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1. qal Capillary 3 130 200 210 220 230 240 250 260 270 280 230 300 310 320 Capillary 4 12004 600 Figure 1 DNA samples amplified using the Yfiler kit 36 User Bulletin Examples of DNA Profiles Generated on the 3130 Genetic Analyzer Figure 2 shows a comparison of signal intensity across a 3130 instrument capillary array using AmpF STR Control DNA 9948 2 ng amplified using the AmpF STR Profiler Plus PCR Amplification Kit 100 110 120 130 140 450 160 170 180 190 200 210 220 230 240 250 260 1600 1600 100 410 120 130 140 450 160 170 190 130 200 210 220 230 240 250 260 3 12004 1600 1200 1600 4 1200 Capillary 1 Capillary 2 400 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 ZER st soo Capillary 3 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 2 M Capillary 4 Figure 2 DNA samples amplified using the Profiler Plus kit Protocols for Processing AmpF4STR PCR Amplification Kit PCR Products 37 Applied Biosystems 3130 3130xl Genetic Analyzers Using Data Collection Software v3 0 Mixture Studies 100 110 In Figure 3 the first and last panels
2. Resolution Figure 5 shows three capillaries of a 3130 instrument 4 capillary array displaying single nucleotide resolution As shown below the 9 3 and 10 alleles were resolved and individually detected in all capillaries in which they were run m Capillary 1 D m E mu SG MM Jm 85 B B 8 bal 1 Capillary 2 ol um SA SN BA Ja is is ls E j s J o B3 1 Capillary 3 40 Figure 5 A 4 capillary array displaying single nucleotide resolution User Bulletin Sensitivity Examples of DNA Profiles Generated on the 3130 Genetic Analyzer Figure 6 shows dilutions for the AmpF STR Control DNA 9948 sample quantified using the Quantifiler Human DNA Quantification Kit and amplified using the AmpF STR Identifiler PCR Amplification Kit 1 0 ng 0 5 ng 0 25 ng and 0 125 ng of input DNA The Y axis is magnified for lower input DNA amounts With 125 pg of input DNA quantified using the Quantifiler kit it was observed at the FGA locus that one of the alleles was not detected at a peak amplitude threshold of 50 RFU 1 0 ng 0 5 ng 0 25 ng 0 125 ng Figure 6 DNA sample amplified using the Identifiler kit Protocols for Processing AmpF4STR PCR Amplification Kit PCR Products 41 Applied Biosystems 3130 3130xl Genetic Analyzers Using Data Collection Software v3 0 Figure 7 shows dilutions for the AmpF STR Control DNA 9948 sample am
3. 2 Select Store Array and follow the prompts 3 Replace the cover over the detection cell 4 Fill a buffer reservoir with fresh 1X running buffer and cover with a septa strip Insert the capillary tips into the buffer 5 Fill the shipping vial with fresh 1X running buffer and insert the detection end of the capillary array 6 Store the capillary array upright 7 Check the 1X running buffer level in the reservoir and tube weekly and replenish the buffer as needed 78 User Bulletin Storing Capillary Arrays Removing Bubbles may be found in the polymer system especially after a Bubbles from the polymer change or array installation Polymer Blocks Remove the bubbles from all parts of the polymer system including the pump chamber the pump block channel polymer supply and interconnect tubing and the lower polymer block channel To clear bubbles 1 Select Wizards gt Bubble Remove Wizard to clear bubbles IMPORTANT Remove bubbles from the interconnect tubing and the channel of the lower polymer block These areas are part of the electrophoresis current path Absence of bubbles in the current path is important for problem free electrophoresis WERE Help Install Array Wizard Change Polymer Type Wizard Replenish Polymer Wizard Bubble Remove Wizard Water Wash Wizard Instrument Shutdown Wizard Autosampler Calibration Wizard Update Cap Array Info 2 Replace the 1X running buffer if excess polymer is expelled
4. Components of the polymer delivery pump PDP are identified in the drawing below Mounting pin PDP motor Syringe fitting PDP motor cover Capillary array tip Water seal Waste fitting Water trap i Capillary array Mounting pin Q K Piston A Capillary array knob Pump chamber Pump block Check valve Double tapered ferrule Array port Interconnect tube Polymer supply tube Buffer valve pin Polymer supply Lower polymer block bottle cap with Mounting pin hole Overflow hole Buffer fill line Polymer Buffer jar 16 mL anode reservoir Electrode GR2459 56 User Bulletin Performing Maintenance Tasks Performing Maintenance Tasks This section lists common tasks required to maintain your Applied Biosystems 3130 3130x Genetic Analyzers in good working condition The tasks are divided into tables based on how often you should perform each task Nee Wear appropriate protection including gloves laboratory goggles and coat whenever you work with the fluids used on this instrument or parts that may come into contact with these fluids Daily Tasks Perform these tasks at least once per day Maintenance Task Frequency Ensure adequate levels of buffer and water in reservoirs Before each run Ensure the plate assemblies are properly assembled Before IMPORTANT
5. The holes in the plate retainer must align each r n with the holes in the septa or the capillary tips will be damaged Ensure the plate assemblies are positioned on the plate Before deck properly Plates should sit snugly on the deck each run IMPORTANT Never use warped plates Check the level of buffer in the buffer jar Ensure that the Before overflow hole faces the front of the instrument and is not each run occluded Replace the water and 1X running buffer in the reservoirs Every 48 on the instrument and make sure that the outside of the hours assemblies are dry Check for bubbles in the pump block lower polymer Daily or block interconnect tube polymer supply tube and before channels each run Remove all bubbles with the Bubble Remove wizard Check the loading end header to ensure the capillary tips Daily or are not crushed or damaged before each run Check the level of polymer in the bottle to ensure sufficient Daily or volume for runs before each run Protocols for Processing AmpF4STR PCR Amplification Kit PCR Products 57 Applied Biosystems 3130 3130xl Genetic Analyzers Using Data Collection Software v3 0 Maintenance Task Frequency Check the pump block and the lower polymer block to Daily ensure they fit securely on the instrument Clean the instrument surfaces Daily Check for leaks around the array knob interconnecting Daily tube nuts and check valve Weekly Tasks
6. 310 320 330 4000 30004 2000 1000 0 A Figure 11 DNA samples amplified using the Identifiler kit Protocols for Processing AmpF4STR PCR Amplification Kit PCR Products 47 Applied Biosystems 3130 3130xl Genetic Analyzers Using Data Collection Software v3 0 In Figure 12 the first and last panels display profiles of each DNA sample amplified individually with a total DNA input of 2 ng using the AmpF STR Profiler Plus PCR Amplification Kit The female AmpF STR Control DNA 9947a sample is in the top panel and the male AmpF STR Control DNA 9948 sample is in the bottom panel The other three panels display the mixtures of DNA at approximate ratios of 1 1 1 5 and 1 10 120 130 140 150 160 170 180 130 200 210 220 230 240 250 260 270 280 230 300 M MM 48 Figure 12 DNA samples amplified using the Profiler Plus kit User Bulletin Examples of DNA Profiles Generated on the 3130xl Genetic Analyzer Resolution Figure 13 shows three capillaries of a 3130xl instrument 16 capillary array displaying single nucleotide resolution As shown below the 9 3 and 10 alleles were resolved and individually detected in all capillaries in which they were run Capillary 1 3000 Capillary 3 o A A DA MA A A AA 7a 0 0 A a 3 Bl lo fo 13 3 THO 3000 k Capillary 5 3 Bl palio Figure 13 A 16 capillary array disp
7. Amplification Kit PCR Products 23 Applied Biosystems 3130 3130xl Genetic Analyzers Using Data Collection Software v3 0 For Dye Set F 1 Follow the procedure on page 22 Prepare the Matrix Standards for Dye Set E 2 In step 3 on page 22 use the following volumes Reagent 3130 etn Nen a System Matrix Standard Set DS 32 5 5 Hi Di Formamide 195 195 Final Volume 200 200 Norme CHEMICAL HAZARD Formamide Exposure causes eye skin and respiratory tract irritation It is a possible developmental and birth defect hazard Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Note These same instructions are present on the Matrix Standard Set DS 32 product insert 24 User Bulletin Performing a Sample Run Performing a Sample Run In order for data collection and autoanalysis to be successful each run of samples must have an instrument protocol and results group assigned within a plate record Workflow To perform a run Step Task See 1 Set up GeneMapper D Software v3 2 that is GeneMapper ID Software v3 1 and import panels and bin sets create analysis v3 2 Human Identification Analysis methods etc Tutorial PN 4357520 Rev A Note This step is only required for autoanalysis 2 Create an instrument protocol for HID analysis page 26 of this user bulletin 3 Create a results group page 11
8. OK within the Plate Editor the completed plate record is stored in the Plate Manager database Once in the Plate Manager database the plate record can be searched for edited exported or deleted In the Sample Name column of a row enter a sample name then click the next cell The value 100 is automatically displayed in the Priority column In the Comment column enter any additional comments or notations for the sample In the Priority column change the priority value if desired In the Sample Type column select a sample type from the drop down list that corresponds to the sample in that well In the Size Standard column select a size standard from the drop down list For four dye AmpF STR kits select GS500 ROX User Bulletin Performing a Sample Run For five dye AmpF STR kits select GS500 LIZ 6 In the Panel column select the appropriate AmpF STR panel from the drop down list 7 In the Analysis Method column select a method from the drop down list 8 Leave the Snp Set column blank 9 Enter text for User Defined columns 1 to 3 10 In the Results Group 1 column select a group from the drop down list 11 In the Instrument Protocol 1 column select a module from the drop down list When you use the 3130 3130x Genetic Analyzers with Data Collection Software v3 0 and the AmpF STR kits Applied Biosystems recommends that you select the HIDFragmentAnalysis36 POP4 1 module 12 To complete
9. Perform these tasks at least once per week Maintenance Task Frequency Replace the polymer using the Replenish Polymer Wizard Weekly or as needed Check the storage conditions of the used arrays Weekly Restart the computer and instrument Weekly Monthly Tasks Perform these tasks at least once per month Maintenance Task Frequency Run the Water Wash Wizard Monthly or as needed Flush the array port during this wizard whether or not bubbles are present in the array port Flush the water trap See Flushing and Filling the Water Monthly or Trap on page 68 as needed Defragment the hard drive Monthly As Needed Tasks Perform these tasks as needed Maintenance Task Frequency Clean the drip tray As needed Change the array As needed Remove any dried polymer from the capillary tips Use a As needed lint free wipe moistened with deionized water 58 User Bulletin Routine Cleaning Routine Cleaning General Cleaning l 2 Ensure the oven and instrument doors are closed Press the Tray button on the front of the instrument to move the autosampler to the forward position IMPORTANT Never use organic solvents to clean the instrument Tray button Wipe off any liquid on or around the autosampler using a lint free tissue Clean off any polymer build up crystals on the instrument including the capillary tips and the stripper plate with deionized w
10. Wizard Instrument Shutdown Wizard Autosampler Calibration Wizard Update Cap Array Info If plates are linked in the Run Scheduler and you complete a wizard the plates automatically unlink You will get a warning dialog box Click OK and then relink the plate if applicable Zoo x A Plate s unlinked The following table lists the wizards and when to use them Wizard Use to Install Array Install a capillary array On a new instrument To reactivate an instrument that has been shut down Replace an installed capillary array with another capillary array User Bulletin Wizards Wizard Use to Change e Change to a different polymer type than the one Polymer Type presently being used Replenish Replenish the polymer supply Polymer Replace the polymer in the PDP with polymer of the same or different lot Enter polymer information when Data Collection software is installed or upgraded Bubble Remove bubbles in the PDP chamber channels and Remove tubing Water Wash Wash the PDP chamber lower polymer block channels and tubing with water As part of a monthly maintenance protocol To remove any suspected contaminants in the PDP To remove persistent bubbles followed by the Bubble Remove Wizard if needed To replace old polymer in the PDP Instrument Prepare the instrument for a period of disuse of Shutdown greater than one
11. array length and serial number information into the database Correct an entry error after using another wizard IMPORTANT The capillary array length defined in the wizard must match the array length you are using Follow these guidelines to properly care for the capillary array and area Wear gloves and handle the capillary array gently Do not touch the detection cell Keep the ends of the capillary array wet at all times Do not overtighten the capillary array knob Clean off any polymer buildup crystals on the instrument including the capillary electrodes and the stripper plate with deionized water and lint free tissue NITE CHEMICAL HAZARD POP polymers cause eye skin and respiratory tract irritation Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Note When cleaning the capillary electrodes be careful not to bend them out of position If the electrodes do get bent follow the procedure Storing Capillary Arrays on page 77 IMPORTANT Never use organic solvents to clean the instrument User Bulletin Filling the Capillary Array Using Manual Control Storing Capillary Arrays See Fill the Capillary With Fresh Polymer Using Manual Control on page 63 Storing Capillary Arrays Storing a Capillary Array on the Instrument Storing a Capillary Array off the Instrument Store the capillary array on the instrumen
12. display profiles of each DNA sample amplified individually with a total DNA input of 1 ng using the AmpF STR Identifiler PCR Amplification Kit The female AmpF STR Control DNA 9947a sample is in the top panel and the male AmpF STR Control DNA 9948 sample is in the bottom panel The other three panels display the mixtures of DNA at approximate ratios of 1 1 1 5 and 1 10 120 130 140 150 160 170 180 190 200 210 220 230 24 250 260 270 290 290 300 310 320 330 340 350 a Ho 2000 1 0 4000 D 400 410 420 130 140 450 460 170 480 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 50 E E A A A A E E A A A A A A A A A A A E A A E A a E A E a E a E E a E I i E A A 4800 1200 1 1 600 D 100 410 420 430 440 150 460 470 480 490 200 210 220 230 240 250 260 270 290 290 300 310 320 330 340 350 i 1200 1 5 600 D 400 410 420 130 140 450 460 470 490 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 50 1800F 4200 1 10 600 D 400 440 420 430 440 150 460 470 480 490 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 o 20004 0 1 10004 D 38 Figure 3 DNA samples amplified using the Iden
13. dyes AmpF STR Yfiler PCR e 6FAM Amplification Kit e VIC AmpF STR SEfiler PCR e NED Amplification Kit e PET e LIZ9 four dye system which Dye Set F Matrix Standard Set AmpF STR Profiler Plus includes the following DS 32 PCR Amplification Kit AmpF STR COfiler PCR e 5FAM Amplification Kit e JOE AmpF STR Profiler Plus ID e NED PCR DE Kit e ROX AmpF STR SGM Plus PCR Amplification Kit Other four dye AmpF STR kits 14 User Bulletin Performing a Spectral Calibration Select a Spectral Calibration Protocol and Create a Plate Record For Dye Set G5 Follow this procedure if you are performing a spectral calibration for kits using a five dye system which includes the 6FAM VIC NED PET and LIZ dyes IMPORTANT For HID applications the default condition number boundaries for the Dye Set G5 spectral calibration protocol should be edited in Data Collection Software v3 0 Instructions for editing the condition number boundaries are provided below you will only need to perform this edit once Edit the Dye Set G5 Spectral Calibration Protocol 1 In the tree pane of the Data Collection software click amp GA Instruments gt E ga3130x or ga3130 gt Protocol Manager to open the Protocol Manager window N GA Instruments gt 9431 gt Protocol Manager Wyttrument Protocols Find Protocol ame Run Medule Spectrat36_P0P4_F ped36_ 0P4_1 J Cex ome mom em
14. in a microcentrifuge Heat the tube at 95 C for 5 min to denature the sample Immediately place the tube on ice for 3 min Protocols for Processing AmpF4STR PCR Amplification Kit PCR Products 21 Applied Biosystems 3130 3130xl Genetic Analyzers Using Data Collection Software v3 0 Prepare the Follow this procedure if you are setting up spectral matrix Matrix Standards for Dye Set F calibration standards for kits using a four dye system which includes the 5 FAM JOE NED and ROX dyes l Thoroughly vortex the Matrix Standard Set DS 32 tube for Dye Set F Spin the tube briefly in a microcentrifuge Prepare Matrix Standard Set DS 32 for Dye Set F by combining the following in a labeled 1 5 mL microcentrifuge tube Reagent 3130 Syren using System Matrix Standard Set DS 32 10 10 Hi Di Formamide 190 190 Final Volume 200 200 ND NET 22 Norme CHEMICAL HAZARD Hi Di Formamide Exposure causes eye skin and respiratory tract irritation It is a possible developmental and birth defect hazard Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Vortex thoroughly Spin the mixture briefly in a microcentrifuge Heat the tube at 95 C for 5 min to denature the sample Immediately place the tube on ice for 3 min User Bulletin Performing a Spectral Calibration Troubleshooting Off Scale Spectral Calibration Peak Height Ne
15. into the anode buffer jar during bubble removal Nri CHEMICAL HAZARD 1X Genetic Analyzer Buffer with EDTA may cause eye skin and respiratory tract irritation Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Protocols for Processing AmpF4STR PCR Amplification Kit PCR Products 79 Applied Biosystems 3130 3130xl Genetic Analyzers Using Data Collection Software v3 0 Autosampler Calibration When to Calibrate Calibrate the autosampler only as needed the Autosampler Calibrating the 1 Autosampler 80 Symptoms of autosampler alignment problems may include Poor injection for a small number of capillaries Low signal strength No evidence of sample Close the oven and instrument doors Select Wizards gt Autosampler Calibration Wizard WERE Help Install Array Wizard Change Polymer Type Wizard Replenish Polymer Wizard Bubble Remove Wizard Water Wash Wizard Instrument Shutdown Wizard Autosampler Calibration Wizard Update Cap Array Info If plates are linked in the Run Scheduler the plates automatically are unlinked In the Warning dialog box click OK Follow the directions given in the wizard to calibrate the autosampler User Bulletin Autosampler Calibration Autosampler Calibration Wizard Step 1 Clear and Initialize Autosampler 1 Push the Tray button on the outside ofthe instrument The autosampler will
16. move forward automatically 2 Open the instrument doors and remove all plate assemblies and fluid reservoirs on the autosampler 3 Close the instrument doors The autosampler will move to the return position automatically 4 When the autosampler stops moving click Initialize to setup the autosampler for calibration 5 When the autosampler stops moving click Next Prey Next Cancel Fin sh 5 Click Finish 6 Turn the instrument power off for 10 sec then on Protocols for Processing AmpF4STR PCR Amplification Kit PCR Products 81 Applied Biosystems 3130 3130xl Genetic Analyzers Using Data Collection Software v3 0 Manual Control Note Manual control is active only if the oven and instrument doors The following table displays the manual control options in the Data are closed Table of Commands Collection software Command Function Command Options Value Electrophoresis Turn On Off power supply On e Off Set voltage A number between 0 0 and 15 kV Read voltage N A Read current Laser Set laser state e Idle e On e Off Set laser power A number between 0 and 25 mW Read laser power N A setting Read laser power Read laser current Open Close shutter Open Closed Oven Set state On e Off Set temperature A number between 18 and 65 C 82 User Bulletin Manual Control Command Function Command Opt
17. the appropriate shutdown procedure based on the information in the following table If the instrument will be unattended Perform this shutdown procedure for no more than 1 week Short term W ai buffer IMPORTANT The key to a successful short term shutdown is keeping the capillary array in 1X running buffer This prevents the polymer from drying in the capillaries Nem CHEMICAL HAZARD 1X Genetic Analyzer Buffer with EDTA may cause eye skin and respiratory tract irritation Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves for more than 1 week Long term 62 User Bulletin Shutting Down the Instrument Performing a Fill the Capillary With Fresh Polymer Using Manual Control Short Term 1 Ensure the oven and instrument doors are closed Shutdown 2 Collect polymer waste a Click A GA Instruments gt EJ ga3130 or ga3130x J instrument name gt Manual Control b In the Send Defined Command drop down menu select Autosampler c In the Command Name drop down menu select Move autosampler to site d In the Value menu select Waste e Click Send Command Wait for the autosampler to stop moving and Send Command becomes active before proceeding 3 Fill the capillaries a In the Send Defined Command for drop down menu select Polymer Delivery Pump b In the Command Name select the appropriate Fill l
18. using run modules created for the POP 7 polymer Applied Biosystems does not recommend activation of the detection cell heater for HID applications Protocols for Processing AmpF4STR PCR Amplification Kit PCR Products 3 Applied Biosystems 3130 3130xl Genetic Analyzers Using Data Collection Software v3 0 Data Collection Software Modifications Data Collection Software v3 0 represents Applied Biosystems latest controlling software for the capillary electrophoresis instruments As in Data Collection Software v2 0 there are software wizards to guide you through maintenance procedures IEEE Help Install Array Wizard Change Polymer Type Wizard Replenish Polymer Wizard Bubble Remove Wizard Water Wash Wizard Instrument Shutdown Wizard Autosampler Calibration Wizard Update Cap Array Info Several of these wizards are new in Data Collection Software v3 0 and support the new pump based PDS of the 3130 3130xl Genetic Analyzers Change Polymer Type Wizard to change to a different polymer type Replenish Polymer Wizard to replenish with the same polymer from the same or different lot Bubble Remove Wizard Water Wash Wizard Instrument Shutdown Wizard User Bulletin Reagents Kits and Run Module for HID Analysis Reagents Kits and Run Module for HID Analysis Reagents The reagents AmpF STR kits and run module listed in this section are available for performing HID analysis using the 3130 3130x Geneti
19. week Autosampler Calibrate the autosampler positions Calibration Update Cap Update the capillary array information and the Array Info serial number Correct an entry mistake after using a wizard a The lower polymer block is cleaned on the instrument using this wizard and should not be removed Protocols for Processing AmpF4STR PCR Amplification Kit PCR Products 67 Applied Biosystems 3130 3130xl Genetic Analyzers Using Data Collection Software v3 0 Flushing and Filling the Water Trap 68 The PDP water trap should be flushed with either distilled or deionized water at least once per month to wash out any diluted polymer and to clear bubbles Leave the trap filled with either distilled or deionized water To flush the water seal trap l Fill the supplied 20 mL all plastic Luer lock syringe PN 4324463 with distilled or deionized water Expel any bubbles from the syringe IMPORTANT Do not use a syringe smaller than 20 mL Doing so may generate excessive pressure within the trap Attach the syringe to the forward facing Luer fitting at the top of the pump block Hold the fitting with one hand while threading the syringe onto the fitting with the other hand Open the Luer fitting by grasping the body of the fitting and turning it and the attached syringe approximately one half turn counterclockwise Open the exit fitting at the top left side of the pump block by turning it approximately one half turn c
20. 2 In the Instrument Protocols pane highlight the spectral calibration protocol for Dye Set G5 Spectral36 POP4 G5 Protocols for Processing AmpF4STR PCR Amplification Kit PCR Products 15 Applied Biosystems 3130 3130xl Genetic Analyzers Using Data Collection Software v3 0 3 Click Edit to open the Protocol Editor Protocol Editor west gs C83 Polymer pop iv Array Length 36 Chemistry Matrix Standard Run Module Spect38 POP4 1 Edit Param OK Cancel 4 Change the condition number boundaries a Click Edit Param The Edit Spectral Params window opens IA Edit Spectral Params Matrix Condition Number Bounds Lower 85 Upper 14 5 Locate Start Point After Scan 300 Before Scan 5000 Limit Analysis scans 20000 Sensitivity 0 4 Minimum Quality Score 0 95 Cancel b Change the condition number boundary in the Lower field from 8 5 to 7 0 c Change the condition number boundary in the Upper field from 14 5 to 12 0 I Edit Spectral Params Matrix Condition Number Bounds Lower 7 0 Upper 12 0 Locate Start Point After Scan 300 Before Scan 5000 Lirnit Analysis scans 20000 Sensitivity 0 4 Minimum Quality Score 0 95 Cancel d Click OK to return to the Protocol Editor 16 User Bulletin Performing a Spectral Calibration 5 Click OK to save your changes and exit the Protocol Editor Note
21. 3130 3130x Genetic Analyzers to perform HID analysis Applied Biosystems Applied Biosystems 3130 3130xl Genetic Analyzers Using Data Collection Software v3 0 Related The following documents are referenced in this user bulletin Be sure Documentation to have these on hand when you are performing experiments with the 3130 3130x Genetic Analyzers Applied Biosystems 3130 3130xl Genetic Analyzers Getting Started Guide PN 4352715 Rev B GeneMapper ID Software v3 1 and v3 2 Human Identification Analysis Tutorial PN 4357520 Rev A Applied Biosystems 3130 3130xl Genetic Analyzers Maintenance Troubleshooting and Reference Guide PN 4352716 Rev B Safety Complete Safety information for the 3130 3130x Genetic Analyzers may be found in the Applied Biosystems 3130 3130xl Genetic Analyzers Getting Started Guide How to Obtain Support For the latest services and support information for all locations go to http www appliedbiosystems com then click the link for Support At the Support page you can Search through frequently asked questions FAQs Submit a question directly to Technical Support Order Applied Biosystems user documents MSDSs certificates of analysis and other related documents Download PDF documents Obtain information about customer training Download software updates and patches In addition the Support page provides access to worldwide telephone and fax numbers to contact App
22. 5 of the 3130 3130xl Genetic Analyzers GSG 4 Create a plate record in GeneMapper D Software page 28 of this user bulletin v3 2 5 Prepare your sample chemistry page 32 of this user bulletin 6 Load your samples page 33 of this user bulletin 7 Place the plate assembly into the instrument page 39 of the 3130 3130xl Genetic Analyzers GSG 8 Run your sample plate which includes pages 128 to 160 of the 3130 3130xl uu irm Genetic Analyzers GSG Linking unlinking a plate Starting the run e Controlling monitoring the run Viewing data Protocols for Processing AmpF4STR PCR Amplification Kit PCR Products 25 Applied Biosystems 3130 3130xl Genetic Analyzers Using Data Collection Software v3 0 Create an Instrument Protocol An instrument protocol contains all the settings necessary to run the instrument Protocol name Typeofrun Run module Dye set Create an 1 In the tree pane of the Data Collection software click Instrument A GA Instruments gt E ga3130x or ga3130 gt amp Protocol Protocol Manager to open the Protocol Manager window Foundation Data Collection Version 3 0fc3 No User is logged in File View Help N GA Instruments ga3130 Protocol Manager Instrument Protocols Find Protocol Create E Run History ji Destin instrument amp Cja1so crsoo2 agmen amp POP4 G5 Fragmen E G5 2 El instrument Status HID_Spectral36_POP4_F Spect36_POP4
23. Data Collection Software v3 0 Mixture Studies In Figure 11 on page 47 the first and last panels display profiles of each DNA sample amplified individually with a total DNA input of 1 ng using the AmpF STR Identifiler PCR Amplification Kit The female AmpF STR Control DNA 9947a sample is in the top panel and the male AmpF STR Control DNA 9948 sample is in the bottom panel The other three panels display the mixtures of DNA at approximate ratios of 1 1 1 5 and 1 10 The panel inset displays the expanded view of the DNA samples mixed at an approximate ratio of 1 10 46 User Bulletin Examples of DNA Profiles Generated on the 3130xl Genetic Analyzer 100 110 120 130 140 150 160 170 180 130 200 210 19 48 s W 8 8 g 5 3 P E 3 N i 2 a E Ei 290 300 310 320 330 340 350 mm 2000 1 0 100 110 120 130 140 150 160 170 180 130 200 210 220 230 240 250 260 270 280 230 300 310 320 330 340 350 4200 1 i 100 110 120 130 140 150 160 170 180 130 200 210 m 1S8 E n3 E 8 240 250 260 270 280 230 300 310 320 330 340 350 1 5 400 110 120 130 140 150 150 170 180 130 200 210 220 230 240 250 260 270 280 p 300 310 320 sd M 340 350 1 10 100 110 120 130 140 150 160 170 180 130 200 210 220 230 240 250 260 270 uu 230 300 310 320 330 340 350 0 1 2000 290 300 5000
24. Once you have saved your changes you will not need to edit the Dye Set G5 spectral calibration protocol again The lower and upper condition number boundaries are now set at 7 0 and 12 0 respectively Continue with Select the Dye Set G5 Spectral Calibration Protocol and Create a Plate Record below Select the Dye Set G5 Spectral Calibration Protocol and Create a Plate Record l In the tree pane of the Data Collection software click amp GA Instruments gt E ga3130x or ga3130 gt Plate Manager to open the Plate Manager window Click New to open the New Plate dialog box Complete the New Plate dialog box New Plate Dialog Name Spectral Cal DyeSetG5 Description Application Spectral Calibration Plate Type 96 VVell M Owner Name AB Operator Name AB a Name Type a name for the plate b Description If desired type a description for the plate c Application Select Spectral Calibration from the drop down list d Plate Type Select 96 Well Protocols for Processing AmpF4STR PCR Amplification Kit PCR Products 17 Applied Biosystems 3130 3130xl Genetic Analyzers Using Data Collection Software v3 0 e Owner Name Type a name for the owner f Operator Name Type a name for the operator 4 Click OK to open the Spectral Calibration Plate Editor 5 Complete the Spectral Calibration Plate Editor a In the Sample Name column enter a sample name then click the next ce
25. User Bulletin Applied Biosystems 3130 3130x Genetic Analyzers Using Data Collection Software v3 0 SUBJECT In This User Bulletin Overview Audience September 9 2010 Protocols for Processing AmpF STR PCR Amplification Kit PCR Products This user bulletin includes the following topics QEL Tc 2 Howto Obtain SUDDOEL 4 2 eee aha CP N W WEBE W al 2 New Features and Design Changes Walk kakil ak a ak a k 3 Reagents Kits and Run Module for HID Analysis 5 Section 1 Performing HID Analysis ss kak a sa xy x an k 7 Section 2 Examples of DNA Profiles Generated on the 3130 3130x1 rene lio ADAE ab oa dh RE RARE DI HEQA GU h QA 6 2 ORE 33 Appendix A Ma mntenal c od gt sk ss kalak a yek da kn 55 This document describes The design changes and new features that differentiate the Applied Biosystems 3130 3130x Genetic Analyzers from the ABI PRISM 3100 3100 Avant Genetic Analyzers The tasks required to perform Human Identification HID analysis with the 3130 3130x Genetic Analyzers using 3130 3130x Genetic Analyzer Data Collection Software v3 0 and GeneMapper ID Software v3 2 Examples of DNA profiles generated on the 3130 3130x Genetic Analyzers Maintenance schedule and maintenance procedures for the 3130 3130x Genetic Analyzers This user bulletin is intended for customers who will be using the
26. _1 protocols Z JEPT Chart HID Spectral36 POP4 65 Spect36 POP4 1 in this event Log Gl spatial Run Schedule pane E be cow edit Delete moo _ Export Brun view capillaries viewer Analysis Protocols Bl Cap Array Viewer spectral viewer Find Pr tocol 4 Manual Control Create cor f service Log Nap Eum analysis 313 POP7 v3 enovo_v5 2 SequencingAnalysis protocols in this pane Eat oeste mpor export Click New here 2 Inthe Instrument Protocols pane click New to open the Protocol Editor 26 User Bulletin 3 Complete the Protocol Editor fields Protocol Editor Name FragmentAnalysis36 POP4 G5 Description Type REGULAR v Run Module HiDFragmentAnalysis36 POP4 1 v e oce Performing a Sample Run Name Type a name for the protocol Description If desired type a description for the protocol Type Select REGULAR from the drop down list Run Module Select HIDFragmentAnalysis36 POPA 1 e Dye Set Per the table below select the correct dye set for your run Matrix Kit Dye Set Standard Set e AmpF STR Identifiler PCR G5 DS 33 Amplification Kit e AmpF STR Yfiler PCR Amplification Kit e AmpF STR SEfiler PCR Amplification Kit Protocols for Processing AmpF4STR PCR Amplification Kit PCR Products 27 Applied Biosystems 3130 3130xl Genetic Analyzers Using Data Collection Software v3 0 Matrix Kit Dy
27. ater and lint free tissue Clean the array port knob plug or opening threads of these parts with moistened lab wipes Clean out the drip trays with deionized water and lint free tissue Resetting the Instrument Reset the instrument when A fatal error as indicated by the red status light The instrument does not respond to the Data Collection software Protocols for Processing AmpF4STR PCR Amplification Kit PCR Products 59 Applied Biosystems 3130 3130xl Genetic Analyzers Using Data Collection Software v3 0 Two procedures can reset the instrument Press the reset button through the pin hole on the front of the instrument to dump and reload the firmware and to reset the electronics Try this method first Shut down and restart the computer and the instrument Resetting With the Reset Button 1 Close the instrument doors 2 Using along narrow implement such as a straightened paper clip press the reset button on the front of the instrument Reset button Resetting by Powering Down 1 Close the instrument doors 2 Power off the instrument by pressing the on off button on the front of the instrument On Off button 3 Restart the computer a Select Start gt Turn off Computer 60 User Bulletin Moving and Leveling the Instrument b In the dialog box select Restart then click OK IMPORTANT Wait until the computer has completely restarted before proceeding 4 Turn on the instrument then wait f
28. birth defect hazard Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves 2 Load 1 uL of the sample or allelic ladder into the wells 3 Cover the reaction plate with an appropriate septa Use Reservoir septa or 96 well plate septa 1f samples for more than one run were prepared 4 Briefly spin the reaction plate in a centrifuge to ensure that the contents of each well are mixed and collected at the bottom 5 To denature heat the reaction plate in a thermal cycler at 95 C for 3 min 6 Place the reaction plate immediately on ice for 3 min Prepare the Plate 1 Assemble the plate assembly Assembly a Place the sample plate into the plate base b Snap the plate retainer onto the plate and plate base 2 Verify that the holes of the plate retainer and the septa strip are aligned If not re assemble the plate assembly IMPORTANT Damage to the array tips will occur if the plate retainer and septa strip holes do not align correctly Protocols for Processing AmpF4STR PCR Amplification Kit PCR Products 33 Applied Biosystems 3130 3130xl Genetic Analyzers Using Data Collection Software v3 0 34 User Bulletin Section 2 Examples of DNA Profiles Generated on the 3130 3130xl Genetic Analyzers Section 2 Examples of DNA Profiles Generated on the 3130 8130x Genetic Analyzers This section covers the following topics Appendix Examples of DNA Profiles Generated on t
29. c Analyzers Part Number Name 3130 Genetic Analyzer 3130x Genetic Analyzer 4345831 Matrix Standard Set DS 32 Dye Set F Note Matrix Standard Set DS 32 is for use on multi capillary instruments that is the 3100 3100 Avant Genetic Analyzers and the 3130 3130x Genetic Analyzers 4345833 Matrix Standard Set DS 33 Dye Set G5 Note Matrix Standard Set DS 33 is for use on multi capillary instruments that is the 3100 3100 Avant Genetic Analyzers and the 3130 3130x Genetic Analyzers 4352755 3130 POP 4 polymer 4311320 Hi Di formamide 402824 10X Genetic Analyzer Buffer with EDTA Note Use 10X Genetic Analyzer Buffer with EDTA to prepare 1X Genetic Analyzer Buffer with EDTA 1X running buffer 4333464 3130 and 3100 Avant Capillary Array 36 cm 4315931 3130x and 3100 Capillary Array 36 cm Protocols for Processing AmpF4STR PCR Amplification Kit PCR Products Applied Biosystems 3130 3130xl Genetic Analyzers Using Data Collection Software v3 0 AmpF STR Kits Matrix Kit Dye Set Standard Set e AmpF STRP Identifiler PCR G5 DS 33 Amplification Kit e AmpF STR Yfiler PCR Amplification Kit e AmpF STR SEfiler PCR Amplification Kit e AmpF STR Profiler Plus PCR F DS 32 Amplification Kit e AmpF STR COfiler PCR Amplification Kit e AmpF STR Profiler Plus ID PCR Amplification Kit A
30. d follow the handling instructions Wear appropriate protective eyewear clothing and gloves 1 Close the oven and instrument doors and then press the Tray button 2 Select Wizards gt Install Array Wizard IMPORTANT The capillary array length defined in the wizard must match the array length you are using irm Help Install Array Wizard Change Polymer Type Wizard Replenish Polymer Wizard Bubble Remove Wizard Water Wash Wizard Instrument Shutdown Wizard Autosampler Calibration Wizard Update Cap Array Info 3 Open instrument and oven doors 4 Follow the directions given in the wizard to install or replace an array 5 Click Finish when done 6 Close and lock the oven door then close the instrument doors IMPORTANT If you installed or replaced an array that is a different length than the one you were using you must reset the active spectral calibration or create a new spectral calibration for the dye set and array length combination see Activating a Spectral Calibration in the Applied Biosystems 3130 3130xl Genetic Analyzers Getting Started Guide 7 Relink plate s if applicable Protocols for Processing AmpF4STR PCR Amplification Kit PCR Products 75 Applied Biosystems 3130 3130xl Genetic Analyzers Using Data Collection Software v3 0 76 Updating Capillary Array Information Caring for the Capillary Array and Work Area Use the Update Cap Array Info wizard to Update the capillary
31. dentifiler kit 972 10096 9948 Yfiler kit 612 10096 9948 Profiler Plus kit 612 10096 Protocols for Processing AmpF4STR PCR Amplification Kit PCR Products 53 Applied Biosystems 3130 3130xl Genetic Analyzers Using Data Collection Software v3 0 54 User Bulletin Genotype Concordance Appendix A Maintenance Note For your convenience this appendix is reproduced from the Applied Biosystems 3130 3130xl Genetic Analyzers Maintenance Troubleshooting and Reference Guide PN 4352716 Rev B This appendix covers the following topics Polymer Delivers P l isses E 4 oe EGER RU OR Re 56 Performing Maintenance Tasks a sc sa sawa kala cee aa dka 57 Routuie CIA i ceed dace ad E E A9 h k 0 4 Ar E 59 Resetting the Instrument i 00 644s a ar RE uA Rd OR gunned 59 Moving and Leveling the Instrument 44 61 Shutting Down the Iustrutient os gt s Ree RR XREREETA 62 Li DD DD rd 66 Flushing and Filling the Water Trap ss ss ala aalk a n aa 68 Fimds and S2 hhh m 70 Capillary ATTIV ci a anka areeni in er a dn Q 24a i 74 Storing Capillary Array amp 2432349 dx b 0 REGERE EG RR T Autosampler Calibration iius 404s sar ao RR a W 80 Mammal Contool Prem 82 Protocols for Processing AmpF4STR PCR Amplification Kit PCR Products 55 Applied Biosystems 3130 3130xl Genetic Analyzers Using Data Collection Software v3 0 Polymer Delivery Pump
32. e Set Standard Set e AmpF STR Profiler Plus PCR F DS 32 Amplification Kit e AmpF STR COfiler PCR Amplification Kit e AmpF STR Profiler Plus ID PCR Amplification Kit AmpF STR SGM Plus PCR Amplification Kit e Other four dye AmpF STR kits 4 Click OK to save your changes and exit the Protocol Editor Create a Plate Record in GeneMapper D Software v3 2 Create a Plate 1 In the tree pane of the Data Collection software click Record amp GA Instruments gt E ga3130x or ga3130 gt Plate Manager to open the Plate Manager window 2 Click New to open the New Plate dialog box 3 Complete the New Plate dialog box New Plate Dialog Name ldertifier Description i Application GeneMapper 3130 CTS002 Plate Type 36 ell v Owner Name AB Operator Name AB NH 28 User Bulletin Performing a Sample Run a Name Type a name for the plate b Description If desired type a description for the plate c In the Application drop down list select GeneMapper Generic or GeneMapper lt Computer Name gt When you are performing autoanalysis you must select the GeneMapper lt Computer Name gt application d Plate Type Select 96 Well e Owner Name Type a name for the owner f Operator Name Type a name for the operator 4 Click OK The GeneMapper Plate Editor opens Pene Oe meses 557 ww 5 5 5 i i i grassssssi kzzzzul 888 8 i
33. ead the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Step Task See 1 Review the instrument parts and functions page 10 of this user bulletin 2 Start the computer workstation then start the page 7 of the 3130 3130xl 3130 3130x Genetic Analyzer Genetic Analyzers GSG 3 Start the 3130 3130x Genetic Analyzer Data page 9 of the 3130 3130xl Collection Software v3 0 Genetic Analyzers GSG 4 Inspect the instrument then perform any e pages 12 to15 of the 3130 3130xl Genetic Analyzers GSG Appendix A on page 55 of this user bulletin User Bulletin Preparing the Instrument Step Task See 5 Use 10X Genetic Analyzer Buffer with EDTA page 16 of the 3130 3130xl PN 402824 to prepare 1X Genetic Analyzer Buffer Genetic Analyzers GSG with EDTA 1X running buffer then fill the instrument reservoirs NIN CHEMICAL HAZARD 10X Genetic Analyzer Buffer with EDTA may cause eye skin and respiratory tract irritation Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves NIN CHEMICAL HAZARD 1X Genetic Analyzer Buffer with EDTA may cause eye skin and respiratory tract irritation Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Protocols for Processing AmpF4STR PCR Amplification Kit PCR Products 9 Applied Biosys
34. ejerce E Deseritke RECHTEN 5 Continue with one of the following procedures as appropriate Complete the Plate Record for Manual Analysis on page 30 Complete the Plate Record for Autoanalysis on page 30 Protocols for Processing AmpF4STR PCR Amplification Kit PCR Products 29 Applied Biosystems 3130 3130xl Genetic Analyzers Using Data Collection Software v3 0 30 Complete the Plate Record for Manual Analysis Complete the Plate Record for Autoanalysis l In the Sample Name column of a row enter a sample name then click the next cell The value 100 is automatically displayed in the Priority column Optional In the Comment column enter any additional comments or notations for the sample In the Results Group 1 column select a group from the drop down list In the Instrument Protocol 1 column select a module from the drop down list When you use the 3130 3130x Genetic Analyzers with Data Collection Software v3 0 and the AmpF STR kits Applied Biosystems recommends that you select the HIDFragmentAnalysis36 POP4 1 module If you want to perform more than one run a Select Edit gt Add Sample Run Additional Results Group Instrument Protocol and Analysis Protocol columns are added to the right end of the plate record b Complete the Results Group and Instrument Protocol columns for the additional runs Click OK to save your changes then close the plate record IMPORTANT After clicking
35. ength cm capillary array length c Click Send Command The array fill is finished when Send Command becomes active d Return the buffer reservoir to the capillaries Cleaning the Reservoirs 1 Press the Tray button to move the autosampler forward 2 Open the doors then remove the Plates Cathode buffer reservoir and water reservoirs 3 Dispose of remaining fluids and rinse out the reservoirs with deionized water Note Follow your company s waste disposal practices for appropriate disposal procedures Protocols for Processing AmpF4STR PCR Amplification Kit PCR Products 63 Applied Biosystems 3130 3130xl Genetic Analyzers Using Data Collection Software v3 0 4 Rinse the cathode reservoir with 1X running buffer and then fill 5 to the line with 1X running buffer about 16 mL Nnm CHEMICAL HAZARD 1X Genetic Analyzer Buffer with EDTA may cause eye skin and respiratory tract irritation Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Fill line GRIB57 Fill the three water reservoirs to the line with quality deionized water about 16 mL N i e Be sure that the septa fit snugly and flush on the tops of the reservoirs in order to prevent damaging the capillary tips 6 64 Place a clean reservoir septa on each reservoir and dry the outside of the reservoirs using a lint free wipe Place the reservoirs into positi
36. ers GSG Consequently this user bulletin is meant to be used in conjunction with the 3 30 3130xl Genetic Analyzers GSG When tasks required to perform HID analysis are the same as those described in the 3 30 3130xl Genetic Analyzers GSG you are referred to the appropriate page number in the 3730 3130xl Genetic Analyzers GSG IMPORTANT Be sure to use the correct revision of the 3130 3130xl Genetic Analyzers GSG Part Number 4352715 Revision B PN 4352715 Rev B If you do not use PN 4352715 Rev B the referenced page numbers may be incorrect If you do not have this revision you can download it from Applied Biosystems website see How to Obtain Support on page 2 Applied Biosystems 3130 3130xl Genetic Analyzers Using Data Collection Software v3 0 When tasks required to perform HID analysis are different from those described in the 3 30 3130xl Genetic Analyzers GSG you are referred to the appropriate page number in this user bulletin The user bulletin references are highlighted in blue text for pdf files or bold text for printed documents Preparing the Instrument Workflow To prepare the instrument for a run necessary maintenance tasks IMPORTANT As part of instrument maintenance you must change or replenish polymer weekly For HID analysis be sure to use 3130 POP 4 polymer PN 4352755 Nee CHEMICAL HAZARD POP 4 polymer causes eye skin and respiratory tract irritation R
37. g 1 0 ng 0 5 ng 0 25 ng Is Figure 16 DNA sample amplified using the Profiler Plus kit 52 User Bulletin Genotype Concordance Genotype Concordance The data summarized in Table 1 below were obtained by amplifying the positive Control DNA 007 9947a and 9948 samples using the AmpF STR Identifiler and Profiler Plus PCR Amplification Kits Additionally Control DNA 007 and 9948 samples were amplified with the AmpF STR Yfiler PCR Amplification Kit The samples were prepared for electrophoresis by combining the following For the four dye kit 1 uL of PCR product with 8 5 uL of Hi Di formamide and 0 5 uL of GeneScan 500 ROX Size Standard For the five dye kits 1 uL of PCR product with 8 7 uL of Hi Di formamide and 0 3 uL of GeneScan 500 LIZ Size Standard Each DNA sample was replicated six times on the plate and injected twice on one 3130 Genetic Analyzer and two 3130x Genetic Analyzers for a total of 36 datapoints per allele The Data Collection Software v3 0 run module for fragment analysis HIDFragmentAnalysis36 POPA4 1 was used to run the samples Data were analyzed using GeneMapper ID Software v3 2 to evaluate genotype concordance Table 1 Genotype concordance a AmpF STR Kit since Ena Evaluated 007 Identifiler kit 1044 10096 007 Yfiler kit 612 10096 007 Profiler Plus kit 648 100 9947a Identifiler kit 936 10096 9947a Profiler Plus kit 540 10096 9948 I
38. he 3130 Genetic Analyzer gt k sa kn K l n n ee ose RR done Examples of DNA Profiles Generated on the 3130xl Genetic AINAT ces Saca ando adobe dolce de dc eee apr 2064i 4 Genotype Concordat 4 seco dik d4 W X EAR ERRARE End Protocols for Processing AmpF4STR PCR Amplification Kit PCR Products 35 Applied Biosystems 3130 3130xl Genetic Analyzers Using Data Collection Software v3 0 Examples of DNA Profiles Generated on the 3130 Genetic Analyzer Overview This section contains examples of data generated on the 3130 Genetic Analyzer during the Verification Study The experiments conducted on the 3130 Genetic Analyzer are as follows Reproducibility page 36 Mixture Studies page 38 Resolution page 40 Sensitivity page 41 All studies were performed using Data Collection Software v3 0 and GeneMapper JD Software v3 2 All DNA samples used in the studies were quantitated using the Quantifiler Human DNA Quantification Kit Reproducibility Figure 1 shows a comparison of signal intensity across a 3130 instrument capillary array using AmpF STR Control DNA 9948 1 ng amplified using the AmpF STR Yfiler PCR Amplification Kit 110 120 130 140 450 160 170 180 130 200 210 220 230 240 250 260 270 280 230 300 310 320 n Capillary 1 Capillary 2 110 120 130 140 450 460 170 180 480 200 210 220 230 240 250 260 270 280 290 300 310 320
39. i6 POP F Note The software automatically fills in the appropriate well numbers for a single run 7 Click OK to save your changes then close the plate record IMPORTANT After clicking OK within the Plate Editor the completed plate record is stored in the Plate Manager database Once in the Plate Manager database the plate record can be searched for edited exported or deleted 20 User Bulletin Performing a Spectral Calibration Prepare the Spectral Calibration Chemistry Prepare the Follow this procedure if you are setting up spectral matrix Matrix Standards for Dye Set G5 calibration standards for kits using a five dye system which includes the 6 FAM VIC NED PET and LIZ dyes 1 Thoroughly vortex the Matrix Standard Set DS 33 tube for Dye Set GS 2 Spin the tube briefly in a microcentrifuge 3 Prepare Matrix Standard Set DS 33 for Dye Set G5 by combining the following in a labeled 1 5 mL microcentrifuge tube Volume pL Volume pL Reagent 3130xl 3130 System System Matrix Standard Set DS 33 10 10 Hi Di Formamide 190 190 Final Volume 200 200 S O i o Norme CHEMICAL HAZARD Hi Di Formamide Exposure causes eye skin and respiratory tract irritation It is a possible developmental and birth defect hazard Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Vortex thoroughly Spin the mixture briefly
40. ions Value Autosampler Initialize autosampler Bring autosampler to forward position Initialize and return to previous position N A Move autosampler up down A number between 500 and 500 steps Move autosampler to site Buffer left front for 1X running buffer home position Water1 left rear for deionized water Water2 right front for deionized water Waste right rear for deionized water Polymer Deliver Pump Initialize polymer delivery pump Home piston Read piston position N A Move piston down 1 to 38000 counts Move piston up 1 to 38000 counts Fill capillary array 1 to 38000 counts heater temperature Buffer Valve Close Open buffer Close valve Open Detection Cell Turn On Off On Heater detection cell heater or Read detection cell N A Protocols for Processing AmpF4STR PCR Amplification Kit PCR Products 83 Applied Biosystems 3130 3130xl Genetic Analyzers Using Data Collection Software v3 0 Command F nctlon Command Options Value Oven Turn On Off oven e On Off Set oven A number between 18 and temperature 70 C Read oven N A temperature Using Manual Note Manual control functions cannot be used during a run Control 1 In the tree pane of the Data Collection software click amp GA Instruments gt J ga3130 or ga3130x EQ instrument name gt amp Manual Co
41. iosystems 3130 31 30x Genetic Analyzers includes patented technology licensed from Hitachi Ltd as part of a strategic partnership between Applied Biosystems and Hitachi Ltd as well as patented tech nology of Applied Biosystems GeneMapper ID Software For Research Forensic or Paternity Use Only Not for use in diagnostic procedures NOTICE TO PURCHASER LICENSE DISCLAIMER Purchase of this software product alone does not imply any license under any process instrument or other apparatus system composition reagent or kit rights under patent claims owned or otherwise controlled by Applied Biosys tems either expressly or by estoppel GeneMapper D Software version 3 2 has undergone a verification process defined by Applied Biosystems However human identification laboratories analyzing forensic paternity databasing and single source samples that choose to use GeneMapper D Software for data analysis should perform their own appropriate validation studies TRADEMARKS ABI PRISM AmpF STR Applied Biosystems COfiler GeneMapper Identifiler LIZ PET Profiler Plus SGM Plus and VIC are registered trademarks and AB Design Applera GeneScan Hi Di iScience Design JOE NED POP POP4 POP 6 POP 7 Quantifiler ROX SEfiler Yfiler SFAM and 6FAM are trademarks of Applied Biosystems or its subsidiaries in the U S and or certain other countries All other trademarks are the sole property of their respective owner
42. laying single nucleotide resolution Protocols for Processing AmpF4STR PCR Amplification Kit PCR Products 49 Applied Biosystems 3130 3130xl Genetic Analyzers Using Data Collection Software v3 0 Sensitivity Figure 14 shows dilutions for the AmpF STR Control DNA 9948 sample amplified using the AmpF STR Identifiler PCR Amplification Kit 1 0 ng 0 5 ng 0 25 ng and 0 125 ng of input DNA The Y axis is magnified for lower input DNA amounts A full profile is presented in each panel a 1 0 ng 0 5 ng 0 25 ng 0 125 ng Figure 14 DNA sample amplified using the Identifiler kit 50 User Bulletin Examples of DNA Profiles Generated on the 3130xl Genetic Analyzer Figure 15 shows dilutions for the AmpF STR Control DNA 9948 sample amplified using the Yfiler kit 1 0 ng 0 5 ng 0 25 ng and 0 125 ng of input DNA The Y axis is magnified for lower input DNA amounts A full profile is presented in each panel 1 0 ng 0 5 ng 0 25 ng 0 125 ng Figure 15 DNA sample amplified using the Yfiler kit Protocols for Processing AmpF4STR PCR Amplification Kit PCR Products 51 Applied Biosystems 3130 3130xl Genetic Analyzers Using Data Collection Software v3 0 Figure 16 shows dilutions for the AmpF STR Control DNA 9948 sample amplified using the Profiler Plus kit 2 0 ng 1 0 ng 0 5 ng and 0 25 ng of input DNA The Y axis is magnified for lower input DNA amounts A full profile is presented in each panel 2 0 n
43. le is used in place of a run module the Calibration Perform a spectral calibration When you use a new dye set on the instrument When you change capillary array length or polymer type Note For every fragment analysis dye set you must create a separate spectral calibration for each capillary array length and polymer type combination you use After the laser or CCD camera has been realigned replaced by a service engineer f you begin to see a decrease in spectral separation pull up and or pull down peaks Workflow To perform a spectral calibration Step Task See Choose the correct dye set and matrix standard set for the AmpF STR kit you are using Note New matrix standard sets are available for all validated multi capillary instruments that is the 3100 3100 Avant Genetic Analyzers and the 3130 3130x Genetic Analyzers page 14 of this user bulletin 12 User Bulletin Performing a Spectral Calibration Step Task See 2 Select a spectral calibration protocol for Dye Set page 15 of this user bulletin G5 or Dye Set F and create a plate record IMPORTANT For HID applications the default condition number boundaries for the Dye Set G5 spectral calibration protocol should be edited in Data Collection Software v3 0 You will only need to perform this edit once See Edit the Dye Set G5 Spectral Calibration Protocol on page 15 of this
44. lied Biosystems Technical Support and Sales facilities 2 User Bulletin New Features and Design Changes New Features and Design Changes Instrument Polymer delivery system PDS Modifications Applied Biosystems has taken a proven PDS technology from the Applied Biosystems 3730 DNA Analyzer and applied it to the 4 and 16 capillary instruments The PDS improves instrument performance and maintenance by reducing manual intervention The primary benefit lies in the automation of the polymer delivery process The automated PDS of the 3130 3130x Genetic Analyzers does not use polymer syringes Eliminating the manual steps for filling priming and loading polymer provides faster easier and more reliable instrument setup You simply load the polymer bottle onto the instrument then perform setup using the software wizards see Data Collection Software Modifications on page 4 Note The positions of the waste and rinse reservoirs are reversed on the 3130 3130x Genetic Analyzers as compared to the 3100 3100 Avant Genetic Analyzers See step 7 on page 64 Oven door Applied Biosystems has changed the composition of the gasket material used to seal the oven door CCD camera shutter This modification affects the CCD camera shutter only It does not alter mechanically or introduce variation to the performance of the CCD camera or optical path components Detection cell heater The detection cell heater is only activated when
45. ll The value 100 automatically displays in the Priority column b Optional In the Comments column enter any additional comments or notations for the sample at the corresponding position of the plate c In the Instrument Protocol 1 column select Spectral36 POP4 G5 6 Highlight the entire row then select Edit gt Fill Down Special The software automatically fills in the appropriate well numbers for a single run W Spectral Calibration Plate Editor Plate Name E Operator AB Plate Sealing Benta Owner E Well Sample Name Comment Priority Instrument Protocol 1 EJ Spectral DyeSetG5 100 Spectrai3 POP4 G5 B01 Spectral_DyeSetGs 400 Spectrai36 POP4 G5 G01 Spectral DyeSetGS 100 Spectrai3 POP4 G5 D01 Spectral DyeSetG5 100 Spectral36_POP4_G5 Em FOI Got HM A0 v Description ox Cancel 18 User Bulletin Performing a Spectral Calibration 7 Click OK to save your changes then close the plate record IMPORTANT After clicking OK within the Plate Editor the completed plate record is stored in the Plate Manager database Once in the Plate Manager database the plate record can be searched for edited exported or deleted For Dye SetF Follow this procedure if you are performing a spectral calibration for kits using a four dye system which includes the 5FAM JOE NED and ROX dyes 1 In the tree pane of the Data Collection software click amp GA Instrume
46. ls for Processing AmpF4STR PCR Amplification Kit PCR Products 71 Applied Biosystems 3130 3130xl Genetic Analyzers Using Data Collection Software v3 0 Before Using the 1 Remove the polymer from 4 C storage Polymer 2 Loosen the cap and bring the polymer to room temperature 3 To dissolve deposits tighten the cap and gently swirl the polymer Replenishing the Polymer Nem CHEMICAL HAZARD POP polymers cause eye skin and respiratory tract irritation Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves IMPORTANT Wear gloves when you handle the polymer 1 Select Wizards gt Replenish Polymer Wizard ILE Help Install Array Wizard Change Polymer Type Wizard Replenish Polymer Wizard Bubble Remove Wizard Water Wash Wizard Instrument Shutdown Wizard Autosampler Calibration Wizard Update Cap Array Info 2 Follow the directions given in the wizard to load fresh polymer on the instrument 3 Relink plate s if applicable 72 User Bulletin Fluids and Waste Changing to a Different Polymer NEE CHEMICAL HAZARD POP polymers Type cause eye skin and respiratory tract irritation Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves IMPORTANT Wear gloves when you handle the polymer 1 Select Wizards gt Change Polymer Type Wizard WERE Help Install Array Wizard Change Pol
47. manual You can prepare the formamide size standard mixture for each individual sample or prepare a bulk for all samples in the run 1 Combine the following in a single microcentrifuge tube Volume pL Preparation Type Reagent Dye Set F Dye Set G5 Individual sample GeneScan 500 ROX Size 0 5 preparation Standard For each sample GeneScan 500 LIZ9 Size Standard 0 3 combine We Hi Di Formamide 8 5 8 7 Bulk sample GeneScan 500 ROX Size Standard 10 ARM GeneScan 500 LIZ Size Standard 6 For 16 samples T combine Hi Di Formamide 170 174 Note Prepare the appropriate size standard formulation for your dye set NITE CHEMICAL HAZARD Hi Di Formamide Exposure causes eye skin and respiratory tract irritation It is a possible developmental and birth defect hazard Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves 2 Vortex the tube to mix then spin briefly in a microcentrifuge 32 User Bulletin Performing a Sample Run Load Your Samples Load the 1 In a 96 well reaction plate Samples and Dispense 9 uL ofthe formamide size standard mixture into Allelic Ladder each well that will contain sample or allelic ladder Add 10 uL of the formamide to each blank well Neem CHEMICAL HAZARD Formamide Exposure causes eye skin and respiratory tract irritation It is a possible developmental and
48. mpF STR SGM Plus PCR Amplification Kit e Other four dye AmpF STR kits Run Module 24 hr ene Capillary Run T TS Run Module Length Polymer Time Reso aion aran cm min 3130 3130x Analyzer Analyzer HIDFragment 36 POP 4 45 2 560 10 240 500 0 15 Analysis36_POP4_1 a 20 GT genotypes capillary injection b 1 bp resolution at 99 99 accuracy User Bulletin Section 1 Section 1 Performing HID Analysis Performing HID Analysis This section covers the following topics How to Perform the Tasks in This Section T Preparing the Instrament auda eese 2 80 4 3 iu ened or eee 8 Performing a Spatial Calibration u A a l k l l wa 11 Performing a Spectral Calibration 12 006 2u a ke rte 12 Performing a Sample Rut sos sxe aca ka a haka RI RR k kd a 25 How to Perform the Tasks in This Section Protocols for Processing AmpF4STR PCR Amplification Kit PCR Products In this section the tasks required to perform HID analysis on the 3130 130x Genetic Analyzers are grouped under four major steps 1 Preparing the Instrument on page 8 2 Performing a Spatial Calibration on page 11 3 Performing a Spectral Calibration on page 12 4 Performing a Sample Run on page 25 Many of the tasks listed under each of these major steps are described in detail in the Applied Biosystems 3130 3130xl Genetic Analyzers Getting Started Guide 3130 3130xl Genetic Analyz
49. n DNA Quantification Kit Reproducibility Figure 9 shows a comparison of signal intensity across a 3130x instrument capillary array using AmpF STR Control DNA 9948 1 ng amplified using the AmpF STR Yfiler PCR Amplification Kit 110 120 430 140 150 160 170 180 430 200 210 220 230 240 250 260 270 280 230 300 310 320 n a oO Capillary 4 Capillary 10 Capillary 12 Capillary 14 Figure 9 DNA samples amplified using the Yfiler kit 44 User Bulletin Examples of DNA Profiles Generated on the 3130xl Genetic Analyzer Figure 10 shows a comparison of signal intensity across a 3130x instrument capillary array using AmpF STR Control DNA 9948 2 ng amplified using the AmpF STR Profiler Plus PCR Amplification Kit 400 110 120 430 140 150 160 170 480 190 200 210 220 230 240 250 260 270 280 290 300 310 320 AAA e ee eei Capillary 4 400 110 120 430 140 450 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 4000 E A M E A A E A A E A E E A E E A M E A E E A A HE A E E e M i i Capillary 10 100 110 120 430 140 450 160 170 480 190 200 210 220 230 240 250 260 270 280 230 300 310 320 9 EM i Capillary 12 Capillary 14 Figure 10 DNA samples amplified using the Profiler Plus kit Protocols for Processing AmpF4STR PCR Amplification Kit PCR Products 45 Applied Biosystems 3130 3130xl Genetic Analyzers Using
50. ntrol f GA trmerts gt gu 51503 Dev gt Manusi Corr Manual Control 2 In the Send Defined Command For drop down list select a function 84 User Bulletin Manual Control 3 In the Command Name drop down list select a command and enter a value if required Note The command names are filtered based the function selected in step 2 4 Click Send Protocols for Processing AmpF4STR PCR Amplification Kit PCR Products 85 Copyright 2005 2010 Applied Biosystems All rights reserved 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 In no event shall Applied Biosystems be liable for incidental special multiple or consequential damages in connection with or arising from the use of this document Applied Biosystems 3130 3130x Genetic Analyzers For Research Use Only Not for use in diagnostic procedures NOTICE TO PURCHASER This instrument is Authorized for use in DNA sequencing and fragment analysis This authorization is included in the purchase price of this instrument and corresponds to the up front fee component of a license under process claims of U S patents and under all process claims for DNA sequence and fragment analysis of U S patents now or hereafter owned or licensable by Applied Biosys
51. nts gt E ga3130x or ga3130 gt Plate Manager to open the Plate Manager window 2 Click New to open the New Plate dialog box 3 Complete the New Plate dialog box New Plate Dialog Name Spectral_Cal_DyeSetF Description Application Spectral Calibration Plate Type 96 well v Owner Name AB Operator Name AB a Name Type a name for the plate b Description If desired type a description for the plate c Application Select Spectral Calibration from the drop down list d Plate Type Select 96 Well e Owner Name Type a name for the owner Operator Name Type a name for the operator Protocols for Processing AmpF4STR PCR Amplification Kit PCR Products 19 Applied Biosystems 3130 3130xl Genetic Analyzers Using Data Collection Software v3 0 4 Click OK to open the Spectral Calibration Plate Editor 5 Complete the Spectral Calibration Plate Editor a In the Sample Name column enter a sample name then click the next cell The value 100 automatically displays in the Priority column b Optional In the Comments column enter any additional comments or notations for the sample at the corresponding position of the plate c In the Instrument Protocol 1 column select Spectral36_POP4_F 6 Highlight the entire row then select Edit gt Fill Down Special A Spectral Calibration Plate Editor Eee Spectral Dyesetl r Spectral DyesntF MD 5pecirai POP4 F Spectral DyeSelF HD Specira
52. on on the autosampler as shown below Water reservoir Water reservoir waste rinse J ua Cathode reservoir i Water reservoir 1X running buffer spare rm Home position Close the instrument doors Note Closing the doors returns the autosampler to the home position placing the tips of the capillaries in buffer Shut down the computer and turn off the instrument User Bulletin Shutting Down the Instrument Performing a IMPORTANT Make sure all parts are completely dry before long Long Term term storage Shutdown Select Instrument Shutdown Wizard and follow the prompts Help Install Array Wizard Change Polymer Type Wizard Replenish Polymer Wizard Bubble Remove Wizard Water Wash Wizard Instrument Shutdown Wizard Autosampler Calibration Wizard Update Cap Array Info Protocols for Processing AmpF4STR PCR Amplification Kit PCR Products 65 Applied Biosystems 3130 3130xl Genetic Analyzers Using Data Collection Software v3 0 Wizards 66 Accessing Wizards General Use Guidelines In the tree pane of the Data Collection software click amp GA Instruments gt ga3130 or ga3130x gt CJ instrument name or any topic name below instrument name to see Wizards in the menu bar The wizards in the Data Collection software guide you through several maintenance procedures Install Array Wizard Change Polymer Type Wizard Replenish Polymer Wizard Bubble Remove Wizard Water Wash
53. or the solid green light 5 Launch the Data Collection software Service Console applications start automatically Moving and Leveling the Instrument N uus PHYSICAL INJURY HAZARD Do not attempt to lift the instrument or any other heavy objects unless you have received related training Incorrect lifting can cause painful and sometimes permanent back injury Use proper lifting techniques when lifting or moving the instrument Two or three people are required to lift the instrument depending upon instrument weight 6 Remove the following components from the instrument Any plate assemblies from the autosampler Water and buffer reservoirs from the autosampler Capillary array by selecting Instrument Shutdown Wizard See Performing a Long Term Shutdown on page 65 Anode buffer reservoir 7 Switch off the breaker on the back of the instrument 8 Disconnect the power cord and the Ethernet cable IMPORTANT While moving the instrument avoid any shock or vibration 9 Move the instrument 10 Place the bubble level on the autosampler deck 11 Turn the instrument legs to level the instrument To move the instrument corner Turn the leg up right clockwise down left counterclockwise Protocols for Processing AmpF4STR PCR Amplification Kit PCR Products 61 Applied Biosystems 3130 3130xl Genetic Analyzers Using Data Collection Software v3 0 Shutting Down the Instrument Perform
54. ounterclockwise Hold an empty tube or beaker under the exit fitting to receive approximately 5 mL of waste Flush the trap by pushing steadily on the syringe plunger IMPORTANT DO NOT USE EXCESSIVE FORCE when you push the syringe plunger as this may damage the trap seals Take approximately 30 seconds to flush 5 mL of either distilled or deionized water through the trap Note Because the water trap volume is approximately 325 uL a relatively small volume of water is adequate for complete flushing However a larger volume only improves flushing as long as force and flow rate are kept within the limits given above User Bulletin Flushing and Filling the Water Trap A 6 Close the fittings in this order by turning each clockwise until the fittings seal against the block a Luer fitting b Exit fitting IMPORTANT Do not over tighten the fittings Very little pressure develops within the trap during pump operation so the fittings require only enough tightening to prevent water leaks Excessive tightening can damage the fittings c Remove the syringe from the Luer fitting Hold the fitting with one hand while turning the syringe counterclockwise with the other hand Protocols for Processing AmpF4STR PCR Amplification Kit PCR Products 69 Applied Biosystems 3130 3130xl Genetic Analyzers Using Data Collection Software v3 0 Fluids and Waste Buffer When to Change inm CHEMICAL HAZARD 10X Gene
55. plified using the Yfiler kit 1 0 ng 0 5 ng 0 25 ng and 0 125 ng of input DNA The Y axis is magnified for lower input DNA amounts A full profile is presented in each panel 1 0 ng 0 5 ng 0 25 ng 0 125 ng Figure 7 DNA sample amplified using the Yfiler kit 42 User Bulletin Examples of DNA Profiles Generated on the 3130 Genetic Analyzer Figure 8 shows dilutions for the AmpF STR Control DNA 9948 sample amplified using the Profiler Plus kit 2 0 ng 1 0 ng 0 5 ng and 0 25 ng of input DNA The Y axis is magnified for lower input DNA amounts A full profile is presented in each panel 2 0 ng tii 1 0 ng 0 5 ng 0 25 ng Figure 8 DNA sample amplified using the Profiler Plus kit Protocols for Processing AmpF4STR PCR Amplification Kit PCR Products 43 Applied Biosystems 3130 3130xl Genetic Analyzers Using Data Collection Software v3 0 Examples of DNA Profiles Generated on the 3130x Genetic Analyzer Overview This section contains examples of data generated on the 3130x Genetic Analyzer during the Verification Study The experiments conducted on the 3130x Genetic Analyzer are as follows Reproducibility page 44 Mixture Studies page 46 Resolution page 49 Sensitivity page 50 All studies were performed using Data Collection Software v3 0 and GeneMapper JD Software v3 2 All DNA samples used in the studies were quantitated using the Quantifiler Huma
56. s Science iScience To better understand the complex interaction of biological systems life scientists are developing revolutionary approaches to discovery that unite technology informatics and traditional laboratory research In partnership with our customers Applied Biosystems provides the innovative products services and knowledge resources that make this new Integrated Science possible 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 Applied Biosystems is committed to providing the world s leading technology and information for life scientists 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 Applied Bipsystems Part Number 4363787 Rev B
57. t only when the capillary array will be unused for less than 1 week To store the capillary array on the instrument follow the instructions to perform a short term shutdown described on page 63 Store the capillary array off of the instrument when the capillary array will be unused for longer than 1 week IMPORTANT Before storing the capillary array for long periods fill the capillaries with fresh polymer IMPORTANT Wear gloves while performing the following procedure and any other time you handle the capillary array septa or buffer reservoirs Nee CHEMICAL HAZARD POP polymers cause eye skin and respiratory tract irritation Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Neen CHEMICAL HAZARD 1X Genetic Analyzer Buffer with EDTA may cause eye skin and respiratory tract irritation Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Protocols for Processing AmpF4STR PCR Amplification Kit PCR Products 77 Applied Biosystems 3130 3130xl Genetic Analyzers Using Data Collection Software v3 0 Remove the capillary array from the instrument by selecting Install Array Wizard IEEE Help Install Array Wizard Change Polymer Type Wizard Replenish Polymer Wizard Bubble Remove Wizard Water Wash Wizard Instrument Shutdown Wizard Autosampler Calibration Wizard Update Cap Array Info
58. tems 3130 3130xl Genetic Analyzers Using Data Collection Software v3 0 10 Instrument Layout Pump block Polymer delivery pump PDP Oven Detection cell block and heater Capillary array Buffer and water reservoirs Autosampler Lower polymer block Anode buffer reservoir Part Function Anode buffer reservoir Contains 16 mL of 1X running buffer Buffer and water reservoirs four Each contain 16 mL of 1X running buffer or water Autosampler Holds the sample plates and reservoirs and moves to align the samples water or buffer with the capillaries Capillary array Enables the separation of the fluorescent labeled DNA fragments by electrophoresis It is a replaceable unit composed of 4 or 16 silica capillaries Detection cell block and heater Holds the capillaries in place for laser detection User Bulletin Performing a Spatial Calibration Part Function Lower polymer block Contains the buffer valve anode electrode and anode buffer reservoir Oven Maintains uniform capillary array temperature Polymer delivery pump PDP Pumps polymer into the array and performs maintenance procedures Pump block Includes the displacement pump chamber piston water seal array attachment point array port and connection to the lower polymer block through the interconnect tube Performing a Spatial Calibra
59. tems for which an Authorization is required and under cor responding process claims in foreign counterparts of the foregoing for which an Authorization is required The running royalty component of licenses may be purchased from Applied Biosystems or obtained by using Authorized reagents purchased from Authorized suppliers in accordance with the label rights accompanying such reagents Purchase of this instrument does not itself convey to the purchaser a complete license or right to perform the above processes This instrument is also licensed under U S patents and apparatus and system claims in foreign counterparts thereof No rights are granted expressly by implication or by estoppel under composition claims or under other process or system claims owned or licensable by Applied Biosystems For more information regarding licenses please contact the Director of Licensing at Applied Biosystems 850 Lincoln Centre Drive Foster City California 94404 USA NOTICE TO PURCHASER The purchase price of the Applied Biosystems 3130 3130x Genetic Analyzers includes a grant of a limited non transferable license under U S Patent and method claims of its foreign counterparts to use this particular instrument for electrophoresis methods employing fluorescence as a means of detection No other licenses or rights are hereby conveyed either expressly by implica tion or estoppel including but not limited to any claims to a composition The Applied B
60. the rest of the plate record based on the samples loaded in your plate do one of the following If the column contains Then the same sample types and highlight the entire column the same protocols then select Edit gt Fill Down Special Based on the plate type 96 well and capillary array 16 or 4 capillaries you are using the software automatically fills in the appropriate well numbers for a single run different sample types and complete the plate record different protocols manually 13 If you want to perform more than one run a Select Edit gt Add Sample Run Additional Results Group Instrument Protocol and Analysis Protocol columns are added to the right end of the plate record b Complete the columns for the additional runs Protocols for Processing AmpF4STR PCR Amplification Kit PCR Products 31 Applied Biosystems 3130 3130xl Genetic Analyzers Using Data Collection Software v3 0 14 Click OK to save your changes then close the plate record IMPORTANT After clicking OK within the Plate Editor the completed plate record is stored in the Plate Manager database Once in the Plate Manager database the plate record can be searched for edited exported or deleted Prepare Your Sample Chemistry Perform PCR Prepare the Formamide Size Standard Mixture To prepare your DNA samples and perform PCR follow the instructions in the appropriate AmpF STR PCR Amplification Kit user
61. tic Analyzer the Buffer Buffer with EDTA may cause eye skin and respiratory tract irritation Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Change the buffer before each batch of runs or at least every 24 hours Making Buffer for To prepare 50 mL of 1X running buffer a Single Run 1 Into a graduated cylinder add a 5mL of 10X Genetic Analyzer buffer b 45 mL of deionized water to bring the total volume up to 50 mL E 50 mL r 45 mL of DI HO Y I 5 mL of 10X Genetic 50 mL graduated cylinder Analyzer Durer 2 Mix well 70 User Bulletin Fluids and Waste Storing the Buffer The 1X running buffer can be stored at 2to 8 C for up to 1 month Room temperature for 1 week Buffer Storage Conditions Option A Option B 2 C to 20 C to 8 C 25 C SuM T WThF S SuM TWThF S 1 month 7 days Polymer Storing Polymer Store any remaining POP polymer at 2 to 8 C until the expiration date printed on the jar Note Excessively hot environments may shorten the working life of the polymer When to Change Change the polymer weekly The polymer is good at 25 C for about the Polymer 7 days POP 4 or POP 6 Polymer Storage Conditions SuM TWThF S 20 C to 25 C 7 days Protoco
62. tifiler kit User Bulletin Examples of DNA Profiles Generated on the 3130 Genetic Analyzer 100 160 170 180 190 200 210 220 230 240 250 260 270 280 230 300 310 320 In Figure 4 the first and last panels display profiles of each DNA sample amplified individually with a total DNA input of 2 ng using the AmpF STR Profiler Plus PCR Amplification Kit The female AmpF STR Control DNA 9947a sample is in the top panel and the male AmpF STR Control DNA 9948 sample is in the bottom panel The other three panels display the mixtures of DNA at approximate ratios of 1 1 1 5 and 1 10 2400 1600 800 0 400 160 170 180 190 200 210 220 230 240 250 260 270 280 280 300 310 320 M E mn Ter Ar 400 0 100 140 2 130 idi MM n 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 amar er rr I A a A A ee a ee eS 800 1 5 1 400 0 400 160 170 180 190 200 210 220 230 240 250 260 270 280 230 300 310 320 800 1 400 0 100 160 170 180 130 200 210 220 230 240 250 260 270 280 230 300 310 320 4 t tt 2400 4600 800 0 n 0 1 Figure 4 DNA samples amplified using the Profiler Plus kit Protocols for Processing AmpF4STR PCR Amplification Kit PCR Products 39 Applied Biosystems 3130 3130xl Genetic Analyzers Using Data Collection Software v3 0 3000 2000 4000
63. tion When to Perform The Data Collection software uses images collected during the spatial calibration to establish a relationship between the signal emitted by each capillary and the position where that signal falls on and is detected by the CCD camera the Calibration You are required to perform a spatial calibration when you Install or replace a capillary array Temporarily remove the capillary array from the detection block Move the instrument Workflow To perform a spatial calibration Step Task See 1 Create a spatial calibration file page 22 of the 3130 3130xl Genetic Analyzers GSG 2 Evaluate the spatial calibration file page 23 of the 3130 3130xl Genetic Analyzers GSG Protocols for Processing AmpF4STR PCR Amplification Kit PCR Products 11 Applied Biosystems 3130 3130xl Genetic Analyzers Using Data Collection Software v3 0 Performing a Spectral Calibration When to Perform A spectral calibration creates a matrix that corrects for the overlapping fluorescence emission spectra of the dyes Although each of these dyes emits its maximum fluorescence at a different wavelength there is some overlap in the emission spectra between the dyes The goal of multicomponent analysis is to effectively correct for spectral overlap Performing a spectral calibration is similar to performing a sample run except that calibration standards are run in place of samples and a spectral calibration modu
64. user bulletin 3 Prepare the spectral calibration chemistry page 21 of this user bulletin 4 Load the spectral calibration samples page 32 of the 3130 3130xl Genetic Analyzers GSG 5 Place the plate assembly into the instrument page 39 of the 3130 3130xl Genetic Analyzers GSG 6 Run the spectral calibration plate which includes pages 40 to 42 of the 3130 3130xl e Genetic Analyzers GSG Linking the plate Starting the run Viewing the pass fail status 7 Evaluate the spectral calibration data page 43 of the 3130 3130xl IMPORTANT If you obtain off scale peaks in a Genetic Analyzers Kreta 3130 3130x Genetic Analyzer spectral calibration when using the new matrix standard sets see Troubleshooting Off Scale Spectral Calibration Peak Height on page 23 of this user bulletin 8 Activate the spectral calibration page 47 of the 3130 3130xl Genetic Analyzers GSG Protocols for Processing AmpF4STR PCR Amplification Kit PCR Products 13 Applied Biosystems 3130 3130xl Genetic Analyzers Using Data Collection Software v3 0 Choose the Dye Set and Matrix Standards Choose the appropriate dye set and matrix standard for the AmpF STR PCR Amplification Kit you are using as shown in the dyes table below For AmpF STR kits that lise a Use And use AmpF STR Kit Examples five dye system which Dye Set G5 Matrix Standard Set AmpF STR Identifiler PCR includes the following DS 33 Amplification Kit
65. w universal matrix standards are available for the multi capillary electrophoresis instruments that 1s the 3100 3100 4vant Genetic Analyzers and the 3130 3130x Genetic Analyzers Matrix Standard Set DS 32 Dye Set F PN 4345831 Matrix Standard Set DS 33 Dye Set G5 PN 4345833 The new matrix standard sets were designed with improved purity signal balance and signal strength If you obtain off scale peaks when using the new matrix standards for spectral calibration follow the procedure below Troubleshoot Ifthe peak height of any one of the matrix standard fragments is Off Scale Peak saturated off scale re run the spectral calibration but use one half Heights the amount of the matrix standards The mixture will be twice as dilute as before For Dye Set G5 1 Follow the procedure on page 21 Prepare the Matrix Standards for Dye Set G5 2 Instep 3 on page 21 use the following volumes Reagent vas yee zi iens x System Matrix Standard Set DS 33 5 5 Hi Di Formamide 195 195 Final Volume 200 200 NIRE CHEMICAL HAZARD Formamide Exposure causes eye skin and respiratory tract irritation It is a possible developmental and birth defect hazard Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Note These same instructions are present on the Matrix Standard Set DS 33 product insert Protocols for Processing AmpF4STR PCR
66. ymer Type Wizard Replenish Polymer Wizard Bubble Remove Wizard Water Wash Wizard Instrument Shutdown Wizard Autosampler Calibration Wizard Update Cap Array Info 2 Follow the wizard prompts Protocols for Processing AmpF4STR PCR Amplification Kit PCR Products 73 Applied Biosystems 3130 3130xl Genetic Analyzers Using Data Collection Software v3 0 Capillary Array When to Change A capillary array should last approximately 100 runs a Capillary Array The following problems may indicate that a new capillary array is required Poor sizing precision or allele calling Poor resolution and or decreased signal intensity Checking the Norme ELECTRICAL SHOCK FIRE HAZARD Do Cathode Bar not leave liquid on the cathode header This can lead to electric shock or even fire if not properly maintained When placing a used array back on the instrument be sure that the cathode bar is dry see figure below A wet bar could lead to arcing Back view Ensure the cathode header is dry especially in the center jo E G S 3130x instrument capillary array 3130 instrument capillary array GR2146 74 User Bulletin Capillary Array Installing IMPORTANT Wear gloves when you handle the polymer blocks Removing or Replacing a N Ecl CHEMICAL HAZARD POP polymers Capillary Array cause eye skin and respiratory tract irritation Read the MSDS an

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