ABI Prism® 7000 System and Applied Biosystems 7500 System
Contents
1. If the Then e Function Test fails The lamp may not be properly illuminated or see Checking for Proper Lamp Illumination on page 11 and or the fuses e Lamp Status Replacement may need to be replaced Test fails 7500 System only Note Refer to the ABI PRISM 7000 Sequence Detection System User Guide or the Applied Biosystems 7300 7500 Real Time PCR System Installation and Maintenance Guide for the proper fuse replacement procedure e Function Test passes Perform a visual inspection of the lamp to 7000 System only make sure the lamp remains illuminated while the instrument is powered on see Checking for Proper Lamp Illumination on page 11 e Lamp Status Replacement The lamp is functioning well Click Reset Test passes Lamp Timer in the Lamp 7500 System only Status Replacement dialog to reset the lamp usage counter in the SDS Software Instrument Calibration and Maintenance Procedures for Use with Quantifiler DNA Quantification Kits 9 ABI Prism 7000 Sequence Detection System and Applied Biosystems 7500 Real Time PCR System Checking for Proper Lamp Illumination 10 After you replace the halogen lamp perform a visual inspection to verify proper lamp illumination For both the 7000 System and the 7500 System the lamp remains illuminated while the instrument is powered on To check lamp illumination on the 7000 System 1 Lift open the lamp access door loca2. e Roll the capping tool across all strips of caps on the short edge then the long edge of the tray e Roll the capping tool around all outer rows of strips of caps If you are using the handle capping tool PN 4330015 Slip your fingers through the handle with the holes in the tool facing down e Place the holes in the tool over the first eight caps in a row e Rock the tool back and forth a few times to seal the caps e Repeat for remaining caps in the row then for all remaining rows Instrument Calibration and Maintenance Procedures for Use with Quantifiler DNA Quantification Kits 17 ABI Prisu 7000 Sequence Detection System and Applied Biosystems 7500 Real Time PCR System Notes 18 User Bulletin Improving Assay Performance Instrument Calibration and Maintenance Procedures for Use with Quantifiler DNA Quantification Kits 19 Copyright 2007 Applied Biosystems All rights reserved For Research Use Only Not for use in diagnostic procedures Information in this document is subject to change without notice Applied Biosystems assumes no responsibility for any errors that may appear in this document This document is believed to be complete and accurate at the time of publication NOTICE TO PURCHASER PLEASE REFER TO THE ABI Prism 7000 Sequence Detection System s and the Applied Biosystems 7500 Real Time PCR System s USER S DOCUMENTATION FOR LIMITED LABEL LICENSE OR DISCLAIMER INFORMAT
3. instrument verification User Bulletin Instrument Calibration Procedures Instrument Calibration Procedures Recommended The 7000 System and the 7500 Systems need to be properly and Calibration routinely calibrated to ensure optimum instrument and assay Schedule performance Applied Biosystems recommends that you perform specific instrument calibration tasks according to the schedule outlined in the table below Note Refer to the ABI PRISM 7000 Sequence Detection System User Guide or the Applied Biosystems 7300 7500 Real Time PCR System Installation and Maintenance Guide for more detailed calibration procedures Schedule Calibration Task Monthly e Perform a background calibration Every 6 Months e Perform an ROI calibration e Perform a background calibration e Perform an optical calibration 7500 System only e Perform a Pure Dye Spectra calibration IMPORTANT You must also perform the following calibrations in this order after successfully replacing the halogen lamp ROI gt background gt optical 7500 System only gt pure dye gt instrument verification Instrument Calibration and Maintenance Procedures for Use with Quantifilef DNA Quantification Kits 11 ABI Prism 7000 Sequence Detection System and Applied Biosystems 7500 Real Time PCR System 12 Verifying Instrument Performance About the TaqMan RNase P Instrument Verification Plate The TaqMan RNase P Instrument Verificat
4. Plates e Tube Strips e Individual Tubes Use the Precision Plate Holder on the 7000 System with the recessed and notched side facing up to ensure proper placement and release of individual reaction tubes tube strips and 96 well reaction plates from the thermal cycler sample block Instrument Calibration and Maintenance Procedures for Use with Quantifiler DNA Quantification Kits 15 ABI Prisu 7000 Sequence Detection System and Applied Biosystems 7500 Real Time PCR System 16 Preventing Sample Loss Note If you do not have the proper Precision Plate Holder for your instrument or run configuration please contact your local Service Engineer HID Technical Support representative or HID Application Specialist see How to Obtain HID Support on page 2 for contact information A tight reaction plate or tube seal provides consistent results and prevents evaporation from and or condensation within the sample wells sample loss as well as well to well contamination Follow the steps below to ensure a proper seal To seal optical plates l Remove the middle portion of the protective backing of an optical adhesive cover and hold the cover by the perforated end tabs taking care not to touch the underside of the cover Place the optical adhesive cover on the plate then rub the flat edge of the applicator back and forth along the long edge length of the plate IMPORTANT Apply significant d
5. Checking the Lamp Current 7500 System only To check whether the halogen lamp on the 7500 System has enough electrical current select Instrument gt Lamp Status Replacement from the SDS Software menu bar The Lamp Status Replacement dialog displays the lamp usage hours current percent and one of three lamp status messages as shown in the table on the next page If the lamp status is Then Good The lamp is functioning well There is no need to replace the bulb at this time Change The lamp bulb usage is either above 2000 hours or it may be Soon dim but functional Applied Biosystems recommends that you replace the lamp soon Failed The lamp bulb must be replaced Refer to the Applied Biosystems 7300 7500 Real Time PCR System Installation and Maintenance Guide for the proper procedure Note The SDS Software for the 7500 System also displays warning messages before or during a run that indicate low lamp current Refer to the Applied Biosystems 7300 7500 Real Time PCR System Installation and Maintenance Guide for more information regarding these messages 8 User Bulletin Instrument Maintenance Procedures Checking the After you replace the halogen lamp run the Function Test and or the Performance of Lamp Status Replacement function 7500 System only again to the Replacement ensure proper lamp performance Follow the appropriate actions for Lamp the performance results as shown in the table below
6. ION TRADEMARKS Applera Applied Biosystems AB Design ABI PRISM AmpF STR and Quantifiler are registered trademarks and MicroAmp is a trademark of Applera Corporation or its subsidiaries in the US and or certain other countries TaqMan is a registered trademark of Roche Molecular Systems Inc All other trademarks are the sole property of their respective owners Worldwide Sales and Support Applied Biosystems vast distribution and service network composed of highly trained support and applications personnel reaches 150 countries on six continents For sales office locations and technical support please call our local office or refer to our Web site at www appliedbiosystems com Applera is committed to providing the world s leading technology and information for life scientists Applera Corporation consists of the Applied Biosystems and Celera businesses Headquarters 850 Lincoln Centre Drive Foster City CA 94404 USA Phone 1 650 638 5800 Toll Free In North America 1 800 345 5224 Fax 1 650 638 5884 04 2007 gt Applied KS Bibsystems Part Number 4374416 Rev B Stock Number 128UB01 02
7. PN 4323032 MicroAmp Cap Installing Tool e Roller e Handle Applied Biosystems e PN N8010438 e PN 4330015 Precision Plate Holder for the 7000 System Applied Biosystems PN 4350105 Powder free gloves MLS Lint free cotton swabs MLS Safety glasses MLS Instrument Calibration and Maintenance Procedures for Use with Quantifiler DNA Quantification Kits 3 ABI Prism 7000 Sequence Detection System and Applied Biosystems 7500 Real Time PCR System Instrument Maintenance Procedures Recommended Maintenance Schedule The 7000 System the 7500 System and the computer that runs the instrument need to be properly and routinely maintained to ensure optimum performance Applied Biosystems recommends that you perform specific instrument maintenance tasks according to the schedule outlined in the table below Note Refer to the ABI PRISM 7000 Sequence Detection System User Guide or the Applied Biosystems 7300 7500 Real Time PCR System Installation and Maintenance Guide for more detailed maintenance procedures Schedule Maintenance Task Weekly e Archive and back up your computer files e Restart the computer e Wipe instrument surfaces with a lint free cloth Monthly e Defragment the computer hard drive e Perform the System Hardware Test Function Test e Check sample wells for contamination Note You can identify contaminated sample wells by either performing a backgr
8. User Bulletin ABI PRISM 7000 Sequence Detection System and Applied Biosystems 7500 Real Time PCR System SUBJECT In This User Bulletin Overview Purpose Safety April 2007 Instrument Calibration and Maintenance Procedures for Use with Quantifiler DNA Quantification Kits COO neces ce Ved EA A dane EE ed ew alee dade 1 Instrument Maintenance Procedures 0 000s eee 5 Instrument Calibration Procedures 00000 cece 12 Improving Assay Performance 2 6 c 5sceeeegne eee vdeeee ines 14 Improving Assay Performance 000 eee eee eee 19 This bulletin provides Human Identification laboratories running Quantifiler DNA Quantification Kits on the ABI PRISM 7000 Sequence Detection System and the Applied Biosystems 7500 Real Time PCR System with a comprehensive plan for instrument calibration and maintenance and recommended laboratory practices to assist in improving Quantifiler assay performance and preventing contamination The information in this bulletin supplements the specific documents available for use with Quantifiler Kits the 7000 System running Sequence Detection System SDS Software v1 0 and v1 2 3 and the 7500 System running SDS Software v1 2 3 see Related Documentation on page 2 For safety guidelines refer to the Safety section in the ABI Prism 7000 Sequence Detection System User Guide and Quantifiler Kits User s Manual and the Safety and EMC Complian
9. alogen lamp if the instrument remains unused for 4 hours e Allow at least 15 minutes for the lamp to cool before removing it from the instrument ANITE PHYSICAL INJURY HAZARD The lamp can become very hot while in use Allow sufficient time for the lamp to cool and put on protective gloves before handling it Wear powder free gloves when handling the lamp When to Replace You should replace the halogen lamp the ne gen e After approximately 2000 hours of use on the 7500 System and amp 1000 hours of use on the 7000 System e Ifunstable signals are generated in all filters component dyes during a run See How to View Component Dye Signals on page 7 for instructions on how to use the SDS Software to view these signals Ifthe instrument fails to produce any raw fluorescence data Ifthe standard curve consistently fails to meet specifications when the quality of the standards themselves is ruled out as a factor Note Refer to the Quantifiler Kits User s Manual for more information on examining the standard curve results If one or more of the above conditions are true for your system check the lamp status to determine whether the lamp needs to be replaced See How to Check Lamp Status on page 8 for the lamp status monitoring procedure appropriate to your system Instrument Calibration and Maintenance Procedures for Use with Quantifiler DNA Quantification Kits 5 ABI Prism 7000 Sequence Detection S
10. ce section in the Applied Biosystems 7300 7500 Real Time PCR System Installation and Maintenance Guide and the SDS Software Online Help Applied KS Biosystems ABI Prism 7000 Sequence Detection System and Applied Biosystems 7500 Real Time PCR System Related Documentation How to Obtain HID Support Required Materials For more detailed instructions on performing the calibrations and routine maintenance discussed throughout this user bulletin refer to the documents listed below Product Documentation 7000 System with SDS Software v1 0 and v1 2 3 ABI Prism 7000 Sequence Detection System User Guide PN 4330228 7500 System with SDS Software v1 2 3 Applied Biosystems 7300 7500 Real Time PCR System Installation and Maintenance Guide PN 4347828 e SDS Software Online Help Quantifiler Kits User s Manual PN 4344790 Quantifiler DNA Quantification Kits PN 43843895 PN 4343906 The online help is embedded in the SDS Software and does not require an Internet connection To access the online help select Help Menu gt Contents and Index from the SDS Software menu bar For Human Identification HID specific support while running Quantifiler Kit assays on the 7000 System and the 7500 System please use the contact information provided below By phone 1 888 821 4HID 4443 select Option 1 By e mail HIDTechSupport appliedbiosystems com The following table lists t
11. es have reported the detection of Cy values lt 40 for extraction blank and negative control samples when performing the Quantifiler Kit assays Applied Biosystems has confirmed that detection of such a low quantity of DNA may vary from amplification to amplification based on stochastic effects Such levels may be considered background and will not produce detectable product when the AmpF4STR Kits are used The Quantifiler Kit reagents undergo rigorous quality control to ensure that the reagents are free of contaminants However due to the extreme sensitivity of the test background DNA from the environment may be detected on rare occasions Each laboratory should take the usual precautions to minimize contamination in their own facility and establish a Cy value above which a positive result represents only background DNA In this way samples that can be successfully amplified using the AmpF STR Kits can be distinguished from those samples lacking sufficient target to generate a meaningful result Establishing the limits of the test is common practice in forensic laboratories when dealing with STR amplification results and Applied Biosystems recommends applying a similar approach when dealing with results generated by the Quantifiler Kit About False It is important to note the following when encountering false positive Positive Results results positive amplification of negative controls The quantities obtained are
12. he user supplied materials required to perform the instrument maintenance and calibration procedures discussed in this bulletin Unless otherwise noted several of the items are available from major laboratory suppliers MLS Material Source Hardware Equipment Applied Biosystems e 7000 System Tungsten halogen lamp 21V 150W PN 4347754 e 7500 System Halogen lamp 12V 75W e PN 4345287 Electronics fuses 12 5A 250V 5 x 20MM MLS User Bulletin Overview Material Source Phillips and flathead screwdrivers MLS Reagents TaqMan RNase P Instrument Verification Plate e 7000 System e 7500 System Applied Biosystems PN 4310982 PN 4350584 Spectral Calibration Kit e 7000 System 8 preloaded and sealed calibration plates 1 Background 7 Spectral e 7500 System 9 preloaded and sealed calibration plates 1 Background 1 Region of Interest ROI 7 Spectral Applied Biosystems e PN 4328895 PN 4349180 10 bleach solution MLS 95 ethanol solution MLS Consumables Optical 96 Well Reaction Plates 500 plates Applied Biosystems PN 4316813 Optical Adhesive Covers Starter Kit 20 covers 1 compression pad 1 applicator Applied Biosystems PN 4313663 Optical Tubes 8 tubes strip 125 strips Applied Biosystems PN 4316567 Optical Caps 8 flat caps strip 300 strips Applied Biosystems
13. ion Plate is used to verify that the instrument meets performance specifications A Service Engineer performs this test at the time of instrument installation after all calibrations have been performed Running the TaqMan RNase P Verification Plate optional Laboratories may choose to implement this procedure as part of their routine maintenance You can also use this test as a troubleshooting tool to identify whether a problem your laboratory is encountering is due to a chemistry or an instrument issue To run this test the TaqMan RNase P Verification Plate must be purchased from Applied Biosystems The instructions for running the plate and analyzing the data can be found on the product insert that comes inside the box containing the plate Please refer to the table below for the appropriate TaqMan RNase P Verification Plate Kit part numbers for your instrument Instrument Kit PN Description 7000 System 4310982 Plate kit includes one MicroAmp Optical 96 Well Reaction Plate pre loaded and sealed with complete TaqMan reagents to 7500 System 4350584 detect and quantitate genomic copies of the human RNase P gene User Bulletin Improving Assay Performance Improving Assay Performance About Assay Real Time PCR assays are extremely sensitive and detection of Cy Sensitivity values greater than 35 may indicate the presence of exceedingly low quantities of DNA lt 3 copies Some user laboratori
14. o Check Checking Lamp Function Lamp Status To check for proper lamp function select Instrument gt Function Test from the SDS Software menu bar The Function Test dialog displays a lamp function status message as shown in the following table If the lamp status Then message is Pass e For the 7500 System The lamp is functioning well There is no need to replace the bulb at this time e For the 7000 System Perform a visual inspection of the lamp to make sure the lamp remains illuminated while the instrument is powered on see Checking for Proper Lamp Illumination on page 11 Fail e For the 7500 System only Check the lamp current using the Lamp Status Replacement function See Checking the Lamp Current 7500 System only on page 9 for more information e For the 7500 System or the 7000 System you can choose to either Perform a visual inspection to verify proper lamp illumination See Checking for Proper Lamp Illumination on page 11 for more information or Replace the halogen lamp Refer to the ABI PRISM 7000 Sequence Detection System User Guide or the Applied Biosystems 7300 7500 Real Time PCR System Installation and Maintenance Guide for the proper procedure Instrument Calibration and Maintenance Procedures for Use with Quantifilerf DNA Quantification Kits 7 ABI Prism 7000 Sequence Detection System and Applied Biosystems 7500 Real Time PCR System
15. ositive displacement pipettes or aerosol resistant pipette tips Never bring amplified PCR products into the PCR setup area Open and close all sample tubes and reaction plates carefully Try not to splash or spray PCR samples When pipetting from a kit component tube hold the cap of the tube in your gloved hand or be sure to set it down on a clean decontaminated surface Keep reactions and components sealed as much as possible Do not open sealed reaction tubes or plates after amplification Clean lab benches and equipment periodically with freshly diluted 10 bleach solution User Bulletin Improving Assay Performance Improving The Precision Plate Holders for the 7000 System and the Replicate 7500 System improve thermal cycler performance and the precision Precision 0f replicates by reducing corner well condensation The Precision Plate Holder fits between individual reaction tubes tube strips and 96 well reaction plates including the TaqMan RNase P Verification Plate and the thermal cycler sample block Note Applied Biosystems recommends using the Precision Plate Holder with every 7000 System and 7500 System run Please refer to the table below for the appropriate Precision Plate Holder for your instrument and run configuration For the Running Use Precision Plate Holder 7000 System 96 Well Reaction Plates Tube Strips Individual Tubes PN 4350105 7500 System 96 Well Reaction
16. ound calibration see Recommended Calibration Schedule on page 12 or using the ROI Inspector to take an image of the thermal cycler sample block Acceptable signal ranges for the background calibration are lt 1 200 Fluorescent Standard Units FSU for the 7000 System and lt 72 000 FSU for the 7500 System lt 90 000 FSU for filter set E If contaminated wells are detected clean the sample wells and recheck them for contamination As Needed e Clean the sample wells e Replace the halogen lamp IMPORTANT You must perform the following calibrations in this order after successfully replacing the halogen lamp ROI gt background gt optical 7500 System only gt pure dye gt instrument verification e Replace the instrument fuses User Bulletin Instrument Maintenance Procedures How to Maintain Fluorescence detection is accomplished in the 7000 System and the the Halogen 7500 System by a halogen lamp which directs light to each well of a Lamp mounted reaction plate and excites the fluorescent dyes therein The average lifetime of the installed halogen lamp is approximately 2000 hours on the 7500 System and 1000 hours on the 7000 System To help maintain the lamp please keep in mind the following The lamp remains illuminated while the instrument is powered on To increase the lifetime of the lamp power down the instrument when not in use Note The 7500 System will automatically turn off the h
17. ownward pressure on the applicator in all steps to form a complete seal on top of the wells Pressure is required to activate the adhesive on the optical cover Rub the flat edge of the applicator back and forth along the short edge width of the plate Rub the end of the applicator horizontally and vertically between all wells Rub the end of the applicator around all outside edges of the plate using small back and forth motions to form a complete seal around the outside wells User Bulletin Improving Assay Performance To seal optical plates continued 6 Use the applicator to apply pressure to the perforated end tabs of the optical adhesive cover then remove the tabs from the cover s protective backing prior to amplification Note Applied Biosystems recommends you place a compression pad on the sealed reaction plate before you insert it into the thermal cycling sample block on the 7000 System IMPORTANT Do not use a compression pad with the 7500 System To seal tubes 1 Place strip caps on the tubes Note To increase the quality of your results Applied Biosystems recommends that you place the reaction tubes toward the center of the reaction tray rather than the edges 2 IMPORTANT Apply significant downward pressure on the capping tool in all steps to form a complete seal on top of the tubes If you are using the roller capping tool PN N8010438
18. ted on the top of the instrument 2 Turn on the 7000 System 3 While the instrument is running look through the access door opening to verify whether the lamp is illuminated To check lamp illumination on the 7500 System 1 Make sure the access door is closed 2 Turn on the 7500 System 3 In the SDS Software select Instrument gt Calibrate 4 Inthe ROI Inspector dialog box select Lamp Control gt Idle 5 While the instrument is running look through the grating of the access door to verify whether the lamp is illuminated then click Done If the halogen lamp is not illuminated the replacement halogen lamp may be defective Replace the lamp again and repeat the visual inspection If the second lamp does not illuminate check the instrument fuses for failure Refer to the ABI PRISM 7000 Sequence Detection System User Guide or the Applied Biosystems 7300 7500 Real Time PCR System Installation and Maintenance Guide for the proper procedure Note Ifthe lamp still does not illuminate after the replacement of the instrument fuses please contact your local Service Engineer HID Technical Support representative or HID Application Specialist see How to Obtain HID Support on page 2 for contact information IMPORTANT Perform the following calibrations in this order after successfully replacing the lamp ROI gt background gt optical 7500 System only gt pure dye gt
19. usually well below the dynamic range of the standard curve Therefore these quantities cannot be used accurately e You may choose to lower the cycle number to overcome such high level sensitivity Note Lowering the cycle number would require further internal validation at your facility e You may choose to set a Cy value threshold for proceeding with STR analysis Note Setting a Cy value threshold would require further internal validation at your facility Instrument Calibration and Maintenance Procedures for Use with Quantifiler DNA Quantification Kits 13 ABI Prism 7000 Sequence Detection System and Applied Biosystems 7500 Real Time PCR System Preventing PCR PCR assays require special laboratory practices to avoid false Contamination positive amplifications The high sensitivity of these assays can lead to amplification of a single DNA molecule To minimize false positives due to amplified material in your work area follow these recommended laboratory practices 14 If possible maintain separate work areas dedicated equipment and supplies for Sample preparation PCR setup PCR amplification Analysis of PCR products Wear a clean lab coat not previously worn while handling amplified PCR products or used during sample preparation and clean gloves when preparing samples for PCR amplification Change gloves whenever you suspect that they are contaminated and before leaving the work area Use p
20. ystem and Applied Biosystems 7500 Real Time PCR System How to View To view the component dye signals from a Quantifiler Kit run Component Dye select the Component sub tab within the Results view of the SDS Signals Software The halogen lamp may need replacement if the lines representing the dye signals contain spikes or appear unstable as shown in the figure below Applied Biosystems does not use the dye signals found in the Component sub tab as a measure of lamp life or need for replacement Fie view Tools instrument Analysis window Help 5 x jb tela a ey Hl Setup Instrum Results Plate ASpectra Component Amplification Plot Standard Curve Dissociation Report Component r Component s f E a rvc 13 00 J Mr ex wow A 2602 00 Fa g awo 2 3 aa g AN L zmo Bs i rd rae Unstable y signals A AN A E ede me N eee a i ee ns li 500 00 a 5 7 3 13 16 7 19 za 2 28 a 29 n 2 35 31 38 Cycles IMPORTANT This signal determination may be implemented as a routine procedure to monitor the life of the halogen lamp However it is critical that the reading is taken from the same sample well to provide a better comparison between determinations 6 User Bulletin Instrument Maintenance Procedures How t
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