DNA polymerase keep PCR reagents on ice Prepare the reactions on ice Usea positive displacement pipette or aerosol resistant pipette tips Follow proper pipette operating techniques to prevent aerosols Wear clean gloves and a clean lab coat not previously worn while handling amplified PCR products or used during sample preparation Change gloves whenever you suspect that they are contaminated Maintain separate areas and dedicated equipment and supplies for Sample preparation PCR setup PCR amplification Analysis of PCR products 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 1096 bleach solution Use DNA Zap Solution PN AM9890 18 AmpliTaq 360 DNA Polymerase Protocol Select the amplicon site Select the amplicon site Using Primer Express Software select an amplicon site within the target sequence refer to the Primer Express Version 3 0 Getting Started Guide and Software Help Guidelines Design primer pairs according to Primer Express Software guidelines Use a primer pair that is specific to the target gene and does not amplify pseudogenes or other related genes
12. speed A solution that contains all components to run the PCR reaction except for the template sample standard or control A set of identical reactions in an experiment See biological replicates or technical replicates An oligonucleotide that flanks the 3 end of the amplicon The reverse primer and the forward primer are used together in PCR reactions to amplify the target An enzyme that converts RNA to cDNA Reverse transcriptase is added to the PCR reaction to perform 1 step RT PCR Definition of the reaction volume and the thermal profile for the instrument run The template that you are testing In the thermal profile a group of one or more steps In core PCR there are two types of stages holding stage including pre PCR read and post PCR read and cycling stage also called amplification stage A component of the thermal profile For each step in the thermal profile you can set the ramp rate ramp increment for melt curve steps hold temperature and hold time duration The nucleic acid sequence that you want to amplify and detect Identical reactions that contain identical components and volumes and that evaluate the same sample See also biological replicates Part ofthe run method that specifies the temperature time and ramp for all steps and stages of the PCR instrument run See melting temperature T AmpliTaq 360 DNA Polymerase Protocol Documentation and Support Related documentation
13. 5 Gently vortex the tube on a low setting for no more than 5 seconds to mix the components 6 Centrifuge the tube briefly to spin down the contents and to eliminate air bubbles from the solution 7 Dispense equal volumes of the PCR reaction mix to the reaction plate or into PCR tubes see Table 1 8 Place the plate in a MicroAmp Splash Free 96 Well Base or place the tubes in a MicroAmp 96 well Base Keep the plate or tubes in their respective bases throughout the remainder of the protocol 9 Seal the plate with MicroAmp Clear Adhesive Film or cap the tubes with MicroAmp 8 Cap Strips 10 Centrifuge the plate or tubes to collect the liquid at the bottom of the wells 11 Put the plate or tubes on ice Prepare the 1 Prepare primers and DNA to their appropriate working dilutions see Table 2 reaction plate or For multiple PCR assays prepare a master mix of components tubes 2 With the plate or tubes in the appropriate base remove the seal from the plate or open the tubes 3 Add primers and DNA to the appropriate wells or tubes according to Table 2 Include the no template controls Table2 Primer and DNA mix for PCR reactions Volume Volume per per Component 25 uL 50 uL Final concentration reaction reaction uL uL Primer 1 0 5t02 5 1to5 0 2 to 1 0 uM Primer 2 0 5t02 5 1to5 0 2 to 1 0 uM DNA Variable Variable 1 pg reaction Total PCR volume 25 50 t If t
14. AmpliTaq 360 DNA Polymerase Protocol Applied Biosystems AmpliTaq 360 DNA Polymerase Protocol AS Beh stems O Copyright 2009 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 APPLIED BIOSYSTEMS DISCLAIMS ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT EXPRESSED OR IMPLIED IN CLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE IN NO EVENT SHALL APPLIED BIOSYSTEMS BE LIABLE WHETHER IN CONTRACT TORT WARRANTY OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL INCIDENTAL INDIRECT PUNITIVE MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT INCLUDING BUT NOT LIMITED TO THE USE THEREOF NOTICE TO PURCHASER LIMITED LICENSE Use of this product is covered by US patent claims and corresponding patent claims outside the US 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 the patented 5 Nuclease Process claims and no right to perform commercial services of any kind including without limitation reporting the results of purchaser s activities for a fee or o
15. Volume Volume Per per Component 25 uL 50 uL Final concentration reaction reaction uL uL PCR grade water Variable Variable AmpliTag 360 Buffer 2 5 5 1x 10X 25 mM Magnesium 1to4 2to8 1 0 to 4 0 mM Chloride dNTP mix 2 4 200 uM each Optional 360 GC 0 5 to 5 1to10 N A Enhancer AmpliTag 360 DNA 0 125 0 25 1 25 Units per 50 uL Polymerase reaction t If the DNA is difficult to amplify in a 25 pL reaction performing the PCR in a 50 uL reaction may give better results 8 The optimal magnesium chloride concentration may vary depending on the primer and template that are used and must be determined by experiment In most cases a final concentration of magnesium chloride at 1 8 mM in the reaction mix works well Contains a 10 mM solution of dNTP 2 5 mM each of dATP dCTP dGTP and dTTP For targets with 65 to 75 GC start with 2 5 uL in a 25 uL reaction or 5 0 uL in a 50 pL reaction 10 v v of the reaction For targets with gt 75 GC start with 5 uL in a 25 pL reaction or 10 uL in a 50 pL reaction 20 v v of the reaction In general if increased specificty is required add 0 5 to 1 uL 360 GC Enhancer per 25 uL reaction or add 1 to 2 uL 360 GC Enhancer per 50 pL reaction 2 to 596 v v of the reaction For some difficult to amplify targets up to 5 0 U per 50 uL of reaction can be added AmpliTaq 360 DNA Polymerase Protocol AmpliTaq 360 DNA Polymerase Protocol 4 Cap the tube
16. You can download the following documents and other product support documents from http docs appliedbiosystems com search taf Document Part number AmpliTaq 360 DNA Polymerase 4398170 Product Insert AmpliTag 360 DNA Polymerase 4398952 Quick Reference Card Applied Biosystems Veriti Thermal 4375799 Cycler User Guide GeneAmp PCR System 9700 Base 4303481 Module User s Manual GeneAmp PCR System 9700 96 4316011 Well Sample Block Module AmpliTaq 360 DNA Polymerase Protocol Documentation and Support How to obtain support 36 Send us your comments For the latest services and support information for all locations go to www appliedbiosystems com then click the link for Support At the Support page you can Access worldwide telephone and fax numbers to contact Applied Biosystems Technical Support and Sales facilities Search through frequently asked questions FAQs Submit a question directly to Technical Support Order Applied Biosystems user documents MSDSs certificates of analysis and other related documents Download PDF documents Obtain information about customer training Download software updates and patches Applied Biosystems welcomes your comments and suggestions for improving its user documents You can e mail your comments to techpubs appliedbiosystems com IMPORTANT The e mail address above is for submitting comments and suggestions relating only to documentatio
17. al environmental and health regulations If potentially hazardous waste is generated when you operate the instrument you must Characterize by analysis if necessary the waste generated by the particular applications reagents and substrates used in your laboratory Ensure the health and safety of all personnel in your laboratory Ensure that the instrument waste is stored transferred transported and disposed of according to all local state provincial and or national regulations IMPORTANT Radioactive or biohazardous materials may require special handling and disposal limitations may apply AmpliTaq 360 DNA Polymerase Protocol 27 Appendix C Safety Biological hazard safety General biohazard WARNING BIOHAZARD Biological samples such as tissues body fluids warning infectious agents and blood of humans and other animals have the potential to transmit infectious diseases Follow all applicable local state provincial and or national regulations Wear appropriate protective equipment which includes but is not limited to protective eyewear face shield clothing lab coat and gloves All work should be conducted in properly equipped facilities using the appropriate safety equipment for example physical containment devices Individuals should be trained according to applicable regulatory and company institution requirements before working with potentially infectious materials Read and follow the applicab
18. col Chemical safety guidelines Chemical safety guidelines To minimize the hazards of chemicals 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 See About MSDSs on page 26 Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals for example 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 for example 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 in the MSDS Comply with all local state provincial or national laws and regulations related to chemical storage handling and disposal AmpliTaq 360 DNA Polymerase Protocol 25 Appendix C Safety MSDSs About MSDSs Chemical manufacturers supply current Material Safety Data Sheets MSDSs with shipments of hazardous chemicals to new customers They also provide MSDSs with the first shipment of a hazardous chemical to a customer after an MSDS has been updated MSDSs provide the safety information you need to store handle transport and dispose of the chemicals safely Each time you receive a
19. emicals not distributed by Applied Biosystems or Ambion contact the chemical manufacturer AmpliTaq 360 DNA Polymerase Protocol How to use this guide Purpose of this guide Audience Assumptions Text conventions User attention words The AmpliTaq 360 DNA Polymerase Protocol provides all the information you need to perform PCR over a wide range of DNA templates including some of the most challenging GC rich sequences This guide is intended for biologists who have had some experience performing PCR This guide assumes that your thermal cycler has been installed by an Applied Biosystems technical representative This guide uses the following conventions Bold text indicates user action For example Type 0 then press Enter for each of the remaining fields e Italic text indicates new or important words and is also used for emphasis For example Before analyzing always prepare fresh matrix Aright arrow symbol gt separates successive commands you select from a drop down or shortcut menu For example Select File gt Open gt Spot Set Right click the sample row then select View Filter View All Runs Two user attention words appear in Applied Biosystems user documentation Each word implies a particular level of observation or action as described below Note Provides information that may be of interest or help but is not critical to the use of the product IMPORTANT Provides i
20. g time when using the Veriti and GeneAmp PCR System thermal cyclers with a calculated in tube temperature Some models of thermal cyclers may require longer annealing times Adjusting The length of the target sequence affects the required extension time Longer extension targets require increased extension times In general allow an extension time of conditions approximately 60 seconds per 1000 bases at 72 C Asthe amount of DNA increases the number of DNA polymerase molecules may become limiting Compensate for this limitation by increasing the extension time in later cycles AmpliTaq 360 DNA Polymerase Protocol 19 Appendix B Guidelines for Designing PCR Assays Optimize the PCR conditions Optimizing The DNA segment to be amplified from the template can be up to 4 kb long template Jeffreys ef al 1988 although 100 to 1000 bases are more typical and easier to concentration amplify Start with enough copies of the template to obtain a signal after 25 to 30 cycles More than 10 copies but less than 1 ug of human genomic DNA per 50 uL reaction is the recommended range Ifthe target DNA concentration is low you may need more than 35 cycles to produce sufficient product for analysis As few as 1 to 10 target copies can be amplified Saiki Gelfand Stoffel 1988 Chou et al 1992 Validation for low copy number amplifications is best done for an average of 5 to 10 target molecules per sample to avoid statistical fal
21. hancer 1 x 1 5 mL e 25 mM Magnesium Chloride 1 x 1 5 mL Reagents sufficient for 800 x 50 pL reactions 4398828 e AmpliTaq 360 DNA Polymerase 1000 U e AmpliTaq 360 Buffer 10X 4 x 1 5 mL e 360 GC Enhancer 4 x 1 5 mL 25 mM Magnesium Chloride 4 x 1 5 mL Reagents sufficient for 1200 x 50 uL reactions 4398891 AmpliTaq 360 DNA Polymerase 6 x 250 U e AmpliTaq 360 Buffer 10X 6 x 1 5 mL 360 GC Enhancer 6 x 1 5 mL e 25 mM Magnesium Chloride 6 x 1 5 mL Reagents sufficient for 2400 x 50 pL reactions 4398893 e AmpliTaq 360 DNA Polymerase 2 x 1500 U e AmpliTag 360 Buffer 10X 12 x 1 5 mL 360 GC Enhancer 12 x 1 5 mL e 25 mM Magnesium Chloride 12 x 1 5 mL Reagents sufficient for 4000 x 50 pL reactions 4398895 AmpliTaq 360 DNA Polymerase 5 x 1000 U e AmpliTaq 360 Buffer 10X 20 x 1 5 mL 360 GC Enhancer 20 x 1 5 mL e 25 mM Magnesium Chloride 20 x 1 5 mL Reagents sufficient for 20 000 x 50 pL reactions 4398897 AmpliTaq 360 DNA Polymerase 25 x 1000 U e AmpliTag 360 Buffer 10X 100 x 1 5 mL 360 GC Enhancer 100 x 1 5 mL e 25 mM Magnesium Chloride 100 x 1 5 mL Reagents sufficient for 20 000 x 50 pL reactions 4398899 AmpliTag 360 DNA Polymerase 1 x 25 000 U e AmpliTaq 360 Buffer 10X 1 x 150 mL e 360 GC Enhancer 1 x 120 mL e 25 mM Magnesium Chloride 1 x 150 mL AmpliTaq 360 DNA Polymerase standalone 25 000 U 4398838 1x5mL AmpliTaq 360 DNA Polymerase
22. he DNA is difficult to amplify in a 25 pL reaction performing the PCR in a 50 uL reaction may give better results Lowering the primer concentration reduces potential secondary products For a no template control add an equivalent volume of water Preferably gt 10 copies of template but 1 ug DNA reaction 4 Seal the plate with MicroAmp Clear Adhesive Film or cap the tubes with MicroAmp 8 Cap Strips 5 Centrifuge the plate or tubes to collect the liquid at the bottom of the wells or the tubes Ensure that the wells are uniformly filled 6 Put the plate or tubes on ice 8 AmpliTaq 360 DNA Polymerase Protocol Set up the run method Load and run the plate or tubes Set the Perform PCR using AmpliTag 360 DNA Polymerase Thermal cycling conditions Table 3 Table 3 Three temperature thermal cycling on a Veriti GeneAmp PCR System 9700 or 2720 Thermal Cycler Stage Step Temp Time Holding Initial denaturation 94 C 3 mint Cycling Denature 95 C 30 sec 25 to 40 cycles Anneal Primer T 30 sec Extend 72 C 60 sec kb Holding Final Extension 72 C 7 min Holding Final hold 4 C co For easy to amplify targets the initial denaturation can be reduced to 2 minutes Although any primer can be used with this product Applied Biosystems recommends using primers with T s gt 55 C Use the Primer T calculator on an Applied Biosystems thermal cycler or go
23. le guidelines and or regulatory requirements in the following US Department of Health and Human Services guidelines published in Biosafety in Microbiological and Biomedical Laboratories stock no 017 040 00547 4 bmbl od nih gov Occupational Safety and Health Standards Bloodborne Pathogens 29 CFR 1910 1030 www access gpo gov nara cfr waisidx 01 29cfr1910a 01 html Your company s institution s Biosafety Program protocols for working with handling potentially infectious materials Additional information about biohazard guidelines is available at www cdc gov 28 AmpliTaq 360 DNA Polymerase Protocol Chemical alerts Chemical alerts General alerts for May cause eye skin and respiratory tract irritation Read the MSDS and follow the all chemicals handling instructions Wear appropriate protective eyewear clothing and gloves Specific Z N CHEMICAL HAZARD AmpliTaq 360 DNA Polymerase chemical alerts Z N CHEMICAL HAZARD AmpliTaq 360 Buffer 10X AAN CHEMICAL HAZARD Ethidium bromide causes eye skin and respiratory tract irritation and is a known mutagen that is it can change genetic material in a living cell and has the potential to cause cancer AmpliTaq 360 DNA Polymerase Protocol 29 Appendix C Safety 30 AmpliTaq 360 DNA Polymerase Protocol Bibliography Birch D E Kolomodin L Laird W J McKinney N Wong J Young K K Y Zangenberg G A and Zoccoli M A
24. n To order documents download PDF files or for help with a technical question see How to obtain support above AmpliTaq 360 DNA Polymerase Protocol www appliedbiosystems com Worldwide Sales and Support Applied Biosystems vast distribution and service network composed of highly trained support and applications personnel reaches 150 countries on six continents For sales office locations and technical support please call our local office or refer to our Web site at www appliedbiosystems com Applied Biosystems is committed to providing the world s leading technology and information for life scientists Headquarters 850 Lincoln Centre Drive Foster City CA 94404 USA Phone 1 650 638 5800 Toll Free In North America 1 800 345 5224 Fax 1 650 638 5884 06 2010 Applied Bibsystems Part Number 4398942 Rev B
25. nactivate enzymes or to incubate a reaction In melt curve experiments the temperature at which 50 of the DNA is double stranded and 50 of the DNA is dissociated into single stranded DNA The T is displayed in the melt curve See ramp speed The task for targets in wells that contain water or buffer instead of sample No amplification of the target should occur in negative control wells Also called no template control NTC See negative control NC In quantitation experiments the amount of target in the samples Absolute quantity can refer to copy number mass molarity or viral load Relative quantity refers to the fold difference between normalized quantity of target in the sample and normalized quantity of target in the reference sample The rate at which the temperature changes during the instrument run The ramp is defined as a percentage The ramp for the melt curve step is defined as a temperature increment In the graphical view of the thermal profile the ramp is indicated by a diagonal line AmpliTaq 360 DNA Polymerase Protocol 33 Glossary ramp speed reaction mix replicate group replicates reverse primer reverse transcriptase run method sample stage step target technical replicates thermal profile 34 Speed at which the temperature ramp occurs during the instrument run For optimal results using the AmpliTaq 360 DNA Polymerase Applied Biosystems recommends using the standard ramp
26. nformation that is necessary for proper instrument operation accurate chemistry kit use or safe use of a chemical AmpliTaq 360 DNA Polymerase Protocol vii How to use this guide viii AmpliTaq 360 DNA Polymerase Protocol AmpliTaq 360 DNA Polymerase Protocol This chapter covers Product information aa oe eet eerte VR Re Eee d NA A ye e e ES WAW UEREIPR UIT an dn n Ae W AA h RN INPS Before you perform PER canica laa ce ttt bask a ek Perform PCR using AmpliTaq 360 DNA Polymerase Analyze the results cuido a e st espace eode Rahal danas Troubleshooting neci da m cle ene Maat RO UR RERO AmpliTaq 360 DNA Polymerase Protocol AmpliTaq 360 DNA Polymerase Protocol Product information Purpose of the product About AmpliTaq9 360 DNA Polymerase About the 360 GC enhancer About this protocol Contents Available kit packaging Use the AmpliTaq 360 DNA Polymerase to amplify a wide range of DNA sequences using PCR AmpliTaq 360 DNA Polymerase is AmpliTaq DNA Polymerase purified by an additional proprietary separation process to reduce contaminating bacterial DNA sequences from the enzyme preparation This ultra pure enzyme reduces false positives and amplifies bacterial templates and very low target sequences The enzyme is quality control tested to ensure that in a 5 unit aliquot of the enzyme bacterial 16S ribosomal RNA gene sequences are present in low copy numbe
27. pears in Nucleic Acids Res 1991 Feb 11 19 3 698 Nucleic Acids Res 18 6409 6412 Saiki R K Gelfand D H Stoffel S etpal 1988 Primer directed enzymatic amplification of DNA with a thermostable DNA polymerase Science 239 487 491 Saiki R K Scharf S J et al 1985 Enzymatic amplification of B globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia Science 230 1350 1354 AmpliTaq 360 DNA Polymerase Protocol Glossary amplicon biological replicates cycling stage diluent forward primer holding stage melting temperature Tm mode negative control NC no template control NTC quantity ramp A segment of DNA amplified during PCR Reactions that contain identical components and volumes but evaluate separate samples of the same biological source for example multiple samples of the same liver tissue See also technical replicates In the thermal profile a stage that is repeated A cycling stage is also called an amplification stage A reagent used to dilute a sample or standard before it is added to the PCR reaction The diluent can be buffer or water Oligonucleotide that flanks the 5 end of the amplicon The reverse primer and the forward primer are used together in PCR reactions to amplify the target In the thermal profile a stage that can include one or more steps You can add a holding stage to the thermal profile to activate enzymes to i
28. r The 360 GC Enhancer is used for difficult to amplify templates especially for templates with high GC content You can adjust the amount of 360 GC Enhancer to optimize the PCR reaction Amplicons that generate nonspecific products may require small amounts of enhancer to improve specificity Amplicons with high GC content require more enhancer This protocol provides Procedures and guidelines on PCR using the AmpliTaq 360 DNA Polymerase Information on troubleshooting PCR results A list of equipment and materials required for using the AmpliTaq 360 DNA Polymerase For more information refer to the documents shipped with your Applied Biosystems PCR System AmpliTaq 360 DNA Polymerase contains AmpliTaq 360 DNA Polymerase AmpliTaq 360 Buffer 10X 25 mM Magnesium Chloride 360 GC Enhancer Note The 360 GC Enhancer AmpliTaq 360 Buffer 10X and 25 mM Magnesium Chloride are also available without enzyme AmpliTaq 360 DNA Polymerase is available in the following packaging AmpliTaq 360 DNA Polymerase Protocol Product information Part Quantity number Reagents sufficient for 80 x 50 pL reactions 4398808 AmpliTaq 360 DNA Polymerase 100 U e AmpliTag 360 Buffer 10X 1 x 1 5 mL e 360 GC Enhancer 1 x 1 5 mL e 25 mM Magnesium Chloride 1 x 1 5 mL Reagents sufficient for 200 x 50 pL reactions 4398818 AmpliTaq 360 DNA Polymerase 250 U e AmpliTaq 360 Buffer 10X 1 x 1 5 mL 360 GC En
29. repeat the PCR e Use 1 to 2 uL of 360 GC Enhancer in a 50 uL reaction Use the 360 GC Enhancer only if there are nonspecific products or products with 26596 GC see Prepare the reaction mix on page 7 Denaturation temperature is too low or too high Adjust the temperature in increments of 1 degree Celsius If you have a Veriti thermal cycler adjust the VeriFlex Block in increments of 1 degree Celsius for up to six different temperatures Nonspecific priming Increase the annealing temperature in increments of 1 to 2 degrees Celsius Primer design is not optimal Review primer design and composition Low levels of PCR product or no product band visible Template concentration is too low Increase the sample concentration Experimental sample DNA is damaged or degraded Add more DNA or use sample that has been processed to minimize shearing and nicking Denaturation time is too short or too long Adjust the time in increments of 5 seconds Denaturation temperature is too low or too high Adjust the temperature in increments of 1 degree Celsius If you have a Veriti thermal cycler adjust the VeriFlex Block in increments of 1 degree Celsius for up to six different temperatures Annealing extension temperature is too high Lower the temperature in increments of 2 degrees Celsius If you have a Veriti thermal cycler adjust the VeriFlex Block in increments of
30. rotocol Safety information Safety information Safety alert words MSDSs vi Note For general safety information see this Preface and Appendix C on page 23 When a hazard symbol and hazard type appear by a chemical name or instrument hazard see the Safety Appendix for the complete alert on the chemical or instrument Four safety alert words appear in Applied Biosystems user documentation at points in the document where you need to be aware of relevant hazards Each alert word IMPORTANT CAUTION WARNING DANGER implies a particular level of observation or action as defined below IMPORTANT Indicates information that is necessary for proper instrument operation accurate chemistry kit use or safe use of a chemical CAUTION Indicates a potentially hazardous situation that 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 that if not avoided could result in death or serious injury DANGER Indicates an imminently hazardous situation that if not avoided will result in death or serious injury This signal word is to be limited to the most extreme situations The MSDSs for any chemicals supplied by Applied Biosystems or Ambion are available to you free 24 hours a day For instructions on obtaining MSDSs see Obtaining MSDSs on page 26 IMPORTANT For the MSDSs of ch
31. rs Adjust the MgCl concentration in parallel with significant changes in the concentration higher or lower of sample DNA or dNTPs to keep the free magnesium ion concentration constant For example reduce the concentration of dNTP from 200 uM each to 40 uM each and reduce the MgCl concentration from the higher concentration to 640 uM The dNTP concentration that is recommended for PCR is 200 uM for each dNTP If the blend of dNTPS is changed and the concentration of any one dNTP is significantly different from the other dNTPs then AmpliTaq 360 DNA Polymerase tends to misincorporate slow down and or terminate prematurely Innis et al 1988 Lower concentrations of dNTPs 40 uM tend to promote polymerase fidelity Eckert and Kunkel 1992 For difficult to amplify targets increasing the AmpliTaq 360 DNA Polymerase up to 5 U per 50 uL reaction may improve the yield AmpliTaq 360 DNA Polymerase Protocol 21 Appendix B Guidelines for Designing PCR Assays 22 AmpliTaq 360 DNA Polymerase Protocol Appendix C Safety This appendix covers Chemical hazard warnings 24 Chemical safety guidelines 25 MID II bs 26 Chemical waste hazards 26 Chemical waste safety guidelines 27 Waste disposal iaces obe Ren E R 27 Biological hazard safety
32. se negatives Optimizing The 360 GC Enhancer helps amplify amplicons that are difficult to amplify enhancer including amplicons that are GC rich or have GC repeats or that generate concentration nonspecific products In a 50 uL reaction for targets with 65 to 75 GC start with 5 uL gt 75 GC start with 10 uL In general if increased specificity is required add 1 to 2 uL per 50 uL reaction The 360 GC Enhancer can reduce nonspecific amplification and improve the yield of specific products However excessive use of the 360 GC Enhancer can reduce yield particularly for non GC rich amplicons 20 AmpliTaq 360 DNA Polymerase Protocol Optimizing Magnesium Chloride concentration Optimizing dNTP concentration Optimizing enzyme concentration Optimize the PCR conditions The magnesium ion concentration that 1s required for optimal PCR amplification depends on the specific set of primers and template Too much or too little MgCl reduces amplification efficiency or results in amplification of non target sequences The optimal MgCl concentration must be determined by experiment To determine the optimum MgCl concentration for each primer set Use the supplied 25 mM MgCl to adjust the magnesium ion concentration Vary the concentration of MgCL around 1 8 mM A typical range is 1 0 to 4 0 mM Raise the MgCl concentration in the reaction mix proportionately if the samples contain EDTA citrate or other chelato
33. sequencing with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction amplified DNA Proc Natl Acad Sci USA 85 9436 9440 Jeffreys A J Wilson V Neumann R and Keyte J 1988 Amplification of human minisatellites by the polymerase chain reaction towards DNA fingerprinting of single cells Nucleic Acids Res 16 10953 10971 Kwok S and Higuchi R 1989 Avoiding false positives with PCR Nature 339 237 238 Kwok S 1990 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 142 145 Mullis K B and Faloona E A 1987 Specific synthesis of DNA in vitro via a polymerase catalyzed chain reaction Methods in Enzymology 155 335 350 AmpliTaq 360 DNA Polymerase Protocol 31 Bibliography 32 Lawyer F C Stoffel S Saiki R K Myambo K Drummond R and Gelfand D H 1989 Isolation characterization and expression in E coli of the DNA polymerase gene from the extreme thermophile Thermus aquaticus J Biol Chem 264 6427 6437 Richardson C C 1966 DNA polymerase from Escherichia coli In Procedures in Nucleic Acid Research Cantoni G L and Davies D R eds New York Harper amp Row 263 276 Rychlik W Spencer W J and Rhoads R E 1990 Optimization of the annealing temperature for DNA amplification in vitro published erratum ap
34. t up the electrophoresis apparatus and running buffer according to the manufacturer s instructions Add an aliquot of the PCR product to a well of a new plate or to an appropriate new tube Add an appropriate volume of gel loading buffer to the PCR product aliquot For example add 1 uL of 10X gel loading buffer to a 9 uL aliquot of PCR reaction Mix the PCR product aliquot and buffer in the wells by pipetting up and down or briefly vortex the samples in the tubes Spin the plate or pulse spin the tubes Dispense the entire volume of the buffer PCR product aliquot from each well or tube into a well of the gel Into one well of the gel load a DNA ladder marker appropriate to the PCR product length Run the gel at the voltage or time appropriate to amplicon length and agarose percentage so that the samples run 1 3 to 1 2 the length of the gel Do not run the dye off the gel Place the gel on a UV transilluminator Verify that each lane with a PCR product aliquot contains one distinct band For more Refer to the getting started guides for your PCR system for information about information analyzing PCR results 10 AmpliTaq 360 DNA Polymerase Protocol Troubleshooting Observation Possible cause Recommended action Nonspecific amplification with or without a product band Carryover contamination Use the GeneAmp PCR Carry Over Prevention Kit PN N8080068 Dispose of reagents make fresh reagents then
35. ther commercial consideration is conveyed expressly by implic ation 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 TRADEMARKS Applied Biosystems AB Design GeneAmp and Primer Express are registered trademarks and Veriti and VeriFlex are trademarks of Applied Biosystems Inc or its subsidiaries in the US and or certain other countries AmpliTaq is a registered trademark of Roche Molecular Systems Inc All other trademarks are the sole property of their respective owners Part Number 4398942 Rev B 06 2010 Contents Preface V Safety ih formation 2233 tees Aa a UE E aa vi How to use this guide vii AmpliTaq 360 DNA Polymerase Protocol 1 Product information Wa e h l a klan ee eee 2 WOrktlow lt 2 feels A Bel tt ee Cais oe one us 5 Before you perform PCR AUA hn 6 Perform PCR using AmpliTag 360 DNA Polymerase 7 Analyze The results coa eke Sao key a eee hae ed 10 Troubleshooting seed RR ee ae ovra ert ober eee ee Pare 11 Appendix A Ordering Information 13 Materials and equipment not included 14
36. to www appliedbiosystems com support techtools calc Thirty seconds for denaturation and annealing is adequate when you use Veriti or GeneAmp PCR System thermal cyclers that display a calculated sample temperature Some models of thermal cyclers may require longer times Ramp speed or mode Standard Reaction volume uL 25 or 50 1 Remove the plate or PCR tubes from the base P Clear Adhesive Film ewe XE om Start the run Load the reaction plate or tubes into a PCR instrument Use a MicroAmp Optical Film Compression Pad when you use a MicroAmp Unload the reaction plate or tubes after the run is complete Store the plate or tubes at 4 C or at 15 to 25 C for long term storage For more Refer to the user guide or getting started guides for your PCR system for more information about setting up the experiment using and maintaining the instrument and performing instrument calibrations information AmpliTaq 360 DNA Polymerase Protocol AmpliTaq 360 DNA Polymerase Protocol Analyze the results Z WA ENI C CHEMICAL HAZARD Ethidium bromide Check the purity Analyze the PCR amplification products by agarose gel electrophoresis of the PCR product IMPORTANT To prevent contamination never bring amplified PCR products into the PCR setup area l Obtain a 1 agarose gel with ethidium bromide stain You can use a gel of up to 396 agarose with ethidium bromide stain Se
37. ulate the number of reactions to perform for each assay In your calculations include extra reactions approximately one extra reaction for every 10 required reactions to provide excess volume for the volume lost during reagent transfers For example for a 96 well plate prepare enough volume for approximately 110 reactions Note You can run multiple PCRs on one reaction plate Include controls for each run on the plate AmpliTaq 360 DNA Polymerase Protocol Perform PCR using AmpliTag 360 DNA Polymerase Perform PCR using AmpliTaq 360 DNA Polymerase Prepare the For the following hazards see the complete safety alert descriptions in Specific reaction mix chemical alerts on page 29 CHEMICAL HAZARD AmpliTaq 360 DNA Polymerase AmpliTaq 360 Buffer 10X IMPORTANT Prepare the reagents on ice Avoid generating bubbles when mixing the enzyme 1 Thaw AmpliTaq 360 Buffer 10X 25 mM Magnesium Chloride dNTP mix primers template and optional 360 GC Enhancer on ice then vortex the reagents before use 2 Thaw AmpliTaq 360 DNA Polymerase on ice Mix the enzyme by gently pipetting it up and down then put the enzyme on ice 3 Combine the following components on ice in an appropriate tube according to the volumes that are shown in Table 1 Multiply the volume for one reaction component Table 1 by the total number of reactions then add that volume to the tube Table1 PCR reaction mix
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