Platinum® Multiplex PCR Master Mix
Contents
1. 20 Platinum Multiplex PCR Master Mix 3 Contents MB APPENDIX B Guidelines for Designing PCR Assays 23 PCR good laboratory practices e 23 Adjust thermal cyeling vecindad 24 Adjust the hold period for activation 24 Adjust denaturation conditions 24 Adjust annealing conditions peace te acd oy kk kk KHK kk kk kk es ee eta ts 24 Adjust extension conditions 24 Optimize the PCR conditions WWW kk kk kk kk kk kk kk kk kk kk kk kK kk kk kk kk kk kk kk kk kk kk kk kk k 24 Optimize template concentration WWW kk kk kk kk kk kk kK KK KK KK KK KK KK KK KK KK KK kK kK kk k 24 Optimize enhancer concentration 25 APPENDIX C Safety sna aa alas ta lios 27 Generalisatet sk daa ta les era le da e e o o de 27 Chemical safety nnr Z s N Re e ea gt te het Pie pre Rare Re 28 Biological haz rd safety 2 22 ss a ss ae Se a nes 29 Documentation and Support 31 Related documentation it AA ted wee ee ae ea 31 Obtaining SDSS mea A ta ta Sey 31 Obtaining support circa a X age deed nay ide be a a re Iu ne ae 31 Platinum Multiplex PCR Master Mix About This Guide IMPORTANT Before using this product read and understand the information in the2. 10 Calculate the number of required reactions 10 Design verify and synthesize the primers kk kk kk kk kk kk kk kk kk kK kk kk kK kk kk kk kk kk 10 Select your amplicon site and design the primers 10 Verify primers for multiplex PCR kk kk kk kk kk kk kk kk kk kk kk kk kk kk kk kk kk kk kk kk k 11 Synthesize the primers ooooccco nana 12 Experimentally verify primers for multiplex PCR cn 12 A at ne ct a i eee A nn aa 12 Prepare and run the PCR reaction kk kk kk kk kk kk kk kk kK kk kk kk kK KK kk kk kk kk kk kk kk kk kk kk k 12 Prepare primer MIX tiara a td cdas ae etl Air 12 Prepare the PCR reaction mix 44 4444 eee 12 Add template DNA to the PCR reaction mx kk kk kk kk kk KK kK KK KK KK KK KK KK KK kk 13 Set up and run POR s n se avana m aa dr 14 Analyze the PCR products s lt s a5 kar Hand ala kin dik nak L Wad kaya EL ui 15 Troubleshooting SOR YR NAY e ose hae es Boa RR un Ln a ce au i 16 S APPENDIX A Ordering Information 19 Kit Contents ita patte Rat da Ea a dete aa tine HHH hate nn gee 19 Materials and equipment not included WWW kk kk kk kk kk kk kk KK KK KK KK KK KK KK KK kK kK kk kk 20 MS eee de at to ai PE AR RE vba tt 20 REA CIS AE iaa AR lb e due ea cala ag at pn a UN eee Seer Sue te de 20 Other equipment and consumables
3. 2 100 bp 2 kb Verify primers for Verify the designed primers for their multiplex PCR compatibility using the in silico multiplex PCR PCR modeling feature at the UCSC Genome Bioinformatics website 1 Go to http genome ucsc edu and select PCR on the top menu bar 2 Enter the sequences of forward and reverse primers 3 Click Submit 4 Verify the following The PCR product is unique The size of the product differentiates from adjacent amplicons e The Tm for each primer is between 59 65 C 5 Copy the sequence of the product then click BLAT 6 Paste the in silico amplicon sequence into the text box in the Blat window and click Submit to conduct Blat 7 Click Details for each retrieved fragment to ensure there is no homology in the primer targeting sites Platinum Multiplex PCR Master Mix 11 Bi Platinum Multiplex PCR Master Mix User Guide Prepare and run the PCR reaction Synthesize the primers Experimentally verify primers for multiplex PCR Store the primers Synthesize primers with a reliable oligo vendor and dissolve them in 0 1X TE buffer 10 mM Tris HCI 0 1 mM EDTA pH 8 for regular oligos and pH 7 for fluorescently labeled oligos De salted primers are generally pure enough for multiplex PCR We recommend measuring the concentrations of each primer with a UV spectrophotometer before use Verify the primers in singleplex PCR and by gel electrophoresis before use in multip
4. Safety appendix of this document Purpose The Platinum Multiplex PCR Master Mix User Guide provides detailed information for performing multiplex end point PCR over a wide range of DNA templates including challenging GC rich sequences using the Platinum Multiplex PCR Master Mix Prerequisites This guide is intended for biologists who have some experience performing PCR User attention words Three user attention words appear in this document Each word implies a particular level of observation or action as described below Note Provides information that may be of interest or help butis not critical to the use of the product IMPORTANT Provides information that is necessary for proper instrument operation or accurate chemistry kit use 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 Platinum Multiplex PCR Master Mix 5 About This Guide User attention words 6 Platinum Multiplex PCR Master Mix Platinum Multiplex PCR Master Mix User Guide Product information Introduction Purpose of the product A variant of PCR end point multiplex PCR enables simultaneous amplification of many targets in a single tube using multiple pairs of primers Multiplex PCR has been applied in research forensic and diagnostic laboratories where simultaneous analysis of multiple markers is required
5. Examples of experiments performed using multiplex PCR Genotyping Deletions mutations and high throughput SNP profiling Detection of pathogens or genetically modified organisms Microsatellites analysis e Forensic analysis Human identification and paternity testing e Quantitative and reverse transcription PCR assays for gene expression The Platinum Multiplex PCR Master Mix contains all of the components except for primers and templates for multiplex end point PCR in a single tube including Platinum DNA Polymerase The kit also includes the GC Enhancer which is used for difficult to amplify templates especially for templates with high GC content This master mix is optimized for simultaneous amplification of multiple targets ranging in size from 50 2500 bp from a DNA template with a wide range of GC content The PCR products are then analyzed by using e Agarose gel electrophoresis Capillary electrophoresis e Agilent Bioanalyzer Instrument s Agilent Lab901 Platinum Multiplex PCR Master Mix 7 Platinum Multiplex PCR Master Mix User Guide Product information Kit contents and storage conditions Part aya i Number Description Storage Conditions 4464268 Platinum Multiplex PCR Master Mix Store unopened at 15 C to 50 reaction kit 25 C until the expiration date on Platinum Multiplex PCR Master Mix 2x the label 1 tube 1 25 mL After opening the master mix _ RO
6. on page 23 and select an instrument and a reaction plate You can perform the PCR amplification with any of the thermal cyclers equipment or materials listed on page 18 Calculate the number of reactions needed to perform each assay including no template control NTC reactions In your calculations account for volume loss from pipetting by adding at least one extra reaction for every 10 required reactions For example for a 96 well plate prepare enough volume for approximately 110 reactions Note Multiple PCRs can be run on one reaction plate but include controls for each run on the plate Design verify and synthesize the primers Select your amplicon site and design the primers 10 IMPORTANT If GC rich targets are unavoidable when selecting your amplicon site and primers keep the amplicon sizes shorter than 300 bp Avoid simultaneous amplification in a single reaction of targets with over 70 GC and those with less than 25 GC when mixing primers GC Enhancer is included as an optional PCR additive for targets with greater than 65 GC content Choose one of the following methods for selecting your amplicon site and designing the primers e Primer Express Software Refer to the Primer Express Version 3 0 Getting Started Guide and Software Help for using this software Primer3Plus Freeware provided at www bioinformatics nl under Useful Links Follow the instructions beginning on this page Note Primer3Plus pr
7. can be up to 2 5 kb long although 100 to 1500 bases are more typical and easier to amplify Start with enough copies of the template to obtain a signal after 25 to 30 cycles More than 30 ng 104 copies but less than 1 ug of human genomic DNA per 50 uL reaction is the recommended range If the target DNA concentration is low you may need more than 35 cycles to produce sufficient product for analysis however this may increase the likelihood of primer dimers Platinum Multiplex PCR Master Mix Appendix B Guidelines for Designing PCR Assays Optimize the PCR conditions Optimize enhancer The GC Enhancer helps amplify challenging amplicons including amplicons that are concentration GC rich have GC repeats or generate nonspecific products GC enhancer is generally not needed if the GC contents of the targets are between 25 and 70 In a 50 uL reaction for targets with e 65 to 75 GC start with 6 uL e gt 75 GC start with 10 uL In general if increased specificity is required add 1 to 2 uL per 50 uL reaction The GC Enhancer may reduce nonspecific amplification and improve the yield of specific products However excessive use of the GC Enhancer may reduce yield particularly for non GC rich amplicons Platinum Multiplex PCR Master Mix 25 Appendix B Guidelines for Designing PCR Assays Optimize the PCR conditions 26 Platinum Multiplex PCR Master Mix Safety This appendix covers General safety ni Ea 27 Ch
8. 375305 Veriti 96 Well Thermal Cycler Part No 4375786 2720 Thermal Cycler Part No 4359659 Aluminum 96 Well GeneAmp PCR System 9700 Part No 4314879 Gold plated 96 Well GeneAmp PCR System 9700 Part No 4314878 Silver 96 Well GeneAmp PCR System 9700 Part No N8050001 T Only one thermal cycler or one PCR system is required Reagents Item Source Nuclease free water not DEPC treated Life Technologies Part No AM9930 500 mL Tris EDTA TE buffer pH 8 0 Life Technologies Part No AM9849 or MLS 10X BlueJuice Gel Loading Buffer Life Technologies Part No 10816015 E Gel 50 bp DNA Ladder Life Technologies Part No 10488099 E Gel Low Range Quantitative DNA Life Technologies Part No 12373031 Ladder Other equipment a Source and consumables 1 5 mL microcentrifuge tubes MLS Agarose MLS Pre cast agarose gels 1 to 4 with ethidium bromide stain MLS Centrifuge with plate adapter MLSt Disposable gloves MLS Electrophoresis apparatus MLS Invitrogen E Gel 4 High Resolution Agarose Gel Invitrogen Part No G5018 04 Invitrogen E Gel 48 4 Agarose Gel Invitrogen Part No G8008 04 MicroAmp Optical 96 Well Reaction Plates Applied Biosystems Part No N8010560 MicroAmp Splash Free 96 Well Base Applied Biosystems Part No 4312063 MicroAmp 8 Tube Strip 0 2 mL Applied Biosystems Part No N8010580 20 Platinum Multiplex PCR Master Mix Appe
9. C Biological hazard safety Biological hazard safety AN AN WARNING Potential Biohazard Depending on the samples used on this instrument the surface may be considered a biohazard Use appropriate decontamination methods when working with biohazards WARNING BIOHAZARD Biological samples such as tissues body fluids 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 In the U S U S Department of Health and Human Services guidelines published in Biosafety in Microbiological and Biomedical Laboratories found at www cdc gov biosafety Occupational Safety and Health Standards Bloodborne Pathogens 29 CFR 1910 1030 found at www access gpo gov nara cfr waisidx_01 29cfr1910a_01 html Your company s institution s Biosafety Program protocols for working with handling potent
10. L reaction Platinum Multiplex PCR Master Mix 17 Platinum Multiplex PCR Master Mix User Guide Troubleshooting 18 Platinum Multiplex PCR Master Mix A Kit Contents Ordering Information Part Number Description Storage Conditions 4464268 Platinum Multiplex PCR Master Mix 50 reaction kit Platinum Multiplex PCR Master Mix 2X 1 tube 1 25 mL GC Enhancer 1 tube 0 3 mL 4464269 Platinum Multiplex PCR Master Mix 250 reaction kit Platinum Multiplex PCR Master Mix 2X 5 tubes 1 25 mL each GC Enhancer 1 tube 1 25 mL 4464270 Platinum Multiplex PCR Master Mix 2000 reaction kit Platinum Multiplex PCR Master Mix 2X 5 bottles 10 mL each GC Enhancer 1 bottle 10 mL Store unopened at 15 C to 25 C until the expiration date on the label After opening the master mix may be stored at 15 C to 25 C until the expiration date on the label or at 4 C for up to 30 days The GC Enhancer must be kept at 15 C to 25 C Platinum Multiplex PCR Master Mix 19 A Appendix A Ordering Information Materials and equipment not included Materials and equipment not included In addition to the supplied reagents the following items are required Thermal cyclers Item Applied Biosystems Part No Veriti 60 Well Thermal Cycler Part No 4384638 Veriti 96 Well Fast Thermal Cycler Part No 4
11. USER GUIDE applied biosystems by Life technologies Platinum Multiplex PCR Master Mix Publication Part Number 4463722 Rev A Revision Date April 2011 6 technologies For Research Use Only Not intended for any animal or human therapeutic or diagnostic use Information in this document is subject to change without notice APPLIED BIOSYSTEMS DISCLAIMS ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT EXPRESSED OR IMPLIED INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE TO THE FULLEST EXTENT ALLOWED BY LAW 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 WHETHER OR NOT FORESEEABLE AND WHETHER OR NOT APPLIED BIOSYSTEMS IS ADVISED OF THE POSSIBILITY OF SUCH DAMAGES 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 w
12. _950 GC Enhancer 1 tube 0 3 mL fe lale n i l i until the expiration date on the 4464269 E PCR Master Mix label or at 4 C for up to 30 days The GC Enhancer must be kept Platinum Multiplex PCR Master Mix 2X at 15 C to 25 C 5 tubes 1 25 mL each GC Enhancer 1 tube 1 25 mL 4464270 Platinum Multiplex PCR Master Mix 2000 reaction kit Platinum Multiplex PCR Master Mix 2X 5 bottles 10 mL each GC Enhancer 1 bottle 10 mL Platinum Multiplex PCR Master Mix Platinum Multiplex PCR Master Mix User Guide Workflow Prepare for PCR Select an instrument and reaction plate page 10 Y Calculate the number of required reactions page 10 Design verify and synthesize the primers Select your amplicon site and design the primers page 10 Y Verify primers for multiplex PCR page 11 Y Synthesize the primers page 12 Y Experimentally verify primers for multiplex PCR page 12 Prepare and run the PCR reaction Prepare primer mix page 12 Y Prepare the PCR reaction mix page 12 Y Add template DNA to the PCR reaction mix page 13 Y Set up and run PCR page 14 Analyze the PCR products page 15 Platinum Multiplex PCR Master Mix Workflow Platinum Multiplex PCR Master Mix User Guide Prepare for PCR Prepare for PCR Select an instrument and reaction plate Calculate the number of required reactions Before you begin PCR review PCR good laboratory practices
13. contact Technical Support and Sales facilities Search through frequently asked questions FAQs Submit a question directly to Technical Support Search for user documents SDSs vector maps and sequences application notes formulations handbooks certificates of analysis citations and other product support documents Obtain information about customer training Download software updates and patches Platinum Multiplex PCR Master Mix 31 HL ZZZ Erk Headquarters 6 5791 Van Allen Way Carlsbad CA 92008 USA Phone 1 760 603 7200 Toll Free in USA 800 955 6288 For support visit www appliedbiosystems com support technologies www lifetechnologies com
14. emical safety ti ad ai 28 Biological hazard safety 29 General safety WARNING GENERAL SAFETY Using this product in a manner not specified in the user documentation may result in personal injury or damage to the instrument Ensure that anyone using this product has received instructions in general safety practices for laboratories and the safety information provided in this document Before using an instrument or device read and understand the safety information provided in the user documentation provided by the manufacturer of the instrument or device Always determine what chemicals have been used in the instrument before changing reagents or instrument components Before handling chemicals read and understand all applicable Safety Data Sheets SDSs and use appropriate personal protective equipment gloves gowns eye protection etc To obtain SDSs see the Documentation and Support section in this document Platinum Multiplex PCR Master Mix 27 Appendix C Safety Chemical safety Chemical safety 28 A WARNING GENERAL CHEMICAL HANDLING To minimize hazards ensure laboratory personnel read and practice the general safety guidelines for chemical usage storage and waste provided below and consult the relevant SDS for specific precautions and instructions Read and understand the Safety Data Sheets SDSs provided by the chemical manufacturer before you store hand
15. ially infectious materials Additional information about biohazard guidelines is available at www cdc gov In the EU Check local guidelines and legislation on biohazard and biosafety precaution and refer to the best practices published in the World Health Organization WHO Laboratory Biosafety Manual third edition found at www who int csr resources publications biosafety WHO_CDS_CSR_LYO_2004_11 en Platinum Multiplex PCR Master Mix 29 E Appendix C Safety Biological hazard safety 30 Platinum Multiplex PCR Master Mix Documentation and Support Related documentation You can download the following documents and other product support documents from www appliedbiosystems com Document Part number Platinum Multiplex PCR Master Mix 2X Product Insert 4463721 Applied Biosystems Veriti Thermal Cycler User Guide 4379799 GeneAmp PCR System 9700 Base Module User s Manual 4303481 GeneAmp PCR System 9700 96 Well Sample Block Module 4316011 Getting Started Guide Primer Express Software Version 3 0 4362460 Obtaining SDSs Safety Data Sheets SDSs are available from www appliedbiosystems com sds Note For the SDSs of chemicals not distributed by Life Technologies contact the chemical manufacturer Obtaining support For the latest services and support information for all locations go to www appliedbiosystems com At the website you can e Access worldwide telephone and fax numbers to
16. ing Veriti and GeneAmp PCR System thermal cyclers with a calculated in tube temperature Some models of thermal cyclers may require longer denaturation times For increased product specificity use annealing temperatures higher than 45 C Determine the optimum annealing temperature by testing at increments of 5 or fewer degrees Celsius until the maximum specificity is reached Use computer programs that calculate primer melting temperatures Tm to help you narrow the range of annealing temperatures to test For such a T calculator go to http www appliedbiosystems com then select Support Tools Research Tools gt T Calculator for PCR Primers The GeneAmp PCR System 9700 Thermal Cycler also contains a T calculator Thirty 30 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 Optimize the PCR conditions Optimize template concentration 24 The DNA segment to be amplified from the template
17. ithout limitation reporting the results of purchaser s activities for a fee or other commercial consideration is conveyed expressly by implication or by estoppel This product is for research use only Diagnostic uses require a separate license from Roche Further information on purchasing licenses may be obtained by contacting the Director of Licensing Applied Biosystems 850 Lincoln Centre Drive Foster City California 94404 USA TRADEMARKS The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners O 2011 Life Technologies Corporation All rights reserved Part Number 4463722 Rev A April 2011 Contents About This Guide cami da 5 PURPOS oa nent pt nn ns ones phir AUS pee af nn a A Ue mm 5 Prerequisites airs case nt do e e a res Gr hr Aus 5 User attention WOrdS is zeitei ne A a eee en 5 B Platinum Multiplex PCR Master Mix User Guide 7 Product informations issus seat dune de neue dila aA dha ete aaa 7 INTFOdUCUON As se ca care i a sa aa decta a Met TARAK eben Gall Ran R es 7 Purpose of the product kk kk kk kk kk kk kK kK kk kK kk kk kk kk kK kk kk kk kk kk kk kk kk kk kk kk k 7 Kit contents and storage conditions 8 NOFKILOWE sanan etats pare EA Xeyal A Abe ai NE ae hg ae 9 Prepare for POR sS sa ias ne a wa ka el teva de e 10 Select an instrument and reaction plate
18. le or work with any chemicals or hazardous materials To obtain SDSs see the Documentation and Support section in this document Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals for example safety glasses gloves or protective clothing Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation for example fume hood Check regularly for chemical leaks or spills Ifa leak or spill occurs follow the manufacturer s cleanup procedures as recommended in the SDS Handle chemical wastes in a fume hood Ensure use of 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 After emptying a waste container seal it with the cap provided Characterize by analysis if necessary the waste generated by the particular applications reagents and substrates used in your laboratory Ensure that the 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 Platinum Multiplex PCR Master Mix Appendix C Safety
19. lex PCR Use only primers that produce single bands with the correct size Note A 4 agarose E gel is recommended If the template DNA is homozygous for the amplified target use only the primers that generate a single band with the correct size for multiplex PCR If a primer pair produces multiple bands when the template DNA is homozygous redesign the primers by changing the location of the targeting site on the genome until a single band is produced Store primers in small aliquots at 20 C and avoid repeated freezing and thawing Prepare and run the PCR reaction Prepare primer mix Prepare the PCR reaction mix 12 1 Combine all primers for the multiplex PCR reaction in one tube and adjust the primer mix to a final concentration of 0 5 uM per primer using 0 1X TE 10 mM Tris HCl 0 1 mM EDTA buffer 2 Dispense the primer mix into smaller aliquots that can be stored at 20 C for one year or 4 C for up to two months IMPORTANT Avoid repeated freezing and thawing 1 Thaw the primer mixes templates GC Enhancer optional and Platinum Multiplex PCR Master Mix 2 Mix the Platinum Multiplex PCR Master Mix by inverting the tube or bottle about 10 times IMPORTANT Avoid generating bubbles when mixing 3 Place the Platinum Multiplex PCR Master Mix tube or bottle on ice 4 Mix the contents of the remaining tubes well by inverting each tube a few times and spinning briefly Place the tubes on ice Platin
20. ndix A Ordering Information A Materials and equipment not included Item Source MicroAmp 8 Cap Strip Applied Biosystems Part No N8010535 MicroAmp 96 well Base Applied Biosystems Part No N8010531 MicroAmp 96 Well Tray Retainer Set 10 sets Life Technologies Part No 403081 MicroAmp Clear Adhesive Film Applied Biosystems Part No 4306311 MicroAmp Optical Film Compression Pad Applied Biosystems Part No 4312639 Microcentrifuge MLS Pipettes positive displacement or air displacement MLS Pipette tips with filter plugs MLS Polypropylene tubes MLS Vortexer MLS 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 See instrument manual for compatibility For more product recommendations visit the PCR technology page at www appliedbiosystems com Platinum Multiplex PCR Master Mix 21 A Appendix A Ordering Information Materials and equipment not included 22 Platinum Multiplex PCR Master Mix Guidelines for Designing PCR Assays This appendix covers PCR good laboratory practices 23 Adjust thermal cycling 24 Optimize the PCR conditions 24 PCR good laborat
21. ory practices When preparing samples for PCR amplification e 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 e Change gloves whenever you suspect that they are contaminated e 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 e Keep reactions and components capped as much as possible e Clean lab benches and equipment periodically with 10 bleach solution Use DNAZap Solution Part no AM9890 Platinum Multiplex PCR Master Mix 23 B Appendix B Guidelines for Designing PCR Assays Adjust thermal cycling Adjust thermal cycling Adjust the hold period for activation Adjust denaturation conditions Adjust annealing conditions Adjust extension conditions For general PCR runs Applied Biosystems recommends a pre PCR activation setup of 95 C for 2 minutes In the early cycles make sure that your DNA template is completely denatured Do not exceed a denaturation temperature of 95 to 96 C A denaturation period of 30 seconds is adequate when us
22. ovides a Basic Local Alignment Search Tool BLAST algorithm which is useful for comparing sequence information Other software options do not provide this algorithm Primer3Plus Freeware 1 In the Main tab paste your sequence ID and the source sequence 2 In the General Settings tab use the following parameters Variable Definition Primer size 21 30 nt Primer Tn 59 65 C Primer GC 40 60 Concentration of monovalent cations 50 mM Annealing oligo concentration 50 nM Mispriming Repeat Library Based on your assay Remaining variables Default or User defined Platinum Multiplex PCR Master Mix Platinum Multiplex PCR Master Mix User Guide Design verify and synthesize the primers 3 Inthe Advanced Settings tab define the product size e Amplicon size differentiation between adjacent amplicons for sufficient separation For GC rich sites amplicon size should be lt 300 bp The table below defines the guidelines for differentiating the sizes if the PCR products are resolved on a 4 High Resolution E gel Size of the adjacent amplicons Minimum difference 50 100 bp 10 bp 100 200 bp 20 bp 200 400 bp 40 bp 400 700 bp 50 bp 700 900 bp 100 bp 900 2500 bp 500 bp If other E gels are to be used the following table gives a rough idea of the resolutions Gel type Agarose ar Resolution 0 8 800 bp 3 kb E gel single comb 1 2 58cm 100bp 3kb
23. run See your instrument user manual for detailed instruction on how to load and run the reactions 14 Platinum Multiplex PCR Master Mix Platinum Multiplex PCR Master Mix User Guide Analyze the PCR products Analyze the PCR products IMPORTANT To prevent contamination never bring amplified PCR products into the PCR setup area or the PCR reagent storage area You may analyze results using gel electrophoresis capillary electrophoresis Lab901 or the Agilent 2100 Bioanalyzer instrument Refer to instrument or PCR system documentation for analysis details For successful analysis using gel electrophoresis follow these guidelines 1 Unload the reaction plate or tubes after the run is complete 2 Optional Store the plate or tubes at 4 C for up to 72 hours or at 15 to 25 C for longer term storage 3 Choose the appropriate agarose concentration according to the total number of amplicons the size difference between adjacent amplicons and the amplicon size range in each reaction 4 Load and run gels according to the manufacturer s instructions to achieve the maximum separation of adjacent amplicons 5 Take pictures of the gels on a UV transilluminator Platinum Multiplex PCR Master Mix 15 Troubleshooting Platinum Multiplex PCR Master Mix User Guide Troubleshooting Observation Possible cause Recommended action Excessive amount of primer dimer Primer design is not optimal Re
24. s Increase the time in increments of 15 seconds too short 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 Increase the pre PCR heat step in increments of 1 minute or time is not sufficient use the Time Release protocol see Adjust the hold period for activation on page 24 16 Platinum Multiplex PCR Master Mix Platinum Multiplex PCR Master Mix User Guide Mm Troubleshooting Observation Possible cause Recommended action Product bands are smeared Potential secondary products Reduce final concentrations of the primers for the select amplicons to 50 nM while maintaining all other at 100 nM Carryover contamination Use the GeneAmp PCR Carry Over Prevention Kit Part no N8080068 e 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 Sample amount is too low Increase the sample input to 0 1 1 ug 50 u
25. um Multiplex PCR Master Mix Add template DNA to the PCR reaction mix Platinum Multiplex PCR Master Mix User Guide Prepare and run the PCR reaction 5 Prepare the PCR reaction mix as follows using your number of calculated reactions as described on page 10 Volume per 50 uL Final Component i reaction pL concentration Platinum Multiplex PCR Master Mix 2X 25 UL 1X Primer mix 5 10 pLt 50 100 nM 0 5 uM each primer each primer GC Enhancer Optional 0 10 uLt 0 20 Variable Use to bring final PCR grade water volume to 50 ul minus the volume of template DNA Use 10 pl of primer mix final concentration 100 nM of each primer when less than 0 1 ug of DNA is used For targets with 65 to 75 GC use 6 uL of the GC Enhancer in a 50 uL reaction For targets with gt 75 GC start with 10 uL in a 50 pL reaction Refer to Optimize enhancer concentration page 25 for more information 0 AI oS Combine the components in an appropriate tube Close the tube and mix the solution well by inverting the tube a few times Centrifuge the tube briefly to spin down the contents Dispense appropriate volumes 50 uL minus the volume of template DNA of the PCR reaction mix to the bottom of the wells of a reaction plate or PCR tubes Add 0 1 0 2 ug of DNA template to the PCR reaction mix to bring the final volume to 50 uL For the no template control reactions adjust the final reaction volume of each
26. view primer design and composition and ensure each primer pair gives a single band and no primer dimer in singleplex PCR Cycle number is too high Reduce the cycle number in increments of three cycles Primer manufacturing error Order primers from a reliable vendor and ensure that a purification process like de salting and HPLC is used Pipetting errors Calibrate pipettes regularly to ensure the accuracy of pipetting Quality of template DNA is too low Always use high quality purified DNA templates Low levels of PCR product or no product Template concentration is too low Increase the sample input to 0 1 1 ug 50 uL reaction Bana visibl Experimental sample DNAis Add more DNA or use sample that has been processed to damaged or degraded minimize shearing and nicking Denaturation time is too Adjust the time in increments of 5 seconds short or too long Denaturation temperature is Adjust the temperature in increments of 1 degree Celsius lf too low or too high you have a Veriti thermal cycler adjust the VeriFlex Blocks in increments of 1 degree Celsius for up to six different temperatures Annealing extension Lower the temperature in increments of 2 degrees Celsius If temperature is too high you have a Veriti thermal cycler adjust the VeriFlex Blocks in increments of 2 degrees Celsius for up to six different temperatures Annealing extension time i
27. well or tube to 50 uL with PCR grade water Seal the plate with MicroAmp Clear Adhesive Film or cap the tubes with MicroAmp 8 Cap Strips Mix the contents well by inverting the plates or tubes a few times then briefly centrifuging Platinum Multiplex PCR Master Mix 13 Set up and run PCR 1 Prepare and run the PCR reaction Platinum Multiplex PCR Master Mix User Guide Choose an amplification protocol based on your analysis method 2 Configure the run method as outlined in your instrument user manual 3 If the products will be analyzed by agarose gel electrophoresis Lab901 or Agilent 2100 Bioanalyzer instruments use the following parameters Stage Step Temperature Time Activation of Platinum Z Holding Multiplex PCR Master Mix cil SHU Denature 95 C 30 sec Cycling Anneal 60 C 90 sec 30 to 40 cycles Extend 72 C 60 sec kb of the largest amplicon Holding Final Extension 72 C 10 min Holding Final Hold 49C co If the products will be analyzed by capillary electrophoresis use the following parameters Stage Step Temperature Time Activation of Platinum zi Poeng Multiplex PCR Master Mix al out Denature 95 C 30 sec Cycling Anneal 60 C 90 sec 25 to 40 cycles Extend 7990 60 sec kb of the largest amplicon Holding Final Extension 60 C 30 min Holding Final Hold 4 C oo 4 Load the reaction plate or tubes into the instrument and start the
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