USER GUIDE GlobalFiler™ Express PCR Amplification Kit for use with: 200 reaction kit (Part no. 4476609) 1000 reaction kit (Part no. 4474665) Publication Part Number 4477672 Rev.
For Forensic or Paternity Use Only. Information in this document is subject to change without notice. DISCLAIMER: LIFE TECHNOLOGIES CORPORATION AND/OR ITS AFFILIATE(S) DISCLAIM ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT, EXPRESSED OR IMPLIED, INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY, FITNESS FOR A PARTICULAR PURPOSE, OR NONINFRINGEMENT.
Contents About This Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Revision history . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Purpose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 ■ CHAPTER 1 Overview . . . . . . . . . . . . . . . . . . .
Contents Prepare the sample lysate (heat protocol) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 Prepare the reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 Store the sample lysate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 Perform PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Contents Peak Detector tab settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Peak Quality tab settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SQ & GQ tab settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Create a size standard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Contents 8 Globalfiler™ Express PCR Amplification Kit User Guide
About This Guide IMPORTANT! Before using this product, read and understand the information the “Safety” appendix in this document. Revision history Revision A Date October 2012 Description New document. Purpose The GlobalFiler™ Express PCR Amplification Kit User Guide provides information about the Life Technologies instruments, chemistries, and software associated with the GlobalFiler™ Express PCR Amplification Kit.
About This Guide Purpose 10 GlobalFiler™ Express PCR Amplification Kit User Guide
1 Overview ■ Product overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 ■ Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 ■ Instrument and software overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 ■ Materials and equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1 Chapter 1 Overview Product overview • 3500/3500xL Genetic Analyzer (requires 3500 Data Collection Software v1 or v2 and HID Updater 3500 Data Collection v2) • 3130/3130xl Genetic Analyzer (requires Data Collection Software v4 and 3130/ 3730 Data Collection v4 6-Dye Module v1) • 3730 Genetic Analyzer (requires Data Collection Software v4 and 3130/3730 Data Collection v4 6-Dye Module v1) • GeneMapper® ID-X Software v1.
Chapter 1 Overview Product overview 1 Table 1 GlobalFiler™ Express PCR Amplification Kit loci and alleles Chromosome location Locus designation Alleles included in Allelic Ladder Dye label 6-FAM™ DNA Control 007 15, 16 D3S1358 3p21.31 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 vWA 12p13.31 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 14, 16 D16S539 16q24.1 5, 8, 9, 10, 11, 12,13, 14, 15 9, 10 CSF1PO 5q33.
1 Chapter 1 Overview Product overview Locus designation Chromosome location Alleles included in Allelic Ladder Dye label SID™ DNA Control 007 D10S1248 10q26.3 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 D1S1656 1q42.2 9, 10, 11, 12, 13, 14, 14.3, 15, 15.3, 16, 16.3, 17, 17.3, 18.3, 19.3, 20.3 13, 16 D12S391 12p13.2 14, 15, 16, 17, 18, 19, 19.
Chapter 1 Overview Product overview Allelic ladder 1 Figure 1 shows the allelic ladder for the GlobalFiler™ Express Kit. See “Allelic ladder requirements” on page 30 for information on ensuring accurate genotyping.
1 Chapter 1 Overview Workflow BSD600 Semi-Automated Dried Sample Punch Instrument Obtain samples Prepare reactions Harris Manual Punch Swab substrates Prepare samples Treated or untreated paper substrates Prepare reactions Prepare samples Perform PCR Obtain samples Workflow Lyse in Prep-n-Go™ Buffer GlobalFiler™ Express PCR Amplification Kit Untreated paper only: Prep-n-Go™ Buffer Perform PCR GlobalFiler™ Express PCR Amplification Kit Veriti® 96-Well Thermal Cycler GeneAmp® PCR System 970
Chapter 1 Overview Instrument and software overview 1 Instrument and software overview This section provides information about the data collection and analysis software versions required to run the this kit on specific instruments. Data collection and analysis software Instrument and software compatibility The data collection software provides instructions to firmware running on the instrument and displays instrument status and raw data in real time.
1 Chapter 1 Overview Materials and equipment Figure 2 Spectral calibration of the six dyes used in the GlobalFiler™ Express Kit LIZ® TAZ™ NED™ VIC® 6-FAM™ SID™ Materials and equipment Kit contents and storage The GlobalFiler™ Express Kit contains sufficient quantities of the following reagents to perform 200 (Part no. 4476609) or 1000 (Part no. 4474665) amplifications at 15 µL/amplification: IMPORTANT! The fluorescent dyes attached to the primers are light-sensitive.
Chapter 1 Overview Materials and equipment Standards for samples 1 For the GlobalFiler™ Express Kit, the panel of standards needed for PCR amplification, PCR product sizing, and genotyping are: • DNA Control 007 – A positive control for evaluating the efficiency of the amplification step and STR genotyping using the GlobalFiler™ Express Allelic Ladder. • GeneScan™ 600 LIZ® Size Standard v2.0 – Used for obtaining sizing results.
1 20 Chapter 1 Overview Materials and equipment GlobalFiler™ Express PCR Amplification Kit User Guide
2 Perform PCR ■ Optimize PCR cycle number. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 ■ Treated paper substrates: prepare reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 ■ Untreated paper substrates: prepare reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 ■ Swab substrates: prepare reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 ■ Perform PCR . . . . . . . . . . .
2 Chapter 2 Perform PCR Treated paper substrates: prepare reactions Note: Our testing has not included blood samples on swab substrates. This sample type is not frequently used for the collection of database or casework reference samples. Note: To minimize the effect of instrument-to-instrument variation, use the same thermal cycler to amplify all three plates. To maximize result quality, prepare and amplify Plate 1 then repeat for Plates 2 and 3. Do not prepare all three plates simultaneously.
Chapter 2 Perform PCR Treated paper substrates: prepare reactions Prepare low-TE buffer 2 You can prepare the buffer as described below or order it from Teknova (Cat # T0223). To prepare low-TE buffer: 1. Mix together: • 10 mL of 1 M Tris-HCl, pH 8.0 • 0.2 mL of 0.5 M EDTA, pH 8.0 • 990 mL glass-distilled or deionized water Note: Adjust the volumes accordingly for specific needs. 2. Aliquot and autoclave the solutions. 3. Store at room temperature. Prepare the reactions 1.
2 Chapter 2 Perform PCR Treated paper substrates: prepare reactions 3. Prepare reagents. Thaw the Master Mix and the Primer Set, then vortex 3 seconds and centrifuge briefly before opening the tubes or bottles. IMPORTANT! Thawing is required only during first use of the kit. After first use, reagents are stored at 2 to 8 °C and, therefore, do not require subsequent thawing. Do not refreeze the reagents. 4. Pipet the required volumes of components into an appropriately sized polypropylene tube. 5.
Chapter 2 Perform PCR Untreated paper substrates: prepare reactions 2 Untreated paper substrates: prepare reactions Sample prep guidelines • If you are processing a blood sample collected on untreated paper substrate, make the punch as close as possible to the center of the sample to ensure optimum peak intensity. Increasing the size of the punch may cause inhibition during PCR amplification.
2 Chapter 2 Perform PCR Untreated paper substrates: prepare reactions 4. Calculate the volume of each component needed to prepare the reactions, using the table below. Reaction component Volume per reaction Master Mix 6.0 µL Primer Set 6.0 µL Note: Include additional reactions in your calculations to provide excess volume for the loss that occurs during reagent transfers.
Chapter 2 Perform PCR Swab substrates: prepare reactions 2 11. Amplify the samples in a Veriti® 96-well Thermal Cycler or PCR System 9700 with the silver or gold-plated silver 96-well block as described in “Perform PCR” on page 28. IMPORTANT! This kit is not validated for use with the GeneAmp PCR System 9700 with the aluminium 96-well block. Use of this thermal cycling platform may adversely affect performance of the this kit.
2 Chapter 2 Perform PCR Swab substrates: prepare reactions 5. Let the lysate stand at room temperature for at least 15 minutes to cool the lysate (for accurate pipetting). 6. Transfer the sample lysate out of the 1.5 mL tubes or sample plate into tubes or plates for storage, then discard the 1.5 mL tubes or deep-well plate containing the swab heads. Note: To minimize the risk of contamination, do not remove the swab heads from the sample lysate plate before transferring the lysate. 7.
Chapter 2 Perform PCR Swab substrates: prepare reactions 2 6. Dispense 12 µL of the reaction mix into each reaction well of a MicroAmp® Optical 96-Well Reaction Plate. The final volume in each well is 15 µL (reaction mix plus Prep-n-Go™ Buffer and sample lysate or positive control). 7. Add samples to the reaction plate: Well(s) Add the following to wells of a MicroAmp® Optical 96-Well Reaction Plate...
2 Chapter 2 Perform PCR Perform PCR These storage recommendations are preliminary pending the results of ongoing stability studies. The effects of multiple freeze-thaw cycles on the lysate have not been fully evaluated. Therefore, multiple freeze-thaw cycles are not recommended. Perform PCR IMPORTANT! The GlobalFiler™ Express Kit is validated for use with the Veriti® 96-well Thermal Cycler Part no. 4375786 NOT the Veriti® 96-Well Fast Thermal Cycler (Part no. 4375305).
3 Perform Electrophoresis Allelic ladder requirements. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .30 ■ Section 3.1 3500/3500xL instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 Set up the 3500/3500xL instruments for electrophoresis . . . . . . . . . . . . . . . . . . . . . 31 Prepare samples for electrophoresis on the 3500/3500xL instruments. . . . . . . . . . 33 ■ Section 3.2 3130/3130xl instruments . . . . . . . . . .
3 Chapter 3 Perform Electrophoresis Allelic ladder requirements Allelic ladder requirements To accurately genotype samples, you must run an allelic ladder sample along with the unknown samples.
Section 3.1 3500/3500xL instruments Set up the 3500/3500xL instruments for electrophoresis 3 Section 3.1 3500/3500xL instruments Set up the 3500/3500xL instruments for electrophoresis Appendix B, “Ordering Information” on page 69 lists the required materials not supplied with this kit. IMPORTANT! The fluorescent dyes attached to the primers are light-sensitive. Protect the primer set, amplified DNA, allelic ladder, and size standard from light when not in use. Keep freeze-thaw cycles to a minimum.
3 Chapter 3 Perform Electrophoresis Set up the 3500/3500xL instruments for electrophoresis Genetic Analyzer 3500† 3500xL† Operating System Windows® 7 Data Collection Software 3500 Data Collection Software v2 Additional software HID Updater 3500 DC v2.0 (Part no. 4480670) Plate templates, assays, run modules, and conditions (installed with the HID Updater) Same as 3500 Data Collection Software v1 listed above References 3500/3500xL Genetic Analyzer User Guide (Pub. no.
Section 3.1 3500/3500xL instruments Prepare samples for electrophoresis on the 3500/3500xL instruments 3 Prepare samples for electrophoresis on the 3500/3500xL instruments Prepare the samples for electrophoresis immediately before loading. 1. Calculate the volume of Hi-Di™ Formamide and GeneScan™ 600 LIZ® Size Reagent Volume per reaction GeneScan™ 600 LIZ® Size Standard v2.0 0.5 µL Hi-Di™ 9.
3 34 Chapter 3 Perform Electrophoresis Prepare samples for electrophoresis on the 3500/3500xL instruments GlobalFiler™ Express PCR Amplification Kit User Guide
Chapter 3 Perform Electrophoresis Set up the 3130/3130xl instruments for electrophoresis 3 Section 3.2 3130/3130xl instruments Set up the 3130/3130xl instruments for electrophoresis Reagents and parts IMPORTANT! The fluorescent dyes attached to the primers are light-sensitive. Protect the primer set, amplified DNA, allelic ladder, and size standard from light when not in use. Keep freeze-thaw cycles to a minimum.
3 Chapter 3 Perform Electrophoresis Set up the 3130/3130xl instruments for electrophoresis 4. Select Tools License Manager to display the Software Activation dialog box. 5. Request the software license file by performing steps 1a, 1b, and 1c as listed on the activation screen. The license file will be emailed to you. 6. Obtain the software license file from your email. 7. Make a copy of the software license file and keep in a safe location. 8.
Section 3.2 3130/3130xl instruments Prepare samples for electrophoresis on the 3130/3130xl instruments Perform spectral calibration 3 Perform a spectral calibration using the DS-36 Matrix Standard (J6 Dye Set) (Part no. 4425042). The following figure is an example of a passing 6-dye spectral calibration. 3730 Instruments Prepare samples for electrophoresis on the 3130/3130xl instruments Prepare the samples for electrophoresis immediately before loading. 1.
3 Chapter 3 Perform Electrophoresis Prepare samples for electrophoresis on the 3130/3130xl instruments Note: For blank wells, add 10 µL of Hi-Di™ Formamide. 5. Seal the reaction plate with appropriate septa, then briefly vortex and centrifuge the plate to ensure that the contents of each well are mixed and collected at the bottom. 6. Heat the reaction plate in a thermal cycler for 3 minutes at 95°C. 7. Immediately place the plate on ice for 3 minutes. 8. Prepare the plate assembly on the autosampler. 9.
Chapter 3 Perform Electrophoresis Set Up the 3730 instrument for electrophoresis 3 Section 3.3 3730 instrument Set Up the 3730 instrument for electrophoresis Reagents and parts Appendix B, “Ordering Information” on page 69 lists the required materials not supplied with the GlobalFiler™ Express Kit.
3 Chapter 3 Perform Electrophoresis Set Up the 3730 instrument for electrophoresis 4. Select Tools License Manager to display the Software Activation dialog box. 5. Request the software license file by performing steps 1a, 1b, and 1c as listed on the activation screen. The license file will be emailed to you. 6. Obtain the software license file from your email. 7. Make a copy of the software license file and keep in a safe location. 8.
Section 3.3 3730 instrument Prepare samples for electrophoresis on the 3730 instrument Perform spectral calibration 3 Perform a spectral calibration using the DS-36 Matrix Standard (J6 Dye Set) (Part no. 4425042). The following figure is an example of a passing 6-dye spectral calibration. 3130/3130xl Instruments Prepare samples for electrophoresis on the 3730 instrument Prepare the samples for electrophoresis immediately before loading. 1.
3 Chapter 3 Perform Electrophoresis Prepare samples for electrophoresis on the 3730 instrument 5. Seal the reaction plate with appropriate septa, then briefly vortex and centrifuge the plate to ensure that the contents of each well are mixed and collected at the bottom. 6. Heat the reaction plate in a thermal cycler for 3 minutes at 95°C. 7. Immediately place the plate on ice for 3 minutes. 8. Place the sample tray on the autosampler. 9. Start the electrophoresis run.
4 Analyze Data ■ Overview of GeneMapper® ID-X Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 ■ Set up GeneMapper® ID-X Software for data analysis . . . . . . . . . . . . . . . . . . . . . . 46 ■ Analyze and edit sample files with GeneMapper® ID-X Software . . . . . . . . . . . 58 ■ Examine and edit a project . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 ■ For more information. . . . . . . . . . . . . . . . . . . . . . . . .
4 Chapter 4 Analyze Data Overview of GeneMapper® ID-X Software • Allelic ladder samples must be labeled as “Allelic Ladder” in the Sample Type column in a project. Failure to apply this setting for ladder samples results in failed analysis. • Injections containing the allelic ladder must be analyzed with the same analysis method and parameter values that are used for samples to ensure proper allele calling. • Alleles that are not in the GlobalFiler™ Express Kit Allelic Ladders do exist.
Chapter 4 Analyze Data Overview of GeneMapper® ID-X Software 4 PQV has been enhanced to evaluate the quality of Y markers designated for analysis in the Panel Manager. Y markers designated in the Panel Manager are excluded from the Mixture Analysis and Statistical Calculations performed in the Mixture Analysis Tool. By default, the software designates two loci in the GlobalFiler™ Express panel, DYS391 and the Y Indel, as Y markers, and processes all other loci as autosomal.
4 Chapter 4 Analyze Data Set up GeneMapper® ID-X Software for data analysis Set up GeneMapper® ID-X Software for data analysis Panel, bin, and stutter file version The file names shown in this section may differ from the file names you see when you download or import files. If you need help determining the correct files to use, contact your local Life Technologies Human Identification representative, or go to www.lifetechnologies.com/support Software, Patches & Updates GeneMapper® ID-X Software.
Chapter 4 Analyze Data Set up GeneMapper® ID-X Software for data analysis 4 3. Check the version of files imported into the Panel Manager: a. Select Panel Manager in the navigation pane. 4. Check the version of files available for import into the Panel Manager: a. Select Panel Manager, then select File Import Panels to open the Import Panels dialog box. b. Navigate to, then open the Panels folder and check the version of panel, bin, and stutter files installed. 5.
4 Chapter 4 Analyze Data Set up GeneMapper® ID-X Software for data analysis 5. Select AmpFLSTR_Panels_v3X (or the version you installed), then click Import. Note: Importing this file creates a new folder in the navigation pane of the Panel Manager “AmpFLSTR_Panels_v3X”. This folder contains panels for multiple kits and associated markers. 6. Import AmpFLSTR_Bins_v3X.txt: a. Select the AmpFLSTR_Panels_v3X folder in the navigation pane. b.
Chapter 4 Analyze Data Set up GeneMapper® ID-X Software for data analysis 4 7. View the imported panels in the navigation pane: a. Double-click the AmpFLSTR_Panels_v3X folder. b. Double-click the GlobalFiler_Express_v1.1X folder to display the panel information in the right pane. GeneMapper® ID-X Software 8. Import AmpFLSTR_Stutter_v3X.txt: a. Select the AmpFLSTR_Panels_v3X folder in the navigation panel. b. Select File Import Marker Stutter to open the Import Marker Stutter dialog box. c.
4 Chapter 4 Analyze Data Set up GeneMapper® ID-X Software for data analysis d. Select AmpFLSTR_Stutter_v3X.txt, then click Import. Note: Importing this file associates the marker stutter ratio with the bin set in the AmpFLSTR_Panels_v3X folder and overwrites any existing stutter ratios associated with the panels and bins in that folder. 9. View the imported marker stutters in the navigation pane: a. Double-click the AmpFLSTR_Panels_v3X folder to display its list of kits in the right pane. b.
Chapter 4 Analyze Data Set up GeneMapper® ID-X Software for data analysis Create an analysis method 4 Use the following procedure to create an analysis method for the GlobalFiler™ Express Kit. IMPORTANT! Analysis methods are version-specific, so you must create an analysis method for each version of the software. For example, an analysis method created for GeneMapper® ID-X version 1.2 is not compatible with earlier versions of GeneMapper® ID-X Software or with GeneMapper® ID Software version 3.2.1.
4 Chapter 4 Analyze Data Set up GeneMapper® ID-X Software for data analysis General tab settings In the Name field, either type the name as shown or enter a name of your choosing. In the Security Group field, select the Security Group appropriate to your software configuration from the dropdown list. The Description and Instrument fields are optional.
Chapter 4 Analyze Data Set up GeneMapper® ID-X Software for data analysis 4 Allele tab settings GeneMapper® ID-X Software The following settings were used during developmental validation of the GlobalFiler™ Express Kit: • In the Bin Set field, select the AmpFLSTR_Bins_v3X bin set. • GeneMapper® ID-X Software v1.0.1 or higher allows you to specify 4 types of marker repeat motifs: tri, tetra, penta and hexa. You can enter parameter values for each type of repeat in the appropriate column.
4 Chapter 4 Analyze Data Set up GeneMapper® ID-X Software for data analysis Perform internal validation studies to determine settings IMPORTANT! Perform the appropriate internal validation studies to determine the appropriate peak amplitude thresholds for interpretation of GlobalFiler™ Express Kit data. Fields include: • Peak amplitude thresholds – The software uses these parameters to specify the minimum peak height, in order to limit the number of detected peaks.
Chapter 4 Analyze Data Set up GeneMapper® ID-X Software for data analysis 4 • Size calling method – The GlobalFiler™ Express Kit has been validated using the 3rd Order Least Squares sizing method. Select alternative sizing methods only after performing the appropriate internal validation studies. • Normalization – A Normalization checkbox is available on this tab in GeneMapper® ID-X Software for use in conjunction with data run on the 3500 Series Genetic Analyzers.
4 Chapter 4 Analyze Data Set up GeneMapper® ID-X Software for data analysis SQ & GQ tab settings IMPORTANT! The values shown are the software defaults and are the values we used during developmental validation. Perform appropriate internal validation studies to determine the appropriate values to use. Set the ACC GQ Weighting according to your laboratory's use of the ACC PQV. For example, set the ACC GQ Weighting to 0.
Chapter 4 Analyze Data Set up GeneMapper® ID-X Software for data analysis 4 GeneScan™ 600 LIZ® Size Standard v2.0 60, 80, 100, 114, 120, 140, 160, 180, 200, 214, 220, 240, 250, 260, 280, 300, 314, 320, 340, 360, 380, 400, 414, 420, 440, and 460 Use the following procedure to create the size standard definition file: 1. Select Tools GeneMapper® ID-X Manager to open the GeneMapper® ID-X Manager. 2. Select the Size Standards tab, then click New.
4 Chapter 4 Analyze Data Set up GeneMapper® ID-X Software for data analysis 3. Complete the Name field as shown below or with a name of your choosing. In the Security Group field, select the Security Group appropriate to your software configuration from the drop-down list. In the Size Standard Dye field, select Orange. In the Size Standard Table, enter the sizes specified on page 56.
Chapter 4 Analyze Data Analyze and edit sample files with GeneMapper® ID-X Software 4 Analyze and edit sample files with GeneMapper® ID-X Software 1. In the Project window, select Edit Add Samples to Project, then navigate to the disk or directory containing the sample files. 2. Apply analysis settings to the samples in the project. Settings Sample Type Select the sample type.
4 Chapter 4 Analyze Data Examine and edit a project 3. Click (Analyze), enter a name for the project (in the Save Project dialog box), then click OK to start analysis. • The status bar displays the progress of analysis as a completion bar extending to the right with the percentage indicated. • The table displays the row of the sample currently being analyzed in green (or red if analysis failed for the sample).
Chapter 4 Analyze Data For more information 4 • GeneMapper® ID-X Software Version 1.0 Reference Guide (Pub. no. 4375671) • GeneMapper® ID-X Software Version 1.1(Mixture Analysis) Getting Started Guide (Pub. no. 4396773) • GeneMapper® ID-X Software Version 1.2 Reference Guide (Pub. no. 4426481) • GeneMapper® ID-X Software Version 1.2 Quick Reference Guide (Pub. no.
4 62 Chapter 4 Analyze Data For more information GlobalFiler™ Express PCR Amplification Kit User Guide
5 Experiments and Results TO BE PROVIDED IN NEXT REVISION GlobalFiler™ Express PCR Amplification Kit User Guide 63
5 64 Chapter 5 Experiments and Results GlobalFiler™ Express PCR Amplification Kit User Guide
A Troubleshooting Follow the actions recommended in this appendix to troubleshoot problems that occur during analysis. Table 3 Troubleshooting Observation Possible causes Recommended actions Faint or no signal from both the DNA Control 007 and the DNA test samples at all loci Incorrect volume or absence of Master Mix or Primer Set Repeat amplification. No activation of DNA Polymerase Repeat amplification, making sure to hold reactions initially at 95°C for 1 minute.
A Appendix A Troubleshooting Observation More than expected number of alleles present at a locus Possible causes Recommended actions Presence of exogenous DNA Use appropriate techniques to avoid introducing foreign DNA during laboratory handling. Amplification of stutter product (–1 repeat unit position) See “Experiments and Results” on page 63. Incomplete 3´ A base addition (n-1 nt position) See “Experiments and Results” on page 63.
Appendix A Troubleshooting Observation Data collected on the 3730 instrument with POP-7™ polymer: the D2S441 and D1S1656 markers in some allelic ladder samples did not pass the base-pair spacing quality assessment Possible causes Data was analyzed using the Light setting for Smoothing GlobalFiler™ Express PCR Amplification Kit User Guide A Recommended actions Use the None setting for smoothing after performing appropriate validation studies.
A 68 Appendix A Troubleshooting GlobalFiler™ Express PCR Amplification Kit User Guide
B Ordering Information Equipment and materials not included Table 4 Equipment Equipment Source 3500/3500xL Genetic Analyzer Contact your local Life Technologies sales representative Veriti® 96-Well Thermal Cycler 4375786 GeneAmp® PCR System 9700 with the Silver 96-Well Block GeneAmp® PCR System 9700 with the Gold-plated Silver 96-Well Block Silver 96-Well Sample Block N8050001 4314878 N8050251 Gold-plated Silver 96-Well Sample Block 4314443 Tabletop centrifuge with 96-Well Plate Adapters (op
B Appendix B Ordering Information Equipment and materials not included Software Source 3130 Data Collection Software v4 4475105 3130xl Data Collection Software v4 4475126 3730/3730xl Data Collection Software v4 4475154 3130/3730 Data Collection Software v4 6-Dye Module v1 Contact your Life Technologies HID representative GeneMapper® ID-X Software v1.4 Full Installation 4479707 GeneMapper® ID-X Software v1.
Appendix B Ordering Information Equipment and materials not included Item† DS-36 Matrix Standard Kit (Dye Set J6) MicroAmp® Optical 96-Well Reaction Plate Hi-Di™ Formamide B Source 4425042 N8010560 4311320 For a complete list of parts and accessories for the 3130xl instrument, refer to Appendix A of the 3130/3130xl Genetic Analyzers Maintenance, Troubleshooting, and Reference Guide (Pub. no. 4352716).
B Appendix B Ordering Information Equipment and materials not included Item† Source Tube, 50-mL Falcon MLS Tube decapper, autoclavable MLS Deionized water, PCR grade MLS Vortex MLS † For the Safety Data Sheet (SDS) of any chemical not distributed by Life Technologies, contact the chemical manufacturer. Before handling any chemicals, refer to the SDS provided by the manufacturer, and observe all relevant precautions.
C Plate Layouts Example PCR plate layout The following layout is recommended for use with the sensitivity experiment on page 19. Create 3 identical plates for amplification at 3 different cycle numbers. Example electrophoresis plate layout The following layout is recommended for use with the sensitivity experiment on page 19.
C 74 Appendix C Plate Layouts Example electrophoresis plate layout GlobalFiler™ Express PCR Amplification Kit User Guide
D PCR Work Areas ■ Work area setup and lab design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75 ■ PCR setup work area . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75 ■ Amplified DNA work area . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76 Work area setup and lab design Many resources are available for the appropriate design of a PCR laboratory.
D Appendix D PCR Work Areas Amplified DNA work area • Tube decapper, autoclavable • Vortex Amplified DNA work area IMPORTANT! Place the thermal cyclers in the Amplified DNA Work Area. You can use the following systems: • Veriti® 96-Well Thermal Cycler (Part no.
E Safety WARNING! GENERAL SAFETY. Using this product in a manner not specified in the user documentation may result in personal injury or damage to the instrument or device. Ensure that anyone using this product has received instructions in general safety practices for laboratories and the safety information provided in this document.
E Appendix E Safety Chemical safety Chemical safety WARNING! GENERAL CHEMICAL HANDLING. To minimize hazards, ensure laboratory personnel read and practice the general safety guidelines for chemical usage, storage, and waste provided below, and consult the relevant SDS for specific precautions and instructions: • Read and understand the Safety Data Sheets (SDSs) provided by the chemical manufacturer before you store, handle, or work with any chemicals or hazardous materials.
Appendix E Safety Biological hazard safety E Biological hazard safety WARNING! Potential Biohazard. Depending on the samples used on this instrument, the surface may be considered a biohazard. Use appropriate decontamination methods when working with biohazards. WARNING! BIOHAZARD. Biological samples such as tissues, body fluids, infectious agents, and blood of humans and other animals have the potential to transmit infectious diseases.
E 80 Appendix E Safety Biological hazard safety GlobalFiler™ Express PCR Amplification Kit User Guide
Bibliography Akane, A., Matsubara, K., Nakamura, H., Takahashi, S., and Kimura, K. 1994. Identification of the heme compound copurified with deoxyribonucleic acid (DNA) from bloodstains, a major inhibitor of polymerase chain reaction (PCR) amplification. J. Forensic Sci. 39:362–372. Bonferroni, C.E. 1936. Teoria statistica delle classi e calcolo Belle probabilita. Publicazioni del R Istituto Superiore di Scienze Economiche e Commerciali di Firenze 8:3–62. Barber, M.D. and Parkin, B.H. 1996.
Bibliography Drabek, J., Chung, D.T., Butler, J.M., McCord, B.R. 2004. Concordance study between Miniplex assays and a commercial STR typing kit. J. Forensic Sci. 49:859–860. Edwards, A., Civitello, A., Hammond, H., and Caskey, C. 1991. DNA typing and genetic mapping with trimeric and tetrameric tandem repeats. Am. J. Hum. Genet. 49:746–756. Edwards, A., Hammond, H.A., Lin, J., Caskey, C.T., and Chakraborty, R. 1992.
Bibliography Levene, H. 1949. On a matching problem in genetics. Ann. Math. Stat. 20:91–94. Li, H. Schmidt, L., Wei, M-H., Hustad, T. Leman, M.I., Zbar, B. and Tory, K. 1993. Three tetranucleotide polymorphisms for loci:D3S1352; D3S1358; D3S1359. Hum. Mol. Genet. 2:1327. Magnuson, V.L., Ally, D.S., Nylund, S.J., Karanjawala, Z.E., Rayman, J.B., Knapp, J.I., Lowe, A.L., Ghosh, S., Collins, F.S. 1996.
Bibliography Sparkes, R., Kimpton, C., Gilbard, S., Carne, P., Andersen, J., Oldroyd, N., Thomas, D., Urquhart, A., and Gill, P. 1996b. The validation of a 7-locus multiplex STR test for use in forensic casework. (II), Artifacts, casework studies and success rates. Int. J. Legal Med. 109:195–204. Straub, R.E., Speer, M.C., Luo, Y., Rojas, K., Overhauser, J., Ott, J., and Gilliam, T.C. 1993. A microsatellite genetic linkage map of human chromosome 18. Genomics 15:48– 56. Suido, H., Nakamura, M., Mashimo, P.
Documentation and Support Related documentation Part number Document title GlobalFiler™ Express PCR Amplification Kit Quick Reference – PCR Amplification and CE 4480794 GlobalFiler™ Express PCR Amplification Kit Quick Reference – PCR Setup – Treated Paper Substrate 4480904 GlobalFiler™ Express PCR Amplification Kit Quick Reference – PCR Setup – Untreated Paper Substrate 4480795 GlobalFiler™ Express PCR Amplification Kit Quick Reference – PCR Setup – Swab Substrate 4477601 3100/3100-Avant Data C
Documentation and Support Obtain SDSs Part number Document title GeneMapper® ID-X Software Version 1.0 Reference Guide 4375671 GeneMapper® ID-X Software Version 1.1 (Mixture Analysis) Getting Started Guide 4396773 GeneMapper® ID-X Software Version 1.1 (Mixture Analysis) Quick Reference Guide 4402094 GeneMapper® ID-X Software Version 1.2 Reference Guide 4426481 GeneMapper® ID-X Software Version 1.
Documentation and Support Limited Product Warranty Limited Product Warranty Life Technologies Corporation and/or its affiliate(s) warrant their products as set forth in the Life Technologies' General Terms and Conditions of Sale found on Life Technologies’ website at www.lifetechnologies.com/termsandconditions. If you have any questions, please contact Life Technologies at www.lifetechnologies.com/support.
Documentation and Support Limited Product Warranty 88 GlobalFiler™ Express PCR Amplification Kit User Guide
Index Numerics D 3130/3130xl instruments 35 3500/3500xL instruments 31 3730 instrument allelic ladder base-pair spacing quality assessment failures 54 set up 31 sizing failures 57 6-dye license activation 35, 39 spectral calibration 32, 37, 41 data collection software 16 DNA negative control sample preparation 21, 23, 26 positive control sample preparation 21, 23, 26, 27 your sample preparation 21, 23 documentation, related 85 DS-33 Matrix Standard 32, 37, 41 Dye Set J6 for 6-dye samples 32, 37, 41 A
Index volume per reaction 33, 37, 41 GS 500 18 GS 600 18 operating systems 16, 31, 35, 39 P H HID Updater 32 Hi-Di formamide, volume per reaction 33, 37, 41 I Instrument and software compatibility 16 K kit contents 17 description 11 fluorescent dyes 16 instruments for use with 11 loci amplified 12 master mix 17 primers 12, 17 purpose 11 reagents 17 storage 17 thermal cyclers for use with 76 L license activation, 6-dye 35, 39 limited product warranty 87 LIZ size standard about 18 volume per reaction 3
Index T technical support 86 thermal cyclers for use with kit 76 programming 28 Veriti Fast model not supported 76 training, information on 86 treated paper 20 PCR 28 prepare reactions 20 sample preparation guidelines 20 U untreated paper 23 PCR 28 prepare reactions 23 sample preparation guidelines 23 W warranty 87 work area amplified DNA 73, 76 PCR setup 75 setup and lab design 75 workflow overview 15 GlobalFiler™ Express PCR Amplification Kit User Guide 91
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