AmpliTaq Gold® 360 Master Mix Protocol
Contents
1. Document Part number AmpliTaq Gold 360 Master Mix 4398275 Product Insert AmpliTaq Gold 360 Master Mix Quick 4398954 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 Gold 360 Master Mix 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 documentation To order documents download PDF files or for help with a technical question see How to obtain support above Am2. Specific reaction mix chemical alerts on page 29 CHEMICAL HAZARD AmpliTaq Gold Master Mix IMPORTANT Avoid generating bubbles when mixing the enzyme 1 Thaw the primers template and optional 360 GC Enhancer then vortex the reagents before use 2 Mix AmpliTaq Gold 360 Master Mix by gently pipetting it up and down 3 Combine the following components 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 Volume Volume Per per Component 25 uL 50 uL Final concentration reaction reaction uL uL PCR grade water Variable Variable Optional 360 GC 0 5 to 5 1to 10 N A Enhancer AmpliTaq Gold 360 12 5 25 1X Master Mix t For targets with 65 to 75 GC start with 2 5 uL in a 25 uL reaction or 5 0 uL in a 50 uL 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 specificity 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 uL reaction 2 to 596 v v of the reaction 4 Cap the tube 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 c
3. User attention words The AmpliTaq Gold 360 Master Mix 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 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 Italic text indicates new or important words and is also used for emphasis For example Before analyzing always prepare fresh matrix A right arrow symbol K separates successive commands you select from a drop down or shortcut menu For example Select File K Open K 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 information that is necessary for proper instrument operation accurate chemistry kit use or safe use of a chemical AmpliTaq Gold 360 Master Mix Protocol vii How to use this guide viii AmpliTaq Gola 360 Master Mix Protocol Ampl
4. content require more enhancer This protocol provides Procedures and guidelines on PCR using the AmpliTaq Gold 360 Master Mix Information on troubleshooting PCR results A list of equipment and materials required for using the AmpliTaq Gold 360 Master Mix For more information refer to the documents shipped with your Applied Biosystems PCR System AmpliTaq Gold 360 Master Mix Protocol Contents AmpliTaq Gold 360 Master Mix contains AmpliTaq Gold 360 Master Mix 360 GC Enhancer Product information Available kit AmpliTaq Gold 360 Master Mix is available in the following packaging packaging A Part Quantity number Reagents sufficient for 40 x 50 pL reactions 4398876 e AmpliTag Gold 360 Master Mix 1 x 1 0 mL e 360 GC Enhancer 1 x 1 5 mL Reagents sufficient for 200 x 50 pL reactions 4398881 s AmpliTaq Gold 360 Master Mix 1 x 5 0 mL e 360 GC Enhancer 1 x 1 5 mL Reagents sufficient for 2000 x 50 uL reactions 4398886 e AmpliTag Gold 360 Master Mix 1 x 50 mL e 360 GC Enhancer 1 x 10 mL Reagents sufficient for 2000 x 50 uL reactions 4398901 s AmpliTaq Gold 360 Master Mix 10 x 5 0 mL e 360 GC Enhancer 10 x 1 5 mL Storage When you receive the AmpliTaq Gold 360 Master Mix store at 15 to 25 C The AmpliTaq Gold 360 Master Mix can be thawed once then stored at 2 to 8 C AmpliTaq Gold 360 Master Mix Protocol AmpliTaq Gold 360 Master Mix Protocol Workflo
5. Gold 360 Master Mix Protocol 12 AmpliTaq Gold 360 Master Mix Protocol Appendix A Ordering Information This appendix covers Materials and equipment not included WAK KK KK KK KK KK KK KK KK KK Thermal yel i unseen T EE A h AA 09002 TAT Other equipment and consumables KK KK KK KK KK KK KK KK KK AmpliTaq Gold 360 Master Mix Protocol Appendix A Ordering Information Materials and 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 Veriti 60 Well Thermal Cycler 4384638 Veriti 96 Well Fast Thermal 4375305 Cycler Veriti 96 Well Thermal Cycler 4375786 Veriti 384 Well Thermal Cycler 4388444 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 N8080
6. gloves when handling reagent and waste bottles 26 AmpliTaq Gold 360 Master Mix Protocol Chemical waste safety guidelines Chemical waste safety guidelines To minimize the hazards of chemical waste L 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 national environmental and health regulations If potentially hazardous waste is generated
7. 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 AmpliTaq Gold 360 Master Mix Protocol AmpliTaq Gold 360 Master Mix Protocol Set up the run method Load and run the plate or tubes Set the Thermal cycling conditions see Table 3 Ne Dn Bo Table 3 Three temperature thermal cycling on a Veriti GeneAmp PCR System 9700 or 2720 Thermal Cycler Stage Step Temp Time Holding Activation of AmpliTaq 95 C 5 to 10 min Gold 360 Master Mix Cycling Denature 95 C 15 to 30 sec 25 to s 40 cycles Anneal Primer T 30 sect Extend 72 C 60 sec kb Holding Final Extension 72 C 7 min Holding Final hold 4 C oo t See Adjusting the hold period for activation in the AmpliTaq Gold 360 Master Mix Protocol Appendix B For amplicons that are gt 2 kb denature for 15 seconds For amplicons that are x2 kb denature for 30 seconds Although any primer can be used with this product Applied Biosystems recommends using primers with T s gt 55 C Use the Primer T ca
8. of the DNA is dissociated into single stranded DNA The Tn 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 Gold 360 Master Mix Protocol 33 Glossary ramp speed reaction mix replicate group replicates reverse primer reverse transcriptase run method sample stage step target technical replicates thermal profile Tm 34 Speed at which the temperature ramp occurs during the instrument run For optimal results using the AmpliTaq Gold 360 Master Mix Applied Biosystems recommends using the standard ramp speed A solution that contains all components to run the PCR reaction except for the template sample standard or control A
9. 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 of the run method that specifies the temperature time and ramp for all steps and stages of the PCR instrument run See melting temperature Tm AmpliTaq Gold 360 Master Mix Protocol Documentation and Support Related documentation You can download the following documents and other product support documents from http docs appliedbiosystems com search taf
10. 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 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 Preincubation activation time is not sufficient Increase the pre PCR heat step in increments of 1 minute or use the Time Release protocol see Adjusting the hold period for activation on page 19 Product band is smeared Carryover contamination Use the GeneAmp PCR Carry Over Prevention Kit PN N8080068 Dispose of reagents make fresh reagents then repeat the PCR Denaturation time is too short or too long Adjust the time in increments of 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 AmpliTaq Gold 360 Master Mix Protocol Troubleshooting 11 AmpliTaq
11. 260 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 Pad Applied Biosystems PN4312639 Centrifuge with plate adapter Major laboratory supplier MLS AmpliTaq Gold 360 Master Mix Protocol Materials and equipment 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 Gold 360 Master Mix Protocol 15
12. 4 PCR good laboratory practices kk kk kk kk KK tee 18 Select the amplicon site xazan yn l gih Wak ala lle eee 19 Adjust thermal CyCliNG ras RS iw EIS Sa ee Ee ee is Ns a ee ea 19 Optimize the PCR conditions lk kk kk kk kK kK KK KK KK KK ee 21 Chemical hazard warnings ooocccooccrr K KI KIR KI KOK KK kK KK KK KK KK kk kk kk 24 Chemical safety guidelines kk kk kk kk kk KK KE eee 25 MSDSS 2 nid duz O A A eee eae de 26 Chemical waste hazards x R R a Xwa kk Kik Ee ak te tees 26 Chemical waste safety guidelines 0 0 2c eee 27 Waste disposal As gron Spas aot WR MR EE le E EID NTC REN REN WE REG 27 Biological hazard safety ss kalek Waa ak ak dek A AR ren 28 Chemical alents 22 3 0 s A baja od RAUS Ava K ya E Eee ER 29 BIDIIGOFA D a PEU 31 Glossary s Ayl O y ese el uut 33 Documentation and Support XAK KK RR KK eee eee 35 Related documentation x gt kan n 301 WEDE eee eee nee 35 AmpliTaq Gold 360 Master Mix Protocol iii Contents Flow to ODtaln SUPpoOlt sica la a 36 iv AmpliTaq Gold 360 Master Mix Protocol Preface This preface covers Safety information How tousetthis guide nes AAA eh CERTI AmpliTaq Gold 360 Master Mix Protocol Safety information Safety information Safety alert words MSDSs vi Note For general safety information see this Preface and Appendix C Safety on page 23 When a hazard symbol and hazard type appear by a chemical na
13. A 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 F A 1987 Specific synthesis of DNA in vitro via a polymerase catalyzed chain reaction Methods in Enzymology 155 335 350 AmpliTaq Gold 360 Master Mix Protocol 31 Bibliography 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 appears 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 Prime
14. AmpliTaq Gold 360 Master Mix Protocol Applied Biosystems AmpliTaq Gold 360 Master Mix Protocol AS SDK 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
15. Appendix A Ordering Information 16 AmpliTaq Gola 360 Master Mix Protocol Appendix B Guidelines for Designing PCR Assays This appendix covers PCR good laboratory practices o o ooooooomooo Select the amplicon SIG KK KK RR RR RR KK KK Adjust thermal cycling 0 0 KK KK RR RR KK KK Optimize the PCR conditions n usssaua seaun AmpliTaq Gold 360 Master Mix Protocol Appendix B Guidelines for Designing PCR Assays PCR good laboratory practices General PCR When preparing samples for PCR amplification practices Use a 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 Gold 360 Master Mix Protocol Select the amplicon site Select the ampli
16. For such a Tp calculator go to http www appliedbiosystems com then select Services amp Support Technical Tools gt T Calculator The GeneAmp PCR System 9700 Thermal Cycler also contains a T calculator Thirty seconds is adequate annealing 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 The length of the target sequence affects the required extension time Longer targets require increased extension times In general allow an extension time of approximately 60 seconds per 1000 bases at 72 C As the 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 Gold 360 Master Mix Protocol Optimize the PCR conditions Optimize the PCR conditions Optimizing The DNA segment to be amplified from the template can be up to 4 kb long template Jeffreys et 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 amp
17. 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 Gola 360 Master Mix 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 N CHEMICAL HAZARD AmpliTaq Gold 360 Master Mix chemical alerts NN CHEMICAL HAZARD Ethidium bromide causes eye skin and respiratory tract irritation and is a known mutagen that is 1t can change genetic material in a living cell and has the potential to cause cancer AmpliTaq Gold 360 Master Mix Protocol 29 Appendix C Safety 30 AmpliTaq Gola 360 Master Mix 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 1996 Simplified Hot Start PCR Product review in Nature 381 445 446 Bost D A Zalloua P and Abramson R D 1997 AmpliTaq G
18. PCR Carry Over Prevention Kit PN N8080068 Dispose of reagents make fresh reagents then 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 6 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 10 AmpliTaq Gold 360 Master Mix Protocol Observation Possible cause Recommended action 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
19. con site Guidelines 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 Design primer pairs according to Primer Express Software guidelines Usea primer pair that is specific to the target gene and does not amplify pseudogenes or other related genes 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 the hold period for activation Adjusting denaturation conditions Adjusting annealing conditions For general PCR runs Applied Biosystems recommends a pre PCR activation setup of 95 C for 10 minutes Perform a titration of pre PCR activation times 2 to 10 minutes in 1 minute intervals to find the best enzyme activity for your reaction You can also activate AmpliTaq Gold 360 DNA Polymerase to release active enzyme slowly over time time release Time release allows enzyme activity to increase with cycle number as the amount of template increases This type of PCR can be accomplished With limited activation during the pre PCR hold period Or Without activation during the pre PCR hold period In a time release protocol the en
20. iTaq Gold 360 Master Mix Protocol This chapter covers Product information a ii EH HHHH NA a ES WOTRT OW o wan nur a TA Before you perform PCR teer Mea ce al a h leche Ghat bask a Perform PCR using AmpliTaq Gold 360 Master Mix W K KK KK KK Analyze tle results td 3 a eh G aka i eb eoe Do RII in Troubleshooting via nes coke ad R E DA MJ reat N EI JI AmpliTaq Gold 360 Master Mix Protocol AmpliTaq Gold 360 Master Mix Protocol Product information Purpose of the product About AmpliTaq Gold 360 Master Mix About the 360 GC enhancer About this protocol Use the AmpliTaq Gold 360 Master Mix Kit to amplify a wide range of DNA fragments with high specificity The AmpliTaq Gold 360 DNA Polymerase in the AmpliTaq Gold 360 Master Mix amplifies a wide range of DNA contexts The AmpliTaq Gold 360 Master Mix contains AmpliTaq Gold 360 DNA Polymerase Heat activates AmpliTaq Gold 360 DNA Polymerase resulting in highly specific robust PCR amplification The ionic strength and the pH of AmpliTaq Gold 360 DNA Master Mix are optimized for use with the AmpliTaq Gold 360 DNA Polymerase enzyme 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
21. lculator on an Applied Biosystems thermal cycler or go 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 Remove the plate or PCR tubes from the base Use a MicroAmp Optical Film Compression Pad when you use a MicroAmp Clear Adhesive Film Load the reaction plate or tubes into a PCR instrument Start the run 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 Gold 360 Master Mix Protocol Analyze the results Analyze the results AN WARNING 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 9 gel with ethidium bromide stainYou can use a gel of up to 3 agarose with ethidium bromide stain Set up the electroph
22. lified 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 false 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 AmpliTaq Gold 360 Master Mix Protocol 21 Appendix B Guidelines for Designing PCR Assays 22 AmpliTaq Gola 360 Master Mix Protocol Appendix C Safety This appendix covers Chemical hazard warnings kk kk kk kk KK KK KK KK KK KK KK KK KK KK KK KK KK 24 Chemical safety guidelines 25 MSDS A abs 26 Chemical waste hazards WWW a kk kk kK KK KK KK KK e 26 Chemical waste safety guidelines 0 KK KK KK KK KK KK KK KK KK KI KK 27 Waste disposals se i hasan RO HR EESTI e PRO don uta 27 Biological hazard safety XA A kk kk kk kK KK KK KK KK KK KK eh 28 Chemical alerts lt sun are tp A EN ee ta 29 AmpliTaq Gold 360 Master Mix P
23. me 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 chemicals not distributed by Applied Biosystems or Ambion contact the chemical manufacturer AmpliTaq Gold 360 Master Mix Protocol How to use this guide Purpose of this guide Audience Assumptions Text conventions
24. old 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 sequencing with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction amplified DNA Proc Natl Acad Sci US
25. ontents 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 6 AmpliTaq Gold 360 Master Mix Protocol Perform PCR using AmpliTaq Gold 360 Master Mix 10 Centrifuge the plate or tubes to collect the liquid at the bottom of the wells 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 HL Primer 1 0 5to2 5 1to5 0 2 to 1 0 uM Primer 2 0 5t02 5 1to5 0 2 to 1 0 M DNA Variable Variable 1 ug reaction Total PCR volume 25 50 t If the DNA is difficult to amplify in a 25 pL reaction performing the PCR in a 50 uL reaction
26. or other 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 MicroAmp Veriti and VeriFlex are trademarks of Applied Biosystems Inc or its subsidiaries in the US and or certain other countries All other trademarks are the sole property of their respective owners AmpliTaq Gold is a registered trademarks of Roche Molecular Systems Inc All other trademarks are the sole property of their respective owners Part Number 4398944 Rev B 06 2010 Contents Preface A V Safety information ere xe E KK Rh KK KIRR ca CREE vi How to use this guide anaana aaa vii AmpliTaq Gold 360 Master Mix Protocol 1 Product information lt xs i gres a niea iira hr 2 WOFK lOW 2 eU EIE ATR AR NA a A ms 4 Before you perform PCR 44 2 KR sx kk R y kal eee n 5 Perform PCR using AmpliTag Gold 360 Master Mix sees 6 Analyze tlie results cure a ERE ue EROR EU EUN D Roni De yn 9 Troubleshooting n lt lt 35 a ay Geram Te aet ree eR ha Re edm 10 Materials and equipment not included kk kk kk kK KK KK KK ee 1
27. oresis 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 more information about information analyzing a PCR results a RS gt CONFIDENTIAL For AB Internal Use Only Do Not Distribute AmpliTaq Gold 360 Master Mix Protocol Troubleshooting Observation Possible cause Recommended action Nonspecific amplification with or without a product band Carryover contamination Use the GeneAmp
28. pliTaq Gold 360 Master Mix 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 4398944 Rev B
29. r 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 32 AmpliTaq Gold 360 Master Mix Protocol Glossary amplicon biological replicates cycling stage diluent forward primer holding stage melting temperature T 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 inactivate enzymes or to incubate a reaction In melt curve experiments the temperature at which 50 of the DNA is double stranded and 50
30. rotocol 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 Gold 360 Master Mix Protocol Chemical safety guidelines Chemical safety guidelines To minimize the hazards of chemical
31. s L 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 Gold 360 Master Mix 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 new MSDS packaged with a hazardous chemical be sure to replace the appropriate MSDS in your file
32. s Obtaining The MSDS for any chemical supplied by Applied Biosystems is available to you free MSDSs 74 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 illness 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 and the handles locked in the upright position Wear appropriate eyewear clothing and
33. w The workflow for PCR using the AmpliTaq Gold 360 Master Mix Prepare the reaction mix 4 Prepare the reaction pam plate or tubes DECR BESSER 384 well plate 96 well plate 4q Set up the run method T _ Load and run the plate or tubes Analyze the results 4 AmpliTaq Gold 360 Master Mix Protocol Before you perform PCR 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 Calculate 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 Gold 360 Master Mix Protocol 5 AmpliTaq Gold 360 Master Mix Protocol Perform PCR using AmpliTaq Gold 360 Master Mix Prepare the For the following hazards see the complete safety alert descriptions in
34. when you operate the instrument you must La 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 Gold 360 Master Mix 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 applicable guidelines and or regulatory requirements in the following US Department of
35. zyme is released slowly so that the enzyme concentration matches the template concentration thereby increasing enzyme specificity When no or limited 1 to 2 minute pre PCR activation is used the enzyme is released gradually during the denaturation step 95 C for 15 to 30 seconds of each cycle Because the enzyme is released slowly up to 5 additional cycles may be required Limiting the amount of active enzyme at the beginning of the amplification reaction enhances specificity in the early PCR cycles Inthe early cycles make sure that your DNA template is completely denatured Do not exceed a denaturation temperature of 95 to 96 C Gelfand and White 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 Forincreased product specificity use annealing temperatures higher than 45 C Saiki Gelfand and Stoffel 1988 Rychlik Spencer and Rhoads 1990 AmpliTaq Gold 360 Master Mix Protocol 19 Appendix B Guidelines for Designing PCR Assays 20 Adjusting extension conditions L 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
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