"user manual"
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1. Electropherogram with raw data of the matrix standard 6 FAM 7 Select an analysis range with a flat baseline and re inject the matrix sample if necessary 8 Record start and end value data points of the analysis range e g start value 3200 end value 5500 9 Calculate the difference between the end and start values e g 5500 3200 2300 data points Generation of a matrix 1 Select New in the File menu and then select Matrix Import the matrix samples for all dyes B G Y R and O Enter a Start At value e g 3200 Under Points enter the calculated difference between end and start values e g 2300 5 Click OK to calculate the new matrix gt CDCOD x Select the Matrix Standard Sample Files Number Of Dyes LB 6FAM_080204 fsa Start At 0 G BTG_080204 fsa Start At RB Y BTY_080204 fsa Statat 00 A BTR_080204 fsa Statt Po O 810_080204 1sa Statat Po Points ET me Matrix sample selection a z gt g jf E m gt pmjrzzum _ _ m zZ _ _ m smn mm m k Investigator Argus Y 12 QS Handbook 02 2015 17 6 Select Save as in the File menu to save the new matrix in the matrix folder Matrix BT5 mtx Reactions B G hf R 0 Josss1 10000 fo 2056 Jo 2258 o o017 fo 1244 joss 9 Jo s311 Ji oooo o 00ss Jootso Jo 020s Jooarr Jozos2 4 0000 Ix I lt n a New matrix BT52. Checking the matrix 1 To check the new matrix with current samples select New in the File menu and then select Project 2 Open the folder of the respective run and select Add Sample Files Select the sample s in the Sample File column 4 Click Sample and then Install New Matrix to open the matrix folder and select the new matrix 5 Re analyze the samples Note There should be no pull up peaks between the dye panels B G Y R O with the new matrix o 18 Investigator Argus Y 12 QS Handbook 02 2015 Sample preparation 1 Set up a mixture of formamide and DNA size standard according to Table 10 Table 10 Setup of formamide and DNA size standard mixture Component Volume per sample Hi Di Formamide 12 0 ul DNA Size Standard 550 BTO 0 5 ul 2 Aliquot 12 pl of the mixture to a tube for each sample to be analyzed 3 Add 1 pl PCR product or allelic ladder diluted if necessary 4 Denature for 3 min at 95 C 5 Snap freeze by placing the plate on ice for 3 min Alternatively the thermal cycler set to 4 C may be used to cool the plate 6 Load the samples on the tray Investigator Argus Y 12 QS Handbook 02 2015 19 Setting up the GeneScan Software Create a Sample Sheet and enter sample designation Table 11 Injection list for the ABI PRISM 310 Genetic Analyzer Component Settings Module File GS STR POP 4 1 ml G5 Matrix File e g Matrix BT5 S
3. DYS389 1 AC004617 TCTG 3 TCTA 12 8 17 DYS389 II AC004617 TCTG TCTA TCTG 29 23 35 TCTA DYS390 ACO011289 TCTG TCTA TCTG 24 12 17 29 TCTA DYS39 1 AC011302 TCTA 11 5 16 DYS392 ACO11745 TAT 3 13 4 20 DYS393 AC006152 AGAT 12 7 18 DYS437 AC002992 TCTA TCTG TCTA 16 4 8 18 DYS438 AC002531 TTTTC o 10 7 18 DYS439 AC002992 GATA 13 5 19 8 Investigator Argus Y 12 QS Handbook 02 2015 Table 2 Chromosomal mapping of Investigator Argus Y 12 QS Kit Locus Chromosomal mapping DYS19 Yp11 2 DYS385 Yq11 222 DYS389 I Yq11 21 DYS389 I Yq11 21 DYS390 Yq 1 221 DYS391 Yq11 21 DYS392 Yq11 222 DYS393 Yp11 31 DYS437 Yq11 21 DYS438 Yq11 21 DYS439 Yq11 21 Investigator Argus Y 12 QS Handbook 02 2015 9 Equipment and Reagents to Be Supplied by User When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information consult the appropriate material safety data sheets MSDSs available from the product supplier EB Hi Di Formamide 25 ml Applied Biosystems cat no 4311320 E Matrix Standards BT5 for single capillary instruments e g ABI PRISM 310 Genetic Analyzer QIAGEN cat no 386113 E Matrix Standards BT5 for multi capillary instruments e g ABI PRISM 3100 and Applied Biosystems 3130 or 3500 Genetic Analyzers QIAGEN cat nos 386123 or 386125 HE Pipets and pipet tips M One of the following DNA
4. Analysis was performed on an Applied Biosystems 3500 Genetic Analyzer with the DNA Size Standard 550 BTO Allele assignment was performed using Investigator IDproof Software 75 in 120 In 150 165 180 1935 210 225 I 255 270 285 300 315 330 345 360 375 390 405 420 435 450 465 480 7500 DY5439 DY5437 DY3390 DYS385 6875 6250 5625 5000 4375 3750 3125 2500 1875 6 Cota Ea alealea fa o en jesesjezealer 2 1 i3 21 23 es 27 10 12 14 16 18 20 13 2 72 15 2 Sean DY5391 DY5369 1 DY519 Dysassn 3200 2800 2400 2000 1600 1200 800 400 0 A am a a AAAS m N d 2 A e Io 2 1 3 12 14 16 8 28 80 62 DY5436 75 90 105 120 135 150 165 180 195 210 225 240 255 270 285 300 35 30 345 360 375 390 405 420 45 40 465 480 Figure 5 Electropherogram of the allelic ladder Argus Y 12 QS analyzed on an Applied Biosystems 3500 Genetic Analyzer Allele assignment was performed using Investigator IDproof Software 58 Investigator Argus Y 12 QS Handbook 02 2015 Interpretation of Results Post PCR analysis and automatic allele assignment with suitable analysis software ensure a precise and reliable discrimination of alleles General procedure for the analysis 1 Check the DNA size standard 2 Ch
5. Plate BT5 Date Select GeneMapper Application 96 Well 7 Click OK and a new table in the Plate Editor opens automatically Table 31 8 Click the column header to select the entire column Select Fill Down from the Edit menu to apply the information to all selected samples Click OK 9 In the Run Scheduler click Find All and select Link to link the reaction plate on the autosampler tray to the newly created plate record position A or B 38 Investigator Argus Y 12 QS Handbook 02 2015 Table 31 GeneMapper Plate Editor II Parameter Settings Sample Name Enter the name for the samples Priority e g 100 Default Sample Type Sample or Allelic Ladder Size Standard e g SST BTO_60 400bp Panel e g Argus Y12_ Panels Analysis Method e g Analysis HID 3130 Snp Set User defined 1 3 Results Group 1 Select results group Instrument Protocol 1 Run36 POP4 BT5 20min setting described before 10 Start the run 11 During the run view Error Status in the Event Log or examine the quality of the raw data for each capillary in the Capillaries Viewer or the Cap Array Viewer 12 View data as an overview in Run History or Cap Array Viewer of the Data Collection Software Run data are saved in the Run Folder of the previously chosen Result Group Investigator Argus Y 12 QS Handbook 02 2015 39 Analysis parameters analysis method Table 32 lists the
6. Polymer POP4 Dye Set e g BT5 Run Module HID36_POPA4A e g Investigator Argus Y 12 QS Default Default Default 3 0 1300 Default 8 0 Default Default Deviating from the standard settings the injection time may range between 1 and 20 s depending on the type of sample If samples with very high signal intensities are recorded a shorter injection time may be selected For samples with low DNA content an injection time of up to 20 s may be necessary 3 Click Save to confirm the changes 4 To set up the Size Standard go to Library select Analyze followed by Size Standards and click Create 5 The parameters in Table 39 must be entered or selected The DNA Size Standard 550 BTO should be used with the following lengths of fragments 60 80 90 100 120 140 160 180 200 220 240 250 260 280 300 320 340 360 380 400 425 450 475 500 525 and 550 bp 48 Investigator Argus Y 12 QS Handbook 02 2015 Table 39 Size standard parameters Parameter Setting Size Standard e g SST BTO_60 550bp Dye Color Orange 6 Click Save to confirm the changes 7 To set up the QC Protocol go to Library and select Analyze followed by QC Protocols and click Create 8 The parameters in Table 40 must be entered or selected Table 40 QC Protocol parameters Parameter Setting Protocol Name e g BTO_550 Size St
7. Y 12 QS Handbook 02 2015 Setup a Dye Set Dye Set Name BT5 Chemistry Matrix Standard z Dye Set Template AnyDye Template NZ Arrange Dyes Dye Selection Reduced Selection Calibration Peak Order 5 v Parameters The parameters will be used for instruments configured with 36cm capillary array and polymer POP4 Matrix Condition Number Upper Limit 20 0 Locate Start Point After Scan 300 Before Scan 5000 Limit Scans To 20000 Sensitivity 0 1 Minimum Quality Score 0 8 Notes Matrix Std BT5 multi cap Setup of dye set BT5 Preparation of the standard calibration plate Example for 8 capillaries Applied Biosystems 3500 Genetic Analyzer 1 Set up a mixture of formamide and Matrix Standard BT5 according to Table 35 eae ans Investigator Argus Y 12 QS Handbook 02 2015 43 Table 35 Setup of formamide and Matrix Standard BT5 mixture for 8 capillaries Component Volume Hi Di Formamide 90 ul Matrix Standard BT5 multi cap 10 ul 2 Load 10 ul of the mixture to a 96 well plate e g positions Al Hl 6 Denature for 3 min at 95 C 4 Snap freeze by placing the plate on ice for 3 min Alternatively the thermal cycler set to 4 C may be used to cool the plate Example for 24 capillaries Applied Biosystems 3500xL Genetic Analyzer 1 Set up a mixture of formamide and Matrix Standard BT5 according to Table 36 Table 36 Setup of formamide and Matrix
8. Y 12 QS Handbook 02 2015 Trademarks QIAGEN QlAamp QlAsymphony EZ1 HotStarTaq Investi Biosystems Avant GeneAmp GeneMapper GeneScan Genotyper 6 gator MinElute QIAGEN Group 3500 ABI PRISM Applied FAM POP 4 Hi Di Applera Corporation or its subsidiaries Eppendorf Mastercycler Eppendorf AG GenBank US Department of Health and Human Services Registered names trademarks etc used in this document even when not specifically marked as such are not to be considered unprotected by law Limited License Agreement for the Investigator Argus Y 12 QS Kit Use of this product signifies the agreement of any purchaser or user of the product to the following terms 1 The product may be used solely in accordance with the protocols provided with the product and this handbook and for use with components contained in the kit only QIAGEN grants no license under any of its intellectual property to use or incorporate the enclosed components of this kit with any components not included within this kit except as described in he protocols provided with the product this handbook and additional protocols available at www giagen com Some of these additional protocols have been provided by QIAGEN users for QIAGEN users These protocols have not been thoroughly tested or optimized by Q not infringe the rights of third parties This kit and its components are licensed for one time
9. analyzers ABI PRISM 310 Genetic Analyzer ABI PRISM 3100 Avant 3100 Genetic Analyzer Applied Biosystems 3130 3130x Genetic Analyzer Applied Biosystems 3500 3500xL Genetic Analyzer BE One of the following PCR thermal cyclers GeneAmp PCR System 9700 Bio Rad PTC 200 Techne TC 512 Biometra T1 Eppendorf Mastercycler ep EB PCR tubes or plates Validity analysis software for human identification products Investigator Human Identification PCR Kits require calibration with an allelic ladder Therefore the software used must be compatible with human identification HID products for forensic applications We recommend GeneMapper ID GeneMapper ID X or Genotyper Software The Investigator Template Files facilitate data analysis and are compatible with the software mentioned above FTYrez 1 op j pzz 9 E 10 Investigator Argus Y 12 QS Handbook 02 2015 Protocol PCR Amplification This protocol is for PCR amplification of STR loci from forensic samples using the Investigator Argus Y 12 QS Kit Important points before starting E Set up all reaction mixtures in an area separate from that used for DNA isolation and PCR product analysis post PCR M Use disposable tips containing hydrophobic filters to minimize cross contamination Things to do before starting E Before opening the tubes with PCR components vortex and then centrifuge briefly to collect contents at the bottom of the tub
10. in the raw data profile M Peak morphology in the spectral profile shows no gross overlaps dips or other irregularities Separate and distinct peaks should be visible If the data for all capillaries meet the criteria above click Accept Results If any capillary data does not meet the criteria above click Reject Results and refer to the spectral calibration troubleshooting section of the Applied Biosystems 3500 3500xL Genetic Analyzers User Guide Sample preparation 1 Set up a mixture of formamide and DNA size standard according to Table 37 Table 37 Setup of formamide and DNA size standard mixture Component Volume per sample Hi Di Formamide 12 0 ul DNA Size Standard 550 BTO 0 5 ul 2 Aliquot 12 pl of the mixture to a tube for each sample to be analyzed Add 1 pl PCR product or allelic ladder diluted if necessary 4 Denature for 3 min at 95 C a 46 Investigator Argus Y 12 QS Handbook 02 2015 5 Snap freeze by placing the plate on ice for 3 min Alternatively the thermal cycler set to 4 C may be used to cool the plate 6 Load the samples on the tray Since injections take place simultaneously on all capillaries 8 or 24 samples must be pipetted onto the plate of multi capillary analyzers If fewer samples are analyzed the empty positions must be filled with 12 ul Hi Di Formamide To ensure a reliable allelic assignment on multi capillary analyzers inject one allelic ladder
11. minimal peak height should be three times as high as the background noise of the baseline t Only the setting for Peak Window Size is different to defaults from Applied Biosystems for HID analysis Note For information on the use of the recommended Template Files as analysis parameters refer to the appropriate Investigator Template Files User Guide Genotyper GeneMapper ID or GeneMapper ID X Investigator Argus Y 12 QS Handbook 02 2015 21 Protocol Electrophoresis Using the ABI PRISM 3100 Avant 3100 Genetic Analyzer For detailed instructions on instrument setup spectral calibration application of the ABI PRISM 3100 Data Collection Software version 1 01 or 1 1 and the GeneScan Software refer to the ABI PRISM 3100 Avant 3100 Genetic Analyzer User s Manual The system with 4 capillaries is the ABI PRISM 3100 Avant Genetic Analyzer and the system with 16 capillaries is the ABI PRISM 3100 Genetic Analyzer The virtual filter set G5 is used for combined application of the 5 fluorescent labels 6 FAM BTG BTY BTR and BTO This matrix standard is known as BT5 The materials required for electrophoresis are given in Table 13 Table 13 Materials required for electrophoresis Material Specifications Capillary 36 cm Capillary Array for ABI PRISM 3100 Avant 3100 Genetic Analyzer Polymer POP 4 Polymer for ABI PRISM 3100 Avant 3100 Genetic Analyzer Buffer 10x Genetic Analyzer Buffer with EDTA Spectral c
12. of the Applied Biosystems 3500 Series Data Collection Software version 1 0 and the GeneMapper ID X Software version 1 2 refer to the Applied Biosystems 3500 3500xL Genetic Analyzers User Guide The system with 8 capillaries is the Applied Biosystems 3500 Genetic Analyzer and the system with 24 capillaries is the Applied Biosystems 3500xL Genetic Analyzer The virtual filter set AnyDye is used for combined application of the 5 fluorescent labels 6 FAM BTG BTY BTR and BTO This matrix standard is known as BT5 The materials required for electrophoresis are given in Table 33 Table 33 Materials required for electrophoresis Material Specifications Capillary 36 cm Array for Applied Biosystems 3500 3500xL Genetic Analyzer Polymer POP 4 for Applied Biosystems 3500 3500xL Genetic Analyzer Buffer Anode Buffer Container ABC 3500 Series Cathode Buffer Container CBC 3500 Series Spectral calibration matrix generation Before conducting DNA fragment size analysis it is necessary to perform a spectral calibration with the 5 fluorescent labels 6 FAM BTG BTY BTR and BTO for each analyzer Table 34 The calibration procedure creates a matrix which is used to correct the overlapping of fluorescence emission spectra of the dyes IMPORTANT Spectral calibration must be performed for each new capillary array Spectral calibration is comprised of the following steps E Preparation of the instrument HE Preparation of dye
13. selected samples 9 Link the reaction plate on the autosampler tray to the created plate ID and start the run 10 Upon completion of the run view the data as Color Data in the Array View of the 3100 Data Collection Software or as Analyzed Sample Files under D AppliedBio 3100 DataExtractor ExtractRuns 28 Investigator Argus Y 12 QS Handbook 02 2015 Analysis parameters Table 20 lists the recommended analysis parameters Table 20 Recommended analysis parameters for the ABI PRISM 3100 Avant 3100 Genetic Analyzer Parameter Settings Analysis Range Data Processing Peak Detection Size Call Range Size Calling Method Split Peak Correction Start 2000 Stop 10 000 Baseline Checked Multi component Checked Smooth options Light Peak Amplitude Thresholds Bir Yet G R O Min Peak Half Width 2 pts Polynomial Degree 3 Peak Window Size 11 ptst Min 60 Max 550 Local Southern Method None The peak amplitude threshold cutoff value corresponds to the minimum peak height that will be detected by the GeneScan or GeneMapper ID Software Thresholds are usually 50 200 RFU and should be determined individually by the laboratory Recommendation The minimal peak height should be three times higher than the background noise of the baseline t Only the setting for Peak Window Size is different to defaults from Applied Biosystems for HID analysis Note For information on the use of t
14. set BT5 HE Preparation of the standard calibration plate E Plate assembly and loading the plate in the instrument Investigator Argus Y 12 QS Handbook 02 2015 41 E Performing a spectral calibration run M Checking the matrix Preparation of the instrument Before the spectral calibration process ensure that the spatial calibration has been performed This process is described in detail in the Applied Biosystems 3500 3500xL Genetic Analyzers User Guide Preparation of dye set BT5 Prior to the spectral calibration a dye set for the Matrix Standard BT5 must be set up Table 34 The fluorescent labels of BT5 Color Matrix standard Blue B 6 FAM Green G BTG Yellow Y BTY Red R BTR Orange O BTO 1 To create a new dye set go to Library and select Analyze followed by Dye Sets and click Create 2 Enter a Dye Set Name e g BT5 3 Select Matrix Standard as a chemistry and AnyDye Template as a dye set template 4 Disable Purple in the field Arrange Dyes Ensure that all other colors are enabled 5 Under Calibration Peak Order the colors need to be arranged as follows 5 blue 4 green 3 yellow 2 red and 1 orange 6 Do not alter the Parameter settings 7 Click Save to confirm the changes e KF f x j x 1 ixj x x t vgjzpzv z rzrz TU EEE 6 42 Investigator Argus
15. 1200 Deviating from the standard settings the injection time may range between 1 and 20 s depending on the type of sample If samples with very high signal intensities are recorded a shorter injection time may be selected For samples with low DNA content an injection time of up to 20 s may be necessary t The run time for Investigator Argus Y 12 QS was modified in order to be able to analyze fragments with lengths of up to 400 bp Starting the run 1 Place the prepared 96 well plate on the autosampler tray 2 Open the Protocol Manager of the Data Collection Software 3 Click New in the Instrument Protocol window to open the Protocol Editor dialog box and enter the information in Table 29 4 Click OK to exit the Protocol Editor Investigator Argus Y 12 QS Handbook 02 2015 37 Table 29 Settings in Instrument Protocol Protocol Editor Settings Name Run36_POP4_BT5_20min Type REGULAR Run Module 3kV_10s_400bp Dye Set Any5Dye See Table 28 Run Module 3kV_10s_ 400bp for the Applied Biosystems 3130 3130x Genetic Analyzer 5 Before each run it is necessary to create a plate definition In the Plate Manager of the Data Collection Software click New to open the New Plate Dialog box 6 Enter the information in Table 30 Table 30 GeneMapper Plate Editor I Protocol Editor Settings Name Application Plate type Owner Name Operator Name e g
16. 360 01380 0 400 0 425 0 450 0 475 0 500 0 525 0 550 0 Figure 2 Electropherogram of the DNA Size Standard 550 BTO fragments with lengths in bp As previously mentioned the Investigator Argus Y 12 QS Kit contains an internal PCR control Quality Sensor QS which provides information about the efficiency of the PCR and the presence of PCR inhibitors A 6 FAM labeled 74 bp fragment is amplified independently of the DNA The PCR control assay without DNA shows only the QS fragment Figure 3 and indicates successful polymerase chain reaction 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 12 QS_Sensor fsa 6 Blue 1500 1000 500 Sensor Figure 3 Electropherogram of the 6 FAM labeled PCR control QS fragment Fragment length in base pairs signal intensities in peak height Investigator Argus Y 12 QS Handbook 02 2015 53 Special features In general the electropherogram displays a single peak for each Y STR locus However locus DYS385 produces 2 peaks of different lengths or of the same length These 2 fragments originate from duplicated and inversed copies of one Y chromosomal locus The primers provided in the Investigator Argus Y 12 QS Kit simultaneously co amplify the 2 homologous loci For separate amplification see reference 5 On locus DYS385 alleles 14 3 15 3 16 3 17 3 and 19 3 represent the alleles 15 16 17 18 and 20 respectively with one thymidine deletion
17. February 2015 Investigator Argus Y 12 QS Handbook For multiplex amplification of 12 STR loci of the Y chromosome QIAGEN Sample amp Assay Technologies QIAGEN Sample and Assay Technologies QIAGEN is the leading provider of innovative sample and assay technologies enabling the isolation and detection of contents of any biological sample Our advanced high quality products and services ensure success from sample to result QIAGEN sets standards in Purification of DNA RNA and proteins Nucleic acid and protein assays microRNA research and RNAi Automation of sample and assay technologies Our mission is to enable you to achieve outstanding success and breakthroughs For more information visit www giagen com Contents Kit Contents 4 Storage 4 Intended Use 4 Safety Information 5 Introduction 6 Equipment and Reagents to Be Supplied by User 10 Protocols MH PCR Amplification 11 E Electrophoresis Using the ABI PRISM 310 Genetic Analyzer 14 E Electrophoresis Using the ABI PRISM 3100 Avant 3100 Genetic Analyzer 22 E Electrophoresis Using the Applied Biosystems 3130 3130x Genetic Analyzer 30 E Electrophoresis Using the Applied Biosystems 3500 3500xL Genetic Analyzer 41 E Analysis 53 Interpretation of Results 59 Troubleshooting Guide 61 References 64 Ordering Information 65 Investigator Argus Y 12 QS Handbook 02 2015 3 Kit Contents Investigator Argus Y 12 QS Kit 100 Catalog no 383615
18. Hi Di Formamide 204 ul Matrix Standard BT5 multi cap 17 ul 2 Load 12 ul of the mixture to 96 well plate e g position A1 H1 and A2 H2 3 Denature for 3 min at 95 C 4 Snap freeze by placing the plate on ice for 3 min Alternatively the thermal cycler set to 4 C may be used to cool the plate a Investigator Argus Y 12 QS Handbook 02 2015 31 Performing spectral calibration run 1 Place the 96 well plate on the autosampler tray 2 In the Protocol Manager of the Data Collection Software open the Instrument Protocol window 3 Click New to open the Protocol Editor dialog box 4 Complete the dialog box with information from Table 24 and click OK Table 24 Instrument protocol for spectral calibration Protocol Editor Settings Name User e g Spectral36 POP4 BT5 Type SPECTRAL Dye Set Any5Dye Polymer User e g POP4 Array Length User e g 36cm Chemistry Matrix Standard Run Module Default e g Spect36_ POP4_1 Depends on the type of polymer and length of capillary used 5 Click New in the Plate Manager of the Data Collection Software to open the New Plate Dialog box 6 Enter information from Table 25 and click OK A new table in the Plate Editor opens automatically Table 26 32 Investigator Argus Y 12 QS Handbook 02 2015 Table 25 Plate Editor for spectral calibration I New plate dialog Settings Name e g Spectral BT5 date Applicatio
19. Number of 25 ul reactions 100 Reaction Mix A 500 ul Primer Mix Argus Y 12 QS 250 ul Multi Taq2 DNA Polymerase 150 U Control DNA 9948 200 ul DNA size standard 550 BTO 50 ul Allelic ladder Argus Y 12 QS 25 ul Nuclease free water 2x I ml Quick Start Protocol 1 Storage All components of the Investigator Argus Y 12 QS Kit should be stored at 20 C Avoid repeated thawing and freezing Primer mix and allelic ladder must be stored protected from the light DNA samples and post PCR reagents allelic ladder and DNA size standard should be stored separately from the PCR reagents Under these conditions the components are stable until the expiration date indicated on the kit Intended Use The Investigator Argus Y 12 QS Kit is intended for molecular biology applications in forensic human identity and paternity testing This product is not intended for the diagnosis prevention or treatment of a disease All due care and attention should be exercised in the handling of the products We recommend all users of QIAGEN products to adhere to the NIH guidelines that have been developed for recombinant DNA experiments or to other applicable guidelines Fe sygwgw y EE e o j m jz lt kZzmzjpje 7 qmjpymprxmprzgpvjrv 4 Investigator Argus Y 12 QS Handbook 02 2015 Safety Information When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information please consult the
20. Standard BT5 mixture for 24 capillaries Component Volume Hi Di Formamide 225 ul Matrix Standard BT5 multi cap 25 ul 2 Load 10 ul of the mixture to a 96 well plate e g positions A1 H1 A2 H2 and A3 H3 3 Denature for 3 min at 95 C 4 Snap freeze by placing the plate on ice for 3 min Alternatively the thermal cycler set to 4 C may be used to cool the plate Plate assembly and loading the plate in the instrument The necessary steps are described in detail in the Applied Biosystems 3500 3500xL Genetic Analyzers User Guide Performing a spectral calibration run Once the multiwell plates containing the spectral calibration mixture are placed in the autosampler tray the spectral calibration process can be started 44 Investigator Argus Y 12 QS Handbook 02 2015 1 To access the Spectral Calibration screen select Maintenance on the Dashboard of the 3500 Series Data Collection software 2 The number of wells in the spectral calibration plate and their location in the instrument must be specified 3 Select Matrix Standard as a chemistry standard and BT5 for dye set 4 Optional Enable Allow Borrowing 5 Click Start Run Checking the matrix Click a capillary in the table in order to display the results for each capillary spectral data Quality value and Condition Number below the run results table HE The quality value Q value of each capillary must be greater t
21. able 45 Recommended Investigator Template Files for GeneMapper ID X File type File name Panels Argus Y12_Panels_x BinSets Argus Y12_Bins_x Stutter Argus _Y12_Stutter_x Size standard SST BTO_60 500bp Analysis Method Analysis HID_310 Analysis HID_3130 Analysis HID_310_50rfu Analysis HID_3130_50rfu Analysis HID_3500 Plot Settings Plots BT5_Adyes Table Settings 310 Data Analysis 31xx Data Analysis Panels and BinSets must always be used other template files are optional Investigator Argus Y 12 QS Handbook 02 2015 55 Controls The alleles listed in Table 46 represent the Control DNA 9948 included in the Investigator Argus Y 12 QS Kit and DNA from other commercially available standard cell lines Table 46 Allele assignment of the Investigator Argus Y 12 QS Kit Locus Control DNA CCR CCR ATCC 9948 9947 3657 K562 DYS19 14 13 z DYS385 11 14 16 19 gt DYS389 I 13 12 E DYS389 I 3 29 DYS390 24 _ 24 E DYS391 10 u 10 p DYS392 13 _ 11 E DYS393 13 13 gt DYS437 15 p 14 J DYS438 11 E 10 u DYS439 12 _ 12 E For further confirmation the table above displays the alleles of the reference DNA purchased from Coriell Cell Repositories CCR as well as 3 reference DNAs purchased from CCR and ATCC up to the standard of Szibor et al 9 Alleles Table 47 shows the alleles of the allelic ladder All analyses have been performed using an Applied Biosystems 3500 Genetic Analyzer with POP 4 polym
22. adder ESSplex SE Plus DNA size standard 550 BTO and nuclease free water Primer mix Fast Reaction Mix including HotStarTaq Plus DNA Polymerase Control DNA allelic ladder IDplex Plus DNA size standard 550 BTO and nuclease free water Primer mix reaction mix DNA Polymerase Control DNA allelic ladder DNA size standard and nuclease free water Primer mix reaction mix DNA Polymerase Control DNA allelic ladder DNA size standard and nuclease free water Primer mix reaction mix DNA Polymerase Control DNA allelic ladder DNA size standard and nuclease free water Larger kit sizes available please inquire Investigator Argus Y 12 QS Handbook 02 2015 Cat no 383615 383213 382415 381535 381545 381625 381215 380317 380327 65 Product Contents Cat no Investigator DIPplex Primer mix reaction mix DNA Polymerase 384013 Kit 25 Control DNA allelic ladder DNA size standard and nuclease free water Investigator Quantification Kits Investigator Primer mix IC FQ reaction mix FQ Control 387016 Quantiplex Kit 200 DNA Z1 dilution buffer Investigator Primer mix IC YQ reaction mix FQ Control 387116 Quantiplex HYres Kt DNA Z1 dilution buffer Investigator Human Identification PCR Kit Accessories DNA Size Standard DNA Size Standard 550 BTO for 100 386015 550 BTO 100 reactions Matrix Standard BT5 Matrix standard 6 FAM BTG BTY BTR 386113 single cap 5 x 25 and BTO Ma
23. adder cannot be used for the analysis Investigator Human Identification PCR Kits gt 4000 RFU for the ABI PRISM 310 Genetic Analyzer gt 5000 RFU for the ABI PRISM 3100 and Applied Biosystems 3130 3500 Genetic Analyzers 62 Investigator Argus Y 12 QS Handbook 02 2015 Comments and suggestions b One peak of the allelic The allelic ladder must be loaded onto the ladder is below the analysis instrument at a higher concentration peak detection value than samples to be analyzed 50 200 RFU of the Alternatively allelic ladder data can be analyzed ee method used with a lower peak detection value in Analysis and thus Is not Software identified c One peak of the allelic Compare the length of the fragments in bp of ladder is not identified the first allele in one color of the allelic ladder because it is outside with the corresponding value in the categories the expected size range Then compare it with the other alleles of the software in bp Point alleles are not Point alleles are i e alleles with at least 1 bp found difference to the next integer allele Check the settings of the analysis method Lower the Peak Window Size value to 11 points KK ts br Investigator Argus Y 12 QS Handbook 02 2015 63 References QIAGEN maintains a large up to date online database of scientific publications utilizing QIAGEN products Comprehensive search options allow you to find the articles you need either by a si
24. alibration matrix generation Proper spectral calibration is critical for evaluation of multicolor systems with the ABI PRISM 3100 Avant 3100 Genetic Analyzer and should be done before conducting fragment length analysis The calibration procedure creates a matrix which is used to correct the overlapping of fluorescence emission spectra of the dyes Spectral calibration comprises the following steps E Preparing the spectral calibration standards E Loading the standards to the 96 well reaction plate one sample per capillary M Entering the plate composition M Performing a spectral calibration run and checking the matrix 22 Investigator Argus Y 12 QS Handbook 02 2015 Preparing the spectral calibration standards Example for 4 capillaries ABI PRISM 3100 Avant Genetic Analyzer 1 Set up a mixture of formamide and Matrix Standard BT5 according to Table 14 Table 14 Setup of formamide and Matrix Standard BT5 mixture for 4 capillaries Component Volume Hi Di Formamide 60 ul Matrix Standard BT5 multi cap 5 pl 2 Load 12 ul of the mixture to 96 well plate e g position A1 D1 Denature for 3 min at 95 C 4 Snap freeze by placing the plate on ice for 3 min Alternatively the thermal cycler set to 4 C may be used to cool the plate ag Example for 16 capillaries ABI PRISM 3100 Genetic Analyzer 1 Set up a mixture of formamide and Matrix Standard BT5 according to Table 15 Table 15 Setup of formamide
25. alyzer The calibration procedure creates a matrix which is used to correct the overlapping of fluorescence emission spectra of the dyes Spectral calibration is comprised of the following steps E Preparing the spectral calibration standards E Loading the standards to the 96 well reaction plate one sample per capillary E Creating the instrument protocol for spectral calibration Protocol Manager E Defining the plate composition in the plate editor Plate Manager E Performing a spectral calibration run and checking the matrix 30 Investigator Argus Y 12 QS Handbook 02 2015 Preparing the spectral calibration standards Example for 4 capillaries Applied Biosystems 3130 Genetic Analyzer 1 Set up a mixture of formamide and Matrix Standard BT5 according to Table 22 Table 22 Setup of formamide and Matrix Standard BT5 mixture for 4 capillaries Component Volume Hi Di Formamide 60 ul Matrix Standard BT5 multi cap 5 ul 2 Load 12 ul of the mixture to 96 well plate e g positions Al D1 Denature for 3 min at 95 C 4 Snap freeze by placing the plate on ice for 3 min Alternatively the thermal cycler set to 4 C may be used to cool the plate ad Example for 16 capillaries Applied Biosystems 3130x Genetic Analyzer 1 Set up a mixture of formamide and Matrix Standard BT5 according to Table 23 Table 23 Setup of formamide and Matrix Standard BT5 mixture for 16 capillaries Component Volume
26. ample name in each well containing a sample or allelic ladder This will identify the well positions of each sample for the data collection and processing SSS EEE ne 50 Investigator Argus Y 12 QS Handbook 02 2015 5 Choose the correct Assay for the analysis If you followed the steps under Setting up the Run this would be Investigator Argus Y 12 QS from step 11 All named wells on the plate must have an assigned assay 6 Select the wells for which to specify an assay Check the box next to the assay name to assign it to the selected wells 7 Optional Repeat for file name conventions and results group 8 If not already done load the assembled plate to the instrument and close the instrument door to re initialize the instrument Then click Link Plate for Run In the next screen enter the desired Run Name and click Start Run oe Investigator Argus Y 12 QS Handbook 02 2015 51 Analysis parameters analysis method Table 43 lists the recommended analysis parameters in the worksheet Peak Detector Table 43 Recommended settings for the Applied Biosystems 3500 3500xL Parameter Settings Peak Detection Algorithm Advanced Ranges Analysis Partial Range Start Point 1000 Stop Point 20 000 Sizing All Sizes Smoothing and Baselining Smoothing Light Baseline Window 51 pts Size Calling Method Local Southern Method Peak Detection Peak Amplitude Thresholds B Xe G R O Min Peak Half Width 2
27. and Matrix Standard BT5 mixture for 16 capillaries Component Volume Hi Di Formamide 204 ul Matrix Standard BT5 multi cap 17 ul 2 Load 12 ul of the mixture to 96 well plate e g position Al H1 and A2 H2 3 Denature for 3 min at 95 C 4 Snap freeze by placing the plate on ice for 3 min Alternatively the thermal cycler set to 4 C may be used to cool the plate a Investigator Argus Y 12 QS Handbook 02 2015 23 Performing a spectral calibration run The parameter file for DyeSetG5 must be modified once to achieve successful calibration with the Data Collection Software version 1 0 1 or 1 1 Spectral parameter 1 To change settings in the parameter file go to the following path D AppliedBio Support Files Data Collection SupportFiles CalibrationData Spectral Calibration ParamFiles 2 Select MtxSTD Genescan_SetG5 to open the PAR file Change Condition Bounds Range to 1 0 20 0 4 If the calibration was unsuccessful also change Sensitivity to 0 1 and Quality to 0 8 5 Select Save As in the File menu and save the parameter file under a new name e g MtxStd Genescan_SetG5_BT5 par Note Always use this parameter file for spectral calibration runs using QIAGEN Matrix Standard BT5 Plate Editor for spectral calibration Place the 96 well plate on the autosampler tray Run the ABI PRISM 3100 Data Collection Software In Plate View click New to open the Plate Editor dial
28. andard SST BTO_60 550 from step 4 Sizecaller SizeCaller v1 1 0 9 Go to Analysis Settings followed by Peak Amplitude Threshold and disable Purple Ensure that all other colors are enabled Check the recommended analysis settings in Table 43 All other settings should remain as Default 10 Click Save to confirm the changes 11 To set up an Assay go to Library and select Manage followed by Assays and click Create a a Investigator Argus Y 12 QS Handbook 02 2015 49 12 To analyze Investigator Argus Y 12 QS fragments the parameters in Table 41 must be selected Table 41 Assay parameters Parameter Setting Assay Name e g Investigator Argus Y 12 QS Color Default Application Type HID Instrument Protocol e g Investigator Argus Y 12 QS from step 1 QC Protocols e g BTO_550 from step 4 13 Click Save to confirm the changes Starting the run 1 In the Dashboard click Create New Plate 2 Go to Define Plate Properties and select Plate Details Select or enter the parameters in Table 42 Table 42 Plate properties Property Setting Name e g Investigator Argus Y 12 QS Number of Wells 96 Plate Type HID Capillary Length 36 cm Polymer POP4 3 Click Assign Plate Contents to confirm the changes 4 Enter the designated s
29. appropriate safety data sheets SDSs These are available online in convenient and compact PDF format at www giagen com safety where you can find view and print the SDS for each QIAGEN kit and kit component os Investigator Argus Y 12 QS Handbook 02 2015 5 Introduction The Investigator Argus Y 12 QS Kit is a multiplex application for 12 Y chromosomal short tandem repeat STR loci The loci comply with the minimal haplotype MH standard as well as recommendations of the Scientific Working Group on DNA Analysis Methods SWGDAM with DYS437 integrated in addition into the multiplex system Figure 1 The primers are fluorescence labeled with one of the following dyes E 6 FAM QS DYS439 DYS437 DYS390 DYS385 M BTG DYS391 DYS389 I DYS19 DYS389 Il M BTY DYS393 DYS438 DYS392 The Investigator Argus Y 12 QS Kit was developed specifically for fast and reliable generation of male DNA profiles from mixtures of male and female DNA up to a ratio of 1 4000 so that separation of sperm from female cells or differential lysis is not required As a special feature the Investigator Argus Y 12 QS Kit contains an internal PCR control Quality Sensor QS which provides helpful information about the efficiency of the PCR and about the presence of PCR inhibitors Generation of DNA profiles using the Investigator Argus Y 12 QS Kit conforms to the guidelines of the International Society for Forensic Genetics 1 3 The optimal a
30. ard BTO 1 0 ul 2 Denature for 3 min at 95 C 3 Snap freeze by placing the plate on ice for 3 min Alternatively the thermal cycler set to 4 C may be used to cool the plate 4 Load the samples on the tray 5 Create a Sample Sheet and enter the sample designation Table 9 shows the injection list for matrix generation a Investigator Argus Y 12 QS Handbook 02 2015 15 Table 9 Injection list for matrix generation Parameter Settings Module File GS STR POP 4 1 ml G5 Matrix File None Size Standard None Injection Time s 5 Injection Voltage kV 15 Run Voltage kV 15 Run Temperature C 60 Run Time min 24 Always prepare matrix standards without DNA Size Standard BTO Analysis of the matrix samples Run the GeneScan Software Select New from the File menu and then select Project Open the folder of the current run and select Add Sample Files Select a matrix sample in the Sample File column Click Sample and then Raw Data Check the matrix samples for a flat baseline As shown in the figure next page there should be at least 5 peaks with peak heights of 1000 4000 RFU for each matrix sample Note The optimal range is 2000 4000 RFU a eS 16 Investigator Argus Y 12 QS Handbook 02 2015 Bre FOE V 3200 data points X ET
31. between the primer binding site and the repeat region 6 This deletion may serve as an additional distinctive feature for differentiation in forensic casework If more than one peak is obtained in the electropherogram for one or several markers this does not necessarily suggest mixed samples Duplications or triplications of STR markers also result in such an effect and have already been observed for DYS385 and DYS19 7 Rarely single systems can also fail because of Y chromosomal deletions as known in azoospermic patients which has already been described for DYS385 and DYS392 8 Analysis software Allele allocation should be carried out with suitable analysis software e g Genotyper GeneMapper ID or GeneMapper ID X Software in combination with the Investigator Template Files available as a download from www qiagen com see Table 44 and Table 45 The recommended Investigator Template File for Genotyper Software is Argus Y 12 QS 54 Investigator Argus Y 12 QS Handbook 02 2015 Table 44 Recommended Investigator Template Files for GeneMapper ID File type File name Panels Argus _Y12_Panels BinSets Argus Y12_Bins Size standard SST BTO_60 500bp Analysis Method Analysis HID_ 310 Analysis HID_3130 Analysis _HID_310_50rfu Analysis HID_3130_50rfu Plot Settings Plots BT5_Adyes Table Settings Table for 2 alleles Table for 10 alleles Panels and BinSets must always be used other template files are optional T
32. eck the allelic ladder 3 Check the positive control 4 Check the negative control 5 Analyze and interpret the sample data Pull up peaks Pull up peaks may occur if peak heights are outside the linear detection range see Troubleshooting Guide page 61 or if an incorrect matrix was applied They appear at positions of specific peaks in other color channels typically with lower signal intensities Peak heights should not exceed 3000 RFU in order to prevent pull up peaks Stutter peaks The occurrence of stutter peaks depends on the sequence of the repeat structure and the number of alleles n 4 peaks are caused by a loss of a repeat unit during amplification of tetranucleotide STR motifs caused by slippage effects of the Taq DNA Polymerase These peaks should be interpreted using the Investigator Template Files for Genotyper GeneMapper ID and GeneMapper ID X Software Template independent addition of nucleotides Because of its terminal transferase activity the Taq DNA Polymerase may cause incomplete adenylation at the 3 end of the amplified DNA fragments The artifact peak is one base shorter than expected 1 peaks All primers included in the Investigator Argus Y 12 QS Kit are designed to minimize these artifacts Artifact formation is further reduced by the final extension step of the PCR protocol at 68 C for 60 minutes Peak height of the artifact correlates with the amount of DNA Laboratories should define their o
33. er Figure 4 and Figure 5 page 58 Different analysis instruments DNA size standards or polymers may result in different fragment lengths In addition a visual alignment with the allelic ladder is recommended Scaling E Horizontal 75 405 bp with Quality Sensor 65 405 bp E Vertical Depending on signal intensity 56 Investigator Argus Y 12 QS Handbook 02 2015 Table 47 Allelic ladder fragments included in the Investigator Argus Y 12 QS Kit Locus Dye label Repeat numbers of allelic ladder DYS439 6 FAM 8 9 10 11 12 13 14 DYS437 6 FAM 12 13 14 15 16 18 DYS390 6 FAM 20 21 22 23 24 25 26 27 DYS385 6 FAM 9 10 11 12 1 132 14 15 15 2 oe 17 17 2 18 19 20 21 DYS391 BTG 8 9 10 11 12 13 DYS389 I BTG 11212 18 14715 DYS19 BTG 11 12 13 14 15 16 17 18 DYS389 I BTG Pia Pr 29 903 1 32 30 DYS393 BTY 11 12 13 14 15 16 DYS438 BTY 8 9410 1i 12 l3 1O DYS392 BTY 7 9 10 11 12 13 14 15 oT Investigator Argus Y 12 QS Handbook 02 2015 57 7500 DYS439 DY5437 DY5390 DY5385 12000 DYS391 DYS389 1 DYS19 DYS389 II 7000 DYS393 DY5438 DYS392 75 90 105 120 135 150 165 180 195 210 225 240 255 270 285 300 315 330 345 360 375 390 405 420 435 450 Figure 4 Electropherogram of the Investigator Argus Y 12 QS Kit using 500 pg Control DNA 9948 The Quality Sensor QS is shown at 74 bp
34. es Procedure 1 Thaw PCR components and template nucleic acid Mix thoroughly before use 2 Prepare a master mix according to Table 3 The master mix contains all of the components needed for PCR except the template sample DNA and nuclease free water Prepare a volume of master mix 10 greater than that required for the total number of PCR assays to be performed This should include positive and negative control reactions 3 Mix the master mix thoroughly and dispense appropriate volumes into PCR tubes or the wells of a PCR plate 4 Add template DNA and nuclease free water to the master mix to give a final sample volume of 25 ul 5 Prepare positive and negative controls Positive control Use 5 ul of the Control DNA Negative control Use nuclease free water instead of template DNA in the reaction Investigator Argus Y 12 QS Handbook 02 2015 11 Table 3 Reaction setup Component Volume per reaction Reaction Mix A 5 0 ul Primer Mix 2 5 ul Multi Taq2 DNA Polymerase 0 6 ul Nuclease free water added in step 4 Variable Template DNA added in step 4 Variable Total volume 25 ul Contains dNTP mix MgCl and bovine serum albumin BSA 6 Program the thermal cycler according to the manufacturer s instructions using the conditions outlined in Table 4 For stains containing small amounts of DNA lt 100 pg 25 ul reaction we recommend using the cycling conditions outlined in Table 5 Note I
35. f using the GeneAmp PCR System 9700 with an Aluminum Block use Std Mode or with a Silver 96 Well Block or Gold plated Silver 96 Well Block use Max Mode Do not use 9600 Emulation Mode Table 4 Standard cycling protocol recommended for all DNA samples Temperature Time Number of cycles 94 C 4 min 94 C 30s 63 C 120s 5 cycles VLG 75s 94 C 30 s 61 C 120s 25 cycles 72 C 75s 68 C 60 min 10 C _ Hot start to activate DNA polymerase 12 Investigator Argus Y 12 QS Handbook 02 2015 Table 5 Optional cycling protocol recommended for stains containing small amounts lt 100 pg of DNA Temperature Time Number of cycles 94 C 4 min 94 C 30 s 63 C 120 s 5 cycles 720 75s 94 C 30 s 61 C 120s 27 cycles 72 C 75s 68 C 60 min 10 C _ Hot start to activate DNA polymerase 7 After the cycling protocol is completed store samples at 20 C protected from the light or proceed directly with running the electrophoresis Investigator Argus Y 12 QS Handbook 02 2015 13 Protocol Electrophoresis Using the ABI PRISM 310 Genetic Analyzer For general instructions on instrument setup matrix generation and application of the GeneScan or GeneMapper ID Software refer to the ABI PRISM 310 Genetic Analyzer User s Manual Electrophoresis using the GeneScan Software is described below The virtual filter set G5 is used for combined application of
36. for each set of 24 samples BE 8 capillary instruments One allelic ladder per 3 injections M 24 capillary instruments One allelic ladder per 1 injection Room temperature may influence the performance of PCR products on multi capillary instruments so that shoulder peaks or split peaks occur especially at low temperatures Ensure ambient conditions are kept as recommended by the instrument manufacturer Setting up a run If you are using the Investigator Argus Y 12 QS Kit for the first time on an Applied Biosystems 3500 Genetic Analyzer you will first need to set up a number of protocols BE Instrument Protocol M Size Standard M QC Protocol E Assay All protocols can be set up via the Dashboard of the 3500 Series Data Collection software 1 To set up the Instrument Protocol go to Library and select Analyze followed by Instrument Protocols and click Create Note Modify the Run Module Default settings from HID36 POP4 as shown in Table 38 2 The parameters in Table 38 must be entered or selected Investigator Argus Y 12 QS Handbook 02 2015 47 Table 38 Instrument Protocol parameters for the Applied Biosystems 3500 Genetic Analyzer Protocol Name Oven Temperature C Run Voltage kV PreRun Voltage kV Injection Voltage kV Run Time s PreRun Time s Injection Time s Data Delay s Advanced Options Parameter Setting Application Type HID Capillary Length 36 cm
37. han 0 8 and the number range C value must be between 1 and 20 BE Check the matrix samples for a flat baseline As shown in the figure there should be 5 peaks with peak heights of about 1000 5000 RFU for each matrix sample Note The optimal range is 2000 4000 RFU v Intensity vs Scan Number Calibrated Data v 5 E nu A E El i 0 400 800 1200 1600 2000 2400 2800 3200 4000 3000 2000 1000 Intensity vs Scan Number Electropherogram of spectral calibration of the matrix standard BT5 on an Applied Biosystems 3500 Genetic Analyzer When a spectral calibration is successfully completed the Overall row displays green results If the Overall row displays red results refer to the Spectral calibration troubleshooting section of the Applied Biosystems 3500 3500xL Genetic Analyzers User Guide Investigator Argus Y 12 QS Handbook 02 2015 45 v Capillary Run Data Capillary j2 3 ja 5 6 7 8 Run L Run 2 Run 3 Passed E Failed Borrowed Not Calibrated Example of successful spectral calibration of the matrix standard BT5 for all capillaries with an Applied Biosystems 3500 Genetic Analyzer For each capillary select and display the spectral and raw data Check that the data meet the following criteria E The order of the peaks in the spectral profile from left to right read orange red yellow green blue M No extraneous peaks appear
38. he recommended Template Files as analysis parameters refer to the appropriate Investigator Template Files User Guide Genotyper GeneMapper ID or GeneMapper ID X Investigator Argus Y 12 QS Handbook 02 2015 29 Protocol Electrophoresis Using the Applied Biosystems 3130 3130x Genetic Analyzer For detailed instructions on instrument setup spectral calibration or application of the ABI PRISM Data Collection Software version 3 0 and the GeneMapper ID Software refer to the Applied Biosystems 3130 3130xl Genetic Analyzers Getting Started Guide The system with 4 capillaries is the Applied Biosystems 3130 Genetic Analyzer and the system with 16 capillaries is the Applied Biosystems 3130x Genetic Analyzer The virtual filter set Any5Dye is used for combined application of the 5 fluorescent labels 6 FAM BTG BTY BTR and BTO This matrix standard is known as BT5 The materials required for electrophoresis are given in Table 21 Table 21 Materials needed for electrophoresis Material Specifications Capillary 36 cm Capillary Array for Applied Biosystems 3130 3130x Genetic Analyzer Polymer POP 4 Polymer for Applied Biosystems 3130 31 30x Genetic Analyzer Buffer 10x Genetic Analyzer Buffer with EDTA Spectral calibration matrix generation Before conducting DNA fragment size analysis it is necessary to perform a spectral calibration with the 5 fluorescent labels 6 FAM BTG BTY BTR and BTO for each an
39. ime s Default Run Time min 20t Deviating from the standard settings the injection time may range between 1 and 20 s depending on the type of sample If samples with very high signal intensities are recorded a shorter injection time may be selected For samples with low DNA content an injection time of up to 20 s may be necessary t The run time for Investigator Argus Y 12 QS was modified in order to be able to analyze fragments with lengths of up to 400 bp Starting the run SCUT eS Place the prepared 96 well plate on the autosampler tray Run the ABI PRISM 3100 Data Collection Software In Plate View click New to open the Plate Editor dialog box Enter a name for the plate see Table 19 Select GeneScan as the application type Select 96 Well as plate type and click Finish Investigator Argus Y 12 QS Handbook 02 2015 27 Table 19 Settings in Plate Editor Parameter Settings Sample Name Enter name for the matrix samples Dyes O Color Info Ladder or sample Project Name e g 3100 Project Dye Set G5 Run Module 3kV_10s 400bp Analysis Module 1 DefaultAnalysis gsp See Table 18 Run Module 3kV_10s_ 400bp for the ABI PRSIM 3100 Avant 3100 Genetic Analyzer 7 Complete the table in the Plate Editor and click OK 8 Click the column header to highlight the entire column and select Fill Down from the Edit menu to apply the information to the
40. ix samples There should be five peaks with heights of 1000 5000 RFU in each matrix sample Note The optimal range is 2000 4000 RFU 4 Check the new matrix with the current samples There should be no pull up peaks between the dye panels B G Y R and O with the new matrix 5 If the calibration failed follow instructions in the section Spectral parameter on page 24 6 If all capillaries have passed the calibration the last calibration file for Dye Set G5 must be activated manually Click Set Active Spectral Calibration under the Tools menu 7 Rename the calibration file under Set Matrix Name e g BT5_Date of calibration Investigator Argus Y 12 QS Handbook 02 2015 25 Sample preparation 1 Set up a mixture of formamide and DNA size standard according to Table 17 Table 17 Setup of formamide and DNA size standard mixture Component Volume per sample Hi Di Formamide 12 ul DNA Size Standard 550 BTO 0 5 ul 2 Aliquot 12 pl of the mixture to a tube for each sample to be analyzed 3 Add 1 pl PCR product or allelic ladder diluted if necessary 4 Denature for 3 min at 95 C 5 Snap freeze by placing the plate on ice for 3 min Alternatively the thermal cycler set to 4 C may be used to cool the plate 6 Load the samples on the tray Since injections take place simultaneously on all capillaries 4 or 16 samples must be pipetted onto the plate of multi capillary analyzers If fewer sam
41. ize Standard e g SST BTO_60 500bp Injection Time s ow Injection Voltage kV 15 Run Voltage kV 15 Run Temperature C 60 Run Time min 26t Deviating from standard settings the injection time may range between 1 and 10 s depending on the type of sample If samples with very high signal intensities are recorded a shorter injection time may be selected For samples with low DNA content an injection time up to 10 s may be necessary t The run time for Investigator Argus Y 12 QS was modified in order to be able to analyze fragments with lengths of up to 400 bp 20 Investigator Argus Y 12 QS Handbook 02 2015 Analysis parameters Table 12 lists the recommended analysis parameters Table 12 Recommended analysis parameters for the ABI PRISM 310 Genetic Analyzer Parameter Settings Analysis Range Start 2000 Stop 10 000 Data Processing Baseline Checked Multi component Checked Smooth options Light Peak Detection Peak Amplitude Thresholds Bit ar G R O Min Peak Half Width 2 pts Polynomial Degree 3 Peak Window Size 11 ptst Size Call Range Min 60 Max 550 Size Calling Method Local Southern Method Split Peak Correction None The peak amplitude threshold cutoff value corresponds to the minimum peak height that will be detected by the GeneScan or GeneMapper ID Software Thresholds are usually 50 200 RFU and should be determined individually by the laboratory Recommendation The
42. mount of DNA under standard conditions is 0 2 0 5 ng Internal validations demonstrated reliable results with lt 0 1 ng DNA The Investigator Argus Y 12 QS Kit was validated using the GeneAmp PCR System 9700 in standard mode ABI PRISM 310 ABI PRISM 3100 and Applied Biosystems 3130 Genetic Analyzers Table 1 and Table 2 show the STR loci with their chromosomal mapping repeat motifs and alleles The most frequent alleles for European populations are included in the allelic ladder Allele ranges include all known alleles of YHRD www yhrd org as of 10 2009 and the current literature e S 6 Investigator Argus Y 12 QS Handbook 02 2015 Yp11 32 Yp11 31 Yp11 2 Yp11 1 Yql1 1 Yq11 21 Yq11 221 Yq11 222 Yq11 223 Yq11 23 Yql2 DYS393 2 8 Mb DYS19 9 2 Mb DYS391 DYS437 DYS439 DYS389 lt 4 Al DYS438 DYS390 DYS385 2 21 5 Mb Figure 1 The ideogram of the Y chromosome describes the physical location of the STR loci that can be analyzed using the Investigator Argus Y 12 QS Kit The positions of the STR loci are shown in Mb www ncbi nlm nih gov genome guide human as of 10 2009 Investigator Argus Y 12 QS Handbook 02 2015 Table 1 Locus specific information of the Investigator Argus Y 12 QS Kit GenBank Accession Repeat motif Ref Allele Locus number Of the reference allele allele range DYS19 AC017019 TAGA TAGG TAGA gt 13 9 19 DYS385 AC022486 GAAA 11 6 28
43. mple keyword search or by specifying the application research area title etc For a complete list of references visit the QIAGEN Reference Database online at www qiagen com RefDB search asp or contact QIAGEN Technical Services or your local distributor Cited references 1 Gill P et al 2001 DNA commission of the International Society of Forensic Genetics recommendations on forensic analysis using Y chromosome STRs Int J Legal Med 114 305 2 Gill P et al 2001 DNA Commission of the International Society of Forensic Genetics recommendations on forensic analysis using Y chromosome STRs Forensic Sci Int 124 5 3 Gusmao L et al 2005 DNA Commission of the International Society of Forensic Genetics ISFG an update of the recommendations on the use of Y STRs in forensic analysis Int J Legal Med 119 1 4 Kittler R Erler A Brauer S Stoneking M and Kayser M 2003 Apparent intrachromosomal exchange on the human Y chromosome explained by population history Eur J Hum Genet 11 304 5 F redi S Woller J Padar Z and Angyal M 1999 Y STR haplotyping in two Hungarian populations Int J Legal Med 113 38 6 Butler J M Decker A E Kline M C and Vallone P M 2005 Chromosomal duplications along the Y chromosome and their potential impact on Y STR interpretation J Forensic Sci 50 1 7 Stein B Willuweit S Nagy M Vogt P H and Roewer L 2005 AZF deletion
44. must be filled with 12 ul Hi Di Formamide To ensure a reliable allelic assignment on multi capillary analyzers several ladders should be run Room temperature may influence the performance of PCR products on multi capillary instruments so that shoulder peaks or split peaks occur especially at low temperatures Ensure ambient conditions are kept as recommended by the instrument manufacturer Setting up the Data Collection Software 1 Edit the Run Module once for the first run In the Module Manager of the Data Collection Software click New to open the Run Module Editor dialog box Note Modify the Run Module Default settings from HIDFragmentAnalysis36_POP4_1 to those shown in Table 28 2 Modify the Injection Voltage to 3 kV and the Injection Time to 10 s Table 28 3 Click Save As enter a name for the new Run Module e g 3kV_10s_400bp and confirm by clicking OK 4 Click Close to exit the Run Module Editor 36 Investigator Argus Y 12 QS Handbook 02 2015 Table 28 Run Module 3kV_10s_400bp for the Applied Biosystems 3130 3130x Genetic Analyzer Parameter Settings Oven Temperature C Default Poly Fill Volume Default Current Stability UA Default Pre Run Voltage kV Default Pre Run Time s Default Injection Voltage kV 3 0 Injection Time s 10 Voltage Number of Steps Default Voltage Step Interval Default Data Delay Time s Default Run Voltage kV Default Run Time s
45. n Spectral Calibration Plate Type 96 well Owner Name Operator Name Investigator Argus Y 12 QS Handbook 02 2015 33 Table 26 Plate Editor for spectral calibration Il Parameter Settings Sample Name Enter name for the matrix samples Priority e g 100 Instrument Protocol 1 Spectral36_POP4_BT5 setting described before 7 Click the column header to select the entire column and select Fill Down from the Edit menu to apply the information to the selected samples Confirm by clicking OK 8 Link the reaction plate on the autosampler tray with the created plate ID position A or B and start the run GA Instruments gt ga3130 gt 3130 1 gt Spectral Viewer 1000 Intensity vs Scan Number Capillary Data Mon May 25 09 21 23 CEST 2009 o R y G B Ant Dye Set AnySDye Active Calibration for Dye Set AnySDye Matrix used for Capillary 4 4 Mon May 25 09 21 23 CEST 2009 Condition 6 937256 Q Value 0 978649 List of Calibrations for Dye Set AnySDye Mon May 25 09 21 23 CEST 2009 Electropherogram of spectral calibration with matrix standard BT5 on an Applied Biosystems 3130 Genetic Analyzer 34 Investigator Argus Y 12 QS Handbook 02 2015 Checking the matrix 1 The quality value Q value of each capillary must be greater than 0 95 and the condition number range C value must be between 1 and 20 2 Check for a flat baseline in the matrix samples As shown in the figu
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47. og box Enter a name for the plate Select a Spectral Calibration Select 96 Well as plate type and click Finish oa ee Table 16 Plate Editor for spectral calibration Parameter Settings Sample Name Enter name for the matrix samples Dye Set G5 Spectral Run Module Default e g Spect36_POP4 Spectral Parameters MtxStd GeneScan_ SetG5 BT5 par parameters created before 7 Click the column header to select the entire column and select Fill Down from the Edit menu to apply the information to the selected samples Confirm by clicking OK 24 Investigator Argus Y 12 QS Handbook 02 2015 8 Link the reaction plate on the autosampler tray with the created plate ID and start the run 9 Upon completion of the run check in the Spectral Calibration Result dialog box that all capillaries have successfully passed calibration label A If individual capillaries are labeled X refer to the ABI PRISM 3100 Avant 3100 Genetic Analyzer User s Manual 10 Click OK to confirm completion of the run Checking the matrix 1 Select Display Spectral Calibration from the Tools menu then Dye Set and G5 to review the spectral calibration profile for each capillary 2 The quality value Q value must be greater than 0 95 and the condition number C value must be between 1 and 20 Both values must be within the pre determined range 3 Check for a flat baseline in the matr
48. ples are analyzed the empty positions must be filled with 12 ul Hi Di Formamide To ensure a reliable allelic assignment on multi capillary analyzers several ladders should be run Room temperature may influence the performance of PCR products on multi capillary instruments so that shoulder peaks or split peaks occur especially at low temperatures Ensure ambient conditions are kept as recommended by the instrument manufacturer Setting up the GeneScan Software 1 Edit the default run module in Dye Set G5 once for the first run Select Module Editor to open the dialog box 2 Select the appropriate Run Module as template from the GeneScan table see Table 18 3 Modify the Injection Voltage to 3 kV and the Injection Time to 10 s 4 Click Save As and enter the name of the new module e g 3kV_10s_400bp Confirm by clicking OK 5 Click Close to exit the Run Module Editor 26 Investigator Argus Y 12 QS Handbook 02 2015 Table 18 Run Module 3kV_10s_400bp for the ABI PRISM 3100 Avant 3100 Genetic Analyzer Parameter Setting Run Temperature C Default Cap Fill Volume Default Maximum Current A Default Current Tolerance A Default Run Current A Default Voltage Tolerance kV Default Pre Run Voltage kV Default Pre Run Time s Default Injection Voltage kV 3 0 Injection Time s 10 Run Voltage kV Default Number of Steps Default Voltage Step Interval Default Data Delay T
49. pts Polynomial Degree 3 Peak Window Size 11 ptst Slope Thresholds 0 0 The peak amplitude threshold cutoff value corresponds to the minimum peak height that will be detected by the GeneMapper ID X Software version 1 2 The thresholds are usually 50 200 RFU and should be determined individually by the laboratory Recommendation The minimal peak height should be three times higher than the background noise of the baseline t Only the setting for Peak Window Size is different to defaults from Applied Biosystems for HID analysis 52 Investigator Argus Y 12 QS Handbook 02 2015 Protocol Analysis For general instructions on automatic sample analysis refer to the appropriate User and or Workflow Guides for GeneScan GeneMapper ID or GeneMapper ID X Software Note The red panel should be faded out Finding the exact lengths of the amplified products depends on the device type the conditions of electrophoresis as well as the DNA size standard used Due to the complexity of some loci determining the size should be based on evenly distributed references The DNA Size Standard 550 BTO should be used with the following lengths of fragments Figure 2 60 80 90 100 120 140 160 180 200 220 240 250 260 280 300 320 340 360 380 400 425 450 475 500 525 and 550 bp 100 200 300 400 500 veu n n a a a n a 60 0 80 0 100 0 120 0140 0 160 0 180 0 200 0 220 0 240 0 260 0 280 0 300 01320 01340 0
50. re on the previous page there should be 5 peaks with peak heights of about 1000 5000 RFU in each matrix sample Note The optimal range is 2000 4000 RFU 3 Check the new matrix with the current samples There should be no pull up peaks between the dye panels B G Y R with the new matrix 4 If calibration failed use the optimized values of the Matrix Standard BT5 and repeat the calibration run 5 If all capillaries have passed the test the last calibration file for the Dye Set Any5Dye is activated automatically in the Spectral Viewer Rename the calibration file e g BT5_Date of calibration Sample preparation 1 Set up a mixture of formamide and DNA size standard according to Table 27 Table 27 Setup of formamide and DNA size standard mixture Component Volume per sample Hi Di Formamide 12 0 ul DNA Size Standard 550 BTO 0 5 ul 2 Aliquot 12 pl of the mixture to a tube for each sample to be analyzed 3 Add 1 pl PCR product or allelic ladder diluted if necessary 4 Denature for 3 min at 95 C 5 Snap freeze by placing the plate on ice for 3 min Alternatively the thermal cycler set to 4 C may be used to cool the plate 6 Load the samples on the tray Investigator Argus Y 12 QS Handbook 02 2015 35 Since injections take place simultaneously on all capillaries 4 or 16 samples must be pipetted onto the plate of multi capillary analyzers If fewer samples are analyzed the empty positions
51. recommended analysis parameters in the worksheet Peak Detector Table 32 Recommended settings for the Applied Biosystems 3130 3130x Genetic Analyzer Parameter Settings Peak Detection Algorithm Advanced Ranges Analysis Partial Range Start Point 2000 Stop Point 10 000 Sizing All Sizes Smoothing and Baselining Smoothing Light Baseline Window 51 pts Size Calling Method Local Southern Method Peak Detection Peak Amplitude Thresholds Bet ye G R O Min Peak Half Width 2 pts Polynomial Degree 3 Peak Window Size 11 ptst Slope Thresholds 0 0 The peak amplitude threshold cutoff value corresponds to the minimum peak height that will be detected by the GeneMapper ID Software The thresholds are usually 50 200 RFU and should be determined individually by the laboratory Recommendation The minimal peak height should be three times higher than the background noise of the baseline t Only the setting for Peak Window Size is different to defaults from Applied Biosystems for HID analysis Note For information on the use of the recommended Template Files as analysis parameters refer to the appropriate Investigator Template Files User Guide Genotyper GeneMapper ID or GeneMapper ID X 40 Investigator Argus Y 12 QS Handbook 02 2015 Protocol Electrophoresis Using the Applied Biosystems 3500 3500xL Genetic Analyzer For detailed instructions on instrument setup spectral calibration or application
52. s of the Y chromosome and failed amplification of commonly used Y STRs 21st Congress of the International Society for Forensic Genetics Ponta Delgada Portugal 8 Szibor R et al 2003 Cell line DNA typing in forensic genetics the necessity of reliable standards Forensic Sci Int 138 37 64 Investigator Argus Y 12 QS Handbook 02 2015 Ordering Information Product Investigator Argus Y 12 QS Kit 100 Related products Contents Primer mix reaction mix DNA Polymerase Control DNA allelic ladder DNA size standard and nuclease free water Investigator Human Identification PCR Kits Investigator Argus X 12 Kit 25 Investigator 24plex QS Kit 100 Investigator ESSplex Plus Kit 100 Investigator ESSplex SE Plus Kit 100 Investigator IDplex Plus Kit 100 Investigator HDplex Kit 100 Investigator Triplex AFS QS Kit 400 Investigator Triplex DSF Kit 400 Primer mix reaction mix DNA Polymerase Control DNA allelic ladder DNA size standard and nuclease free water Primer Mix Fast Reaction Mix 2 0 including Taq DNA Polymerase Control DNA allelic ladder 24plex DNA size standard 550 BTO and nuclease free water Primer mix Fast Reaction Mix including HotStarTagq Plus DNA Polymerase Control DNA allelic ladder ESSplex Plus DNA size standard 550 BTO and nuclease free water Primer mix Fast Reaction Mix including HotStarTaq Plus DNA Polymerase Control DNA allelic l
53. the 5 fluorescent labels 6 FAM BTG BTY BTR and BTO This matrix standard is known as BT5 The materials required for electrophoresis are given in Table 6 Table 6 Materials required for electrophoresis Material Specifications Capillary 47 cm 50 um green Polymer POP 4 for ABI PRISM 310 Genetic Analyzer Buffer 10x Genetic Analyzer Buffer with EDTA Matrix generation Before conducting DNA fragment size analysis with the filter set G5 a matrix with the 5 fluorescent labels 6 FAM BTG BTY BTR and BTO must be generated Table 7 Table 7 The fluorescent labels of BT5 Color Matrix standard Blue B 6 FAM Green G BTG Yellow Y BTY Red R BTR Orange O BTO 14 Investigator Argus Y 12 QS Handbook 02 2015 1 Five electrophoresis runs should be conducted one for each fluorescent label under the same conditions as for the samples and allelic ladders of the Investigator Argus Y 12 QS Kit in order to generate suitable matrix files Table 8 Table 8 Matrix setup for single capillary instruments ABI PRISM 310 Genetic Analyzer Matrix sample Component Volume Matrix sample 1 Hi Di Formamide 12 0 ul Matrix standard 6 FAM 1 0 ul Matrix sample 2 Hi Di Formamide 12 0 ul Matrix standard BTG 1 0 ul Matrix sample 3 Hi Di Formamide 12 0 ul Matrix standard BTY 1 0 ul Matrix sample 4 Hi Di Formamide 12 0 ul Matrix standard BTR 1 0 ul Matrix sample 5 Hi Di Formamide 12 0 ul Matrix stand
54. ties are Reduce the injection time in increments to a too high If the peak minimum of 1 s reduce the amount of the PCR heights of the samples amplification product for analysis or reduce the are outside the linear quantity of DNA for PCR detection range gt 4000 RFU gt 5000 RFU stutters split peaks and artifacts may be increased c Bubbles in the capillary Repeat electrophoresis to confirm results lead to pull up peaks in all color panels spikes that result in allele misnomer d Differences in the run For reliable allelic assignment on multi capillary performance among analyzers a number of allelic ladders should be the capillaries of a run multi capillary analyzer may result in allelic assignment shift e Use of 32 cycle PCR Too small amounts of DNA may result in allelic program for small dropouts and imbalances of the peaks amounts of DNA Furthermore unspecific amplification products may appear By increasing the number of cycles there is a risk of cross contamination due to impurities Injection file of the allelic ladder is not appropriate a An additional signal Use a different injection file of the allelic ladder can be identified as and check the data of the analyzed sizes from peak of the allelic the Size Standard in bp of the allelic ladder ladder because of Always use the DNA Size Standard 550 for dysfunctions during the electrophoresis If peaks of the allelic ladder are miscalled the l
55. trix Standard BT5 Matrix standard 6 FAM BTG BTY BTR 386123 multi cap 25 and BTO Matrix Standard BT5 Matrix standard 6 FAM BTG BTY BTR 386125 multi cap 50 and BTO Multi Taq2 DNA 100 Units Multi Taq2 DNA Polymerase 386315 Polymerase 100 DNA extraction and purification QlAamp DNA 50 QlAamp MinElute Columns Proteinase K 56504 Investigator Kit 50 Carrier RNA Buffers Collection Tubes 2 ml EZ1 DNA Reagent Cartridges Disposable Filter Tips 952034 Investigator Kit 48 Disposable Tip Holders Sample Tubes 2 ml Elution Tubes 1 5 ml Buffer G2 Proteinase K Carrier RNA QlAsymphony For 192 preps of 200 ul each from 931436 DNA Investigator Kit casework and reference samples includes 192 2 reagent cartridges and enzyme racks and accessories MinElute PCR 50 MinElute Spin Columns Buffers 28004 Purification Kit 50 Collection Tubes 2 ml Larger kit sizes available please inquire 66 Investigator Argus Y 12 QS Handbook 02 2015 For up to date licensing information and product specific disclaimers see the respective QIAGEN kit handbook or user manual QIAGEN kit handbooks and user manuals are available at www qiagen com or can be requested from QIAGEN Technical Services or your local distributor eo e Investigator Argus Y 12 QS Handbook 02 2015 67 Notes 68 Investigator Argus Y 12 QS Handbook 02 2015 Notes Investigator Argus Y 12 QS Handbook 02 2015 69 Notes 70 Investigator Argus
56. use and may not be OU Se BOE gto The purchaser and user of the kit agree not to take or permit anyone else IAGEN QIAGEN neither guarantees them nor warrants that they do Other than expressly stated licenses QIAGEN makes no warranty that this kit and or its use s do not infringe the rights of third parties reused refurbished or resold QIAGEN specifically disclaims any other licenses expressed or implied other than those expressly stated to take any steps that could lead to or facilitate any acts prohibited above QIAGEN may enforce the prohibitions of this Limited License Agreement in any Court and shall recover all its investigative and Court costs including attorney fees in any action to enforce this Limited License and or its components For updated license terms see www qiagen com 2010 2015 QIAGEN all rights reserved Agreement or any of its intellectual property rights relating to the kit www giagen com Australia techservice au qiagen com Austria techservice at giagen com Belgium techservice bnI qiagen com Brazil suportetecnico brasil qiagen com Canada techservice ca qiagen com China techservice cn qiagen com Denmark techservice nordic qiagen com Finland techservice nordic giagen com France techservice fr qiagen com Germany techservice de qiagen com Hong Kong techservice hk giagen com India techservice india qiagen com Ireland techservice uk qiage
57. wn limits for analysis of the peaks KK D aEz5w m ne_m xmpnk Investigator Argus Y 12 QS Handbook 02 2015 59 Quality Sensor to check PCR results The Investigator Argus Y 12 QS Kit contains an internal PCR check QS which provides information about PCR efficiency and presence of PCR inhibitors Figure 3 page 53 Complete QS failure indicates total inhibition of the PCR or errors in the assay If the sensor signal is amplified in presence of DNA either in the negative control or in the positive control the PCR is not inhibited Samples that contain sufficient DNA and no inhibiting substances result in a DNA profile according to the kit and the sensor fragment Reduced sensor peak heights in forensic samples indicate partial PCR inhibition If only the QS is amplified the sample contains very little only female or degraded DNA Artifacts Room temperature may influence the performance of PCR products on multi capillary instruments so that shoulder peaks or split peaks occur If shoulder or split peaks appear we recommend injecting the sample again Ze m v v EEE 60 Investigator Argus Y 12 QS Handbook 02 2015 Troubleshooting Guide This troubleshooting guide may be helpful in solving any problems that may arise For more information see also the Frequently Asked Questions page at our Technical Support Center w
58. ww qgiagen com FAQ FAQList aspx The scientists in QIAGEN Technical Services are always happy to answer any questions you may have about either the information and protocols in this handbook or sample and assay technologies for contact information see back cover or visit www giagen com Comments and suggestions Sample preparation Sample signal intensity Reduce the volume of the DNA Size Standard must be increased 550 BTO to peak heights of about 500 RFU Purify the PCR products before starting the analysis We recommend the MinElute PCR Purification Kit for rapid and effective purification see Ordering Information Matrix spectral calibration is not appropriate There are pull up This matrix cannot be used for the analysis peaks between the dye Repeat the matrix generation spectral panels B G Y R O calibration Be sure to carefully follow the correct with the current matrix protocol for the specific analysis instrument spectral calibration Many peaks are labeled as off ladder OL alleles in the samples a DNA Size Standard Click the orange Size Match Editor icon in the 550 BTO was not upper toolbar or the GeneMapper ID or defined or identified GeneMapper ID X Software Mark the orange correctly fragments of all samples Always use the DNA Size Standard 550 included in Investigator Human Identification PCR Kits Investigator Argus Y 12 QS Handbook 02 2015 61 Comments and suggestions b Signal intensi
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