PowerPlex(R) Matrix Standards, 310 Technical Bulletin
Contents
1. bath or thermal cycler crushed ice or ice water bath 310 capillaries 47cm x 50um performance optimized polymer 4 POP 4 polymer 10X genetic analyzer buffer sample tubes and septa aerosol resistant pipette tips Hi Di formamide Applied Biosystems Cat 4311320 The quality of formamide is critical Use Hi Di formamide Freeze formamide in aliquots at 20 C Multiple OQ freeze thaw cycles or long term storage at 4 C may cause breakdown of formamide Poor quality formamide may contain ions that compete with DNA during injection which results in lower peak heights and reduced sensitivity A longer injection time may not increase the signal Formamide is an irritant and a teratogen avoid inhalation and contact with skin Read the warning label and take the necessary precautions when handling this substance Always wear gloves and safety glasses when working with formamide 2 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TBD021 Revised 10 15 www promega com Promega 3 A Instrument Preparation Refer to the ABI PRISM 310 Genetic Analyzer User s Manual for instructions on cleaning the pump block installing the capillary calibrating the autosampler and adding polymer to the syringe 1 2 eS oS 10 Open the ABI PRISM 310 Data Collection Software Version 3 1 0 To preheat the ABI PRI2. SM 310 Genetic Analyzer to 60 C select Manual Control in the Window menu In the Function menu select Temperature Set Set Value to 60 0 and then select Execute Close the Manual Control screen In the File menu select New to open the Create New menu Open a GeneScan sample sheet either 48 Tube or 96 Tube In the upper right corner of the sample sheet 4 Dyes should be selected Enter the appropriate sample information in the Sample Name field Matrix sample names should be descriptive for example add the color to the sample name Label tubes with the corresponding sample names To save the sample sheet select Save As in the File menu Assign a name to the file and save to the Sample Sheet folder Close the file In the File menu select New to open the Create New menu Open the GeneScan injection list Select the sample sheet i e the gss file that was created in Step 5 Choose the GS STR POP4 1mL F md4 module from the drop down menu The settings should be Injection Time 3 seconds Injection Voltage 15 0kV Run Voltage 15 0kV Run Temperature 60 C Run Time 30 minutes Note The injection time may need to be increased or decreased depending on instrument sensitivity Peak heights of 1 000 4 000RFU are optimal for matrix generation Select none for the matrix file Matrix Sample Preparation Thaw the matrix standards For each matrix
3. T IEE EIE TA TE T A A S TA AT 7 0 Summary of Chang CS ssia E E cvacvecesacueecsnaseaasamnevaceccossccnnecexeseiccassanoneniuentsanoens 7 1 Description Proper generation of a matrix file is critical to evaluate multicolor STR data with the ABI PRISM 310 Genetic Analyzer To prepare a matrix four standards are analyzed using the same capillary electrophoresis CE conditions as those for samples and allelic ladders The PowerPlex Matrix Standards 310 consists of DNA fragments labeled with four fluorescent dyes one tube contains DNA fragments labeled with fluorescein one tube contains DNA fragments labeled with JOE one tube contains DNA fragments labeled with TMR and one tube contains DNA fragments labeled with CXR Use the Fluorescein Matrix JOE Matrix TMR Matrix and CXR Matrix for the blue green yellow and red standards respectively The PowerPlex Matrix Standards 310 can be used with any of the 4 dye Promega STR amplification systems A matrix must be generated for each individual instrument A new matrix should be run after major maintenance on the system such as changing the laser calibrating or replacing the CCD camera or changing the polymer type or capillary array We also recommend that you generate a new matrix after the instrument is moved to a new location In some instances a software upgrade may necessitate generation of a new matrix Individual labs should determine the frequency of matrix generation Protocols to op
4. TECHNICAL BULLETIN PowerPlex Matrix Standards 310 Instructions for Use of Product DG4640 All technical literature is available at www promega com protocols Visit the web site to verify that you are using the most current version of this Technical Manual E mail Promega Technical Services if you have questions on use of this system genetic promega com De DCS CUO coc csnce te sccanacs voces tcocaastun bia sosecunn cavaectudicauasenesssactmsraseanteceuneuudestsasavancdenseasduacsuootnnteasaumecweiuadenss 1 2 Product Components and Storage Conditions ccsccssscssccssccsscsccsccnsccsscesccesccesccnscenscssccescesscesscenss 2 3 Detection of Matrix Fragments Using the ABI PRISM 310 Genetic Analyzer and GeneMapper ID or GeneMapper ID X Software sessessessessosecsecsseosesscoseseoscoscsscsecsecseosesecsecseoseoseoseos 2 LAs MMoiument Prepar alioi ccisocusantunansaucceescuvencneiseacenatencsseadauencusestacdouenanteesssecedasatwocseseveruaterncesuentauaees 3 3 B Matrix Sample Preparation esssseeseesecsecscsecsecsecsecseoseoscsecseoseoseoscoecoeoscoscoscsecsecsecsesecsecsecsecseoseos 3 3 C Capillary Electrophoresis and Detection ccssccssscsscssccssscnsscnsccnscosscnsccnsccnsccnscesccesccescesscesses 4 3 D Matrix Generation for the ABI PRISM 310 Genetic Analyzer cssccssccssccssccsscesccsscesscesccesees 4 A TOUTES MOOG ING sorrisi EE a E ENRE 6 T E A e A EE A E A E A AEAT E
5. ce or Distributor Contact information available at www promega com E mail genetic promega com S toms Causes and Comments Unable to generate a matrix due to faint or no peaks Poor capillary electrophoresis CE injection Re inject the sample Check the syringe for leakage Check the laser power Poor quality formamide was used Use only fresh Hi Di formamide Samples were degraded due to improper storage Store matrix standards at 30 C to 10 C protected from light Do not store in the freezer door or in a frost free freezer We recommend using these matrix standards once and then discarding them Peak heights were too low Peak heights should be 1 000 4 000RFU for the ABI PRISM 310 Genetic Analyzer To increase peak heights increase injection time or loading volume Samples were not denatured Heat denature samples and immediately chill on crushed ice or in an ice water bath before loading the capillary Denature samples just prior to loading Poor quality matrix extra peaks visible in CE related artifacts spikes Minor voltage changes or urea one or all color channels crystals passing by the laser can cause spikes or unexpected peaks Spikes sometimes appear in one color but often are easily identified by their presence in more than one color Re inject the samples to confirm S CE related artifacts contaminants Contaminants in the water used with the ABI PRISM 310 Genetic Analyzer and fo
6. erate the fluorescence detection instrumentation should be obtained from the manufacturer Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 www promega com TBDO21 Revised 10 15 Promega 2 Product Components and Storage Conditions Product Size Cat PowerPlex Matrix Standards 310 50ul each dye DG4640 Not for Medical Diagnostic Use Includes e 50ul Fluorescein Matrix e 50ul JOE Matrix e 50ul TMR Matrix e 50ul CXR Matrix Storage Conditions Upon receipt store all components at 30 C to 10 C in a nonfrost free freezer protected from light Do not store reagents in the freezer door where the temperature can fluctuate The fragments in the matrix standards are light sensitive and must be stored in the dark We strongly recommend that the matrix standards be stored with the post amplification reagents away from pre amplification materials and used separately with different pipettes tube racks etc We recommend using these matrix standards once and then discarding them Additional product information and ordering information for accessory components and related products are available upon request from Promega or at www promega com 3 Detection of Matrix Fragments Using the ABI PRISM 310 Genetic Analyzer and GeneMapper ID or GeneMapper ID X Software Materials to Be Supplied by the User 95 C dry heating block water
7. eviously generated matrix no longer Changes to or aging of instrument components Instrument performs optimally sensitivity can change if the instrument was moved or recently serviced replacement or realignment of the laser CCD camera power supply or mirrors The sensitivity also can change over time due to aging of the instrument These changes can result in poor matrix performance Generate a new matrix 5 Related Products Product PowerPlex 16 HS System PowerPlex 16 System PowerPlex CS7 System PowerPlex S5 System Size 100 reactions 400 reactions 100 reactions 400 reactions 100 reactions 100 reactions 400 reactions MSI Analysis System Version 1 2 100 reactions 50 reaction pairs Not for Medical Diagnostic Use For Research Use Only Not for Use in Diagnostic Procedures 6 Summary of Changes The following changes were made to the 10 15 revision of this document 1 Instructions for the ABI PRISM 377 DNA Sequencer were deleted 2 Information about the use of GeneMapper ID X software was added 3 The technical bulletin was moved into the new format Cat DC2101 DC2100 DC6531 DC6530 DC6613 DC6951 DC6950 MD1641 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 7 TBDO21 Revised 10 15 www promega com 2006 2010 2015 Promega Corporation All Rights Reserved PowerPlex is a regis
8. it to add the sample file Repeat this step for each matrix standard Note To find the fsa files in the default location go to My Computer AB SW8DATA D Applied Bio 310 and then Runs and locate the correct run folder 4 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TBDO21 Revised 10 15 www promega com yg Matrix Editor trix Description Matrix Name 4dye_310_POP4 SSUES Description trix Settings Select the Matrix Standard Sample File FAM3 31 15 3 45 AM fsa s JOE3 31 15 4 22 AM fsa TMR3 31 15 2 32 AM fsa Re CXR3 31 15 3 09 AM fsa Figure 1 The Matrix Editor d Enter the data point value recorded from Step 4 in the Start at field Repeat this step for each matrix standard e Click on the Create button The Matrix Result should give a value of 1 000 when comparing a dye to itself Typically all other values will be less than 1 000 Select OK and the matrix will be created in the Matrices tab of the GeneMapper Manager Select Done Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 5 www promega com TBDO21 Revised 10 15 Promega 4 Troubleshooting For questions not addressed here please contact your local Promega Branch Offi
9. older containing the fsa files from Section 3 C Highlight the run folder and select Add To List and then Add 2 To open the raw data for a specific matrix sample file locate Project in the upper left corner of the screen and double click on the run folder to reveal the fsa files 3 Choose a single fsa file to observe the raw data While viewing the raw data move the cursor to the region that is to the right of the primer peak and to the left of at least five peaks Choose a region in a flat part of the baseline 4 Record the data point value found at the lower left portion of the screen for use in Step 6 Repeat this step for each matrix standard Dye Color Corresponding Matrix Start At Value Blue Fluorescein Matrix Green JOE Matrix Yellow TMR Matrix Red CXR Matrix 5 To create a new matrix select GeneMapper Manager in the Tools menu Select the Matrices tab and then New 6 Define the new matrix in the Matrix Editor Figure 1 Note The Matrix Name Start At values and Matrix Result values shown in Figure 1 are instrument specific and will change depending on your instrument a Assign a matrix name in the Matrix Name field b Set Number of Dyes to 4 c To select each matrix standard sample file click on the dye color for each matrix B for fluorescein G for JOE Y for TMR and R for CXR Navigate to the fsa sample file that corresponds to that dye and double click on
10. r diluting the 10X genetic analyzer buffer can generate peaks in the blue and green dye colors Use autoclaved water to clean the pump block and prepare sample dilutions Change vials and wash the buffer reservoir Poor quality matrix elevated baseline and or Matrix used was generated on another instrument A matrix inverted peaks in analyzed samples must be generated for each instrument Wrong dye was used Generate the matrix using the same dyes as those in the samples Oversubtraction of signal occurred because signal was saturated When generating a matrix avoid choosing samples with peak heights that are higher than the recommended RFU values as this can result in a matrix that causes inverted peaks or elevated baseline Analyzed sample results may be improved by diluting the matrix samples in water before preparing them for use Alternatively decrease the injection time 6 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TBD021 Revised 10 15 www promega com S toms Causes and Comments O Promega Inverted peaks in the matrix baseline Inappropriate or no Start At value was entered The Start At value chosen in Section 3 D should be chosen in a region with a flat baseline Wrong colors were assigned to the dyes Confirm the dye and color selection Fluorescein Blue JOE Green TMR Yellow CXR Red Pr
11. standard vortex the tube for 5 10 seconds to mix and then add 2ul of matrix standard to 25ul of Hi Di formamide Denature each sample for 3 minutes at 95 C and immediately chill on crushed ice or in an ice water bath for 3 minutes Denature samples just prior to loading Place tubes in the appropriate autosampler tray 48 tube or 96 tube Place the autosampler tray in the instrument and close the instrument doors Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 3 www promega com TBDO21 Revised 10 15 3 C Capillary Electrophoresis and Detection 1 After loading the sample tray and closing the doors select Run to start the capillary electrophoresis system 2 Monitor the electrophoresis by observing the raw data and status windows Each sample will take approximately 40 minutes for syringe pumping sample injection and electrophoresis Note The matrix files that are created will be fsa files After the run is finished save or transfer the fsa files to a secure location where they can be opened in a GeneMapper project 3 D Matrix Generation for the ABI PRISM 310 Genetic Analyzer 99 1 Open a new GeneMapper project To add matrix sample files to the new project select Add Samples to Project in the File menu for GeneMapper ID software or the Edit menu for GeneMapper ID X software Choose the appropriate run f
12. tered trademark of Promega Corporation ABI PRISM and GeneMapper are registered trademarks of Applied Biosystems GeneScan is a registered trademark of Eurofins Genescan Holding GmbH Hi Di is a trademark of Applera Corporation POP 4 is a registered trademark of Life Technologies Corporation Products may be covered by pending or issued patents or may have certain limitations Please visit our Web site for more information All prices and specifications are subject to change without prior notice Product claims are subject to change Please contact Promega Technical Services or access the Promega online catalog for the most up to date information on Promega products 8 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TBDO21 Revised 10 15 www promega com
Download Pdf Manuals
Related Search