Complete Protocol
Contents
1. Name Description Action Figure 1 The Dashboard The arrow highlights the preheat options Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TBD024 Revised 7 12 Page 5 9247TA Promega 3 B Instrument Preparation continued i Dashboard Edit Library Resources 2 To perform a spectral calibration with the Promega 5 dye chemistry a new dye set should be created If a new dye set was created previously proceed to Section 3 B Step 2 c a To create this new dye set navigate to the Library highlight Dye Sets and select Create b The Create a New Dye Set tab will appear Figure 2 Name the Dye Set select Matrix Standard for the Chemistry and select G5 Template for the Dye Set Template Select Save gt Library Maintenance Tools Manage Preferences Help Log Out E Create A Edit EQ Duplicate g Delete pM Import Export sey E Signature View Filter All x Manage ae Plates Assays Dye Set Chemistry Standard Calibration Date Capillary Array Serial Number i Signed File Name Conventions REAE Group SetupaD lilli Arasyze Instrument Protocols Size Standards HID Analysis Protocols Dye Set Name Promega G5 D Locked Sespcaing ROC Chemistry Matrix Standard gt Sizecaling Protocol2. 7 12 Page 17 10609TA 5 B Instrument Preparation continued 1 Prepare matrix samples as previously described in Section 5 A Note Differences in instrument sensitivity may result in peak imbalance or reduced peak height of the matrix standards You may need to adjust injection time or voltage to achieve a passing spectral calibration You also may need to prepare a new plate and adjust the dilution of individual matrix standards Peak heights in the range of 1 000 4 000RFU are ideal Peak heights above 750RFU and below the saturation point of the instrument are required 2 Perform the spectral calibration as described in the Applied Biosystems 3130 or 3130x1 Genetic Analyzer User s Manual with the following modifications a In the Module Manager select New Select Spectral in the Type drop down list and select Spect36_POP7 in the Template drop down list Confirm or change the following settings Inj kV 1 2 Inj Secs 18 Data Delay Time 275 Run time 700 seconds b Create a new name for the run module then select OK c In the Protocol Manager under Instrument Protocols select New Type a name for your protocol Make the following selections in the Protocol Editor Spectral in the Type drop down list e G5 in the DyeSet drop down list e POP for the polymer e 36 in the Array Length drop down list e Matrix Standard for the chemistry Select
3. Road gt Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TBD024 Printed in USA Page 2 Revised 7 12 3 Instrument Preparation and Spectral Calibration Using the Applied Biosystems 3500 and 3500xL Genetic Analyzers Materials to Be Supplied by the User e 95 C dry heating block water bath or thermal cycler e crushed ice or ice water bath e centrifuge compatible with 96 well plates e aerosol resistant pipette tips e 3500 3500xL capillary array 36cm e performance optimized polymer 4 POP 4 in a pouch for the 3500 or 3500xL e anode buffer container with 1X buffer e cathode buffer container with 1X buffer e conditioning reagent pouch for the 3500 or 3500xL e MicroAmp optical 96 well plate and septa e Hi Di formamide Applied Biosystems Cat 4311320 The quality of formamide is critical Use Hi Di formamide Freeze formamide in aliquots at 20 C Multiple freeze thaw cycles or long term storage at 4 C may cause breakdown of formamide Poor quality formamide may contain ions that compete with DNA during injection which results in lower peak heights and reduced sensitivity A longer injection time may not increase the signal Formamide is an irritant and a teratogen avoid inhalation and contact with skin Read the warning label and take the necessary precautions when handling this substance Always wear gloves and safety glasse
4. herein End Users may not use sequence s in an attempt to reverse engineer parameters of any of GE Healthcare Bio Sciences Corp proprietary products or services Disclaimer of Warranties GE Healthcare Bio sciences Corp provides no warranties to end user statutory or implied including without limitation as to product quality condition description merchantability or fitness for a particular purpose and all such warranties are hereby expressly disclaimed GE Healthcare Bio Sciences Corp hereby expressly disclaims any warranty regarding results obtained through the use of the products including without limitation any claim of inaccurate invalid or incomplete results Exclusion of Liability GE Healthcare Bio Sciences Corp and its affiliates shall have no liability to an End User including without limitation for any loss of use or profits business interruption or any consequential incidental special or other indirect damages of any kind regardless of how caused and regardless of whether an action in contract tort strict product liability or otherwise TMR ET CXR ET and CC5 dyes are proprietary 2009 2011 2012 Promega Corporation All Rights Reserved PowerPlex is a registered trademark of Promega Corporation ABI PRISM and MicroAmp are registered trademarks ofApplera Corporation Hi Di POP 4 and POP 7 are trademarks of Applera Corporation Products may be covered by pending or issued patents or may have certain limitatio
5. the matrix standards You may need to adjust injection time or voltage to achieve a passing spectral calibration You also may need to prepare a new plate and adjust the dilution of individual matrix standards Peak heights in the range of 1 000 4 000RFU are ideal Peak heights above 750RFU and below the saturation point of the instrument are required 2 Perform the spectral calibration as described in the ABI PRISM 3100 or 3100 Avant or Applied Biosystems 3130 or 3130x Genetic Analyzer User s Manual with the following modifications a In the Module Manager select New Select Spectral in the Type drop down list and select Spect36_POP4 in the Template drop down list Confirm or change the following settings Inj kV LZ Inj Secs 12 Data Delay Time 400 Run time 700 seconds Promega Corporation gt 2800 Woods Hollow Road gt Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TBD024 Printed in USA Page 12 Revised 7 12 8198TA b Create a new name for the run module then select OK c In the Protocol Manager under Instrument Protocols select New Type a name for your protocol Make the following selections in the Protocol Editor Spectral in the Type drop down list e G5 in the DyeSet drop down list e POP4 for the polymer e 36 in the Array Length drop down list e Matrix Standard f
6. the spectral module you created in the previous step in the Run Module drop down list Finally select Edit Parameters and make the following modifications e Change the lower condition bound to 4 0 and change the upper condition bound to 12 0 e Confirm that the Minimum Quality Score is 0 95 Select OK in the Edit Parameters window and select OK in the Protocol Editor Note The condition number C value obtained when generating a spectral calibration will vary with the instrument After obtaining a spectral calibration that performs acceptably the condition bounds range in the previous step may be narrowed to more critically evaluate C values for subsequent spectral calibrations Promega Corporation gt 2800 Woods Hollow Road gt Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TBD024 Printed in USA Page 18 Revised 7 12 d In the Plate Manager create a new plate record as described in the instrument user s manual In the dialog box that appears select Spectral Calibration in the Application drop down list and select 96 well as the plate type Add entries in the owner and operator windows name the plate and select OK e In the spectral calibration plate editor dialog box place sample names in the appropriate cells In the Instrument Protocol column select the protocol you created in Step 2 c Ensure th
7. 30 Genetic Analyzers four wells are used for spectral calibration on four capillaries wells A1 through D1 in a 96 well plate Load 25ul of fragment mix into each of the four wells Briefly centrifuge the plate to remove any bubbles 5 Denature samples at 95 C for 3 minutes then immediately chill on crushed ice or in an ice water bath for 3 minutes Denature samples just prior to loading the instrument Promega Corporation gt 2800 Woods Hollow Road gt Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TBD024 Revised 7 12 Page 15 Promega 5 B Instrument Preparation We have found that the use of fresh polymer and new capillary array results in an optimal spectral Representative raw data are shown in Figure 7 Another example of a passing spectral calibration in the Spectral Viewer is shown in Figure 8 1 5 10 15 Select capillaries bo dissa AH MMOMOOOOOOOOOOOTC Fo 1 2400 1800 20 1200 6 600 0 60 800 400 B00 100 1200 1400 1500 1800 27000 2200 Intensity s Scan Number Copliary 2 3200 2400 1600 300 0 300 0 Coptiary 3 1000 1200 intensity ys Sean Number Ceopliary 4 50 800 n J00 400 B00 nou 1000 1200 1400 1800 1800 2000 2900 intensity xs Scan Number Figure 7 Representative data for the PowerPlex 5 Dye Matrix Standards 3100 3130 on the Applied Biosystems 3130 Genetic Analyzer
8. 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TBD024 Revised 7 12 Page 11 Promega 4 B Instrument Preparation continued eo GA hetruments ga31 JO 3130 Spectra Viewer S Brun Hstary BSJEPT Viewer E Evart Log fe Instrument Protocol GM spatial Calibration vi Gi spectra Calbration ireextraction e E2320 EHJEPT Chart 40 100 140 200 250 m Intansity vs Pixel Number Spatial Run Schecuk B Run Schadder plate view run Yew By Capileries Viewer E cap array Viewer v Manua Contral EB Service Log 800 400 0 400 0 1000 2000 Intensityvs Scan Number 3000 4000 5000 Capillary Data Thu May 07 13 29 49 CDT 2009 AD Dye Set GS bd peer Active Calibretian for Dye Set GS E Matrix used far Capllary 3 3 Thu May 07 1229 49 CDT 2009 O ee 7 J x Condition 7 00167 a Yaue 0 396953 List of Cathrations for Dye Set GS Figure 6 Representative data for the PowerPlex 5 Dye Matrix Standards 3100 3130 on the Applied Biosystems 3130 Genetic Analyzer using POP 4 polymer and Data Collection Software Version 3 0 Figure shows the CC5 orange CXR ET red TMR ET yellow JOE green and fluorescein blue peaks in the Spectral Viewer from one of the four capillaries 1 Prepare matrix samples as previously described in Section 4 A Note Differences in instrument sensitivity may result in peak imbalance or reduced peak height of
9. Mix each matrix standard by vortexing for 5 10 seconds prior to use Do not centrifuge the matrix standards as this may cause the DNA to be concentrated at the bottom of the tube Place on ice Promega Corporation gt 2800 Woods Hollow Road gt Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TBD024 Printed in USA Page 14 Revised 7 12 2 Initial dilution of concentrated fragments Before combining the matrix standards dilute the individual matrix standards 1 10 in Nuclease Free Water as described below Vortex for 5 10 seconds to mix Place on ice Component Fluorescein JOE TMR ET CXR ET COS Concentrated Dye 5ul 5ul 5ul 5ul 5ul Nuclease Free Water 45ul 45ul 45 ul 45ul 45ul 3 Fragment mix using 1 10 dilutions of matrix standards After the initial dilution in Step 2 combine the 1 10 dilutions as directed below Vortex for 5 10 seconds to mix Component Volume Hi Di formamide A75ul Fluorescein from initial dilution SOM JOE from initial dilution 5 0ul TMR ET from initial dilution DOM CXR ET from initial dilution 5 0ul CC5 from initial dilution 5 0ul 4 On the Applied Biosystems 3130x Genetic Analyzer 16 wells are used for spectral calibration on 16 capillaries wells A1 through H2 of a 96 well plate Load 25u1 of fragment mix prepared in Step 3 into each of the 16 wells Briefly centrifuge the plate to remove bubbles On the Applied Biosystems 31
10. Technical Bulletin PowerPlex 5 Dye Matrix Standards 3100 3130 INSTRUCTIONS FOR USE OF PRODUCTS DG4700 Note These matrix fragments are compatible with the ABI PRISM 3100 and 3100 Avant and Applied Biosystems 3130 3130x1 3500 and 3500xL Genetic Analyzers PRINTED IN USA Revised 7 12 Part TBD024 PowerPlex 5 Dye Matrix Standards 3100 3130 All technical literature is available on the Internet at www promega com protocols Please visit the web site to verify that you are using the most current version of this Technical Bulletin Please contact Promega Technical Services if you have questions on use of this system E mail genetic promega com TSS CE UE GIN AAE A E E essa vacua maven E E 2 2 Product Components and Storage Conditions 0 00 esseseeeeeeneeneees 2 3 Instrument Preparation and Spectral Calibration Using the Applied Biosystems 3500 and 3500xL Genetic Analyzers 3 As Mairi nample Preparata aiie a Ea E a a 3 D loctenent Repara IOTE aiseria oenn E A E a 3 4 Instrument Preparation and Spectral Calibration Using POP 4 Polymer and Data Collection Software Version 2 0 or Version 3 0 ABI PRISM 3100 and 3100 Avant and Applied Biosystems 3130 and 3130x Genetic Analyzers eessessseeeesesrress 9 A Matix hample Trepat 16 roserne ERRi J De ASPECT PE parao siinne anpa E 11 5 Instrument Preparation and Spectral Calibration Using POP 7 Polymer and Data Collection Softwar
11. amatic increase or change in the C value between spectral runs on a single instrument that has not been serviced between the runs may indicate an instrument problem that requires service Spectral failure due to improper Clean the instrument and install a new array Q value and fresh polymer 7 Related Products Product Size Cat PowerPlex Y23 System 50 reactions DC2305 200 reactions DC2320 PowerPlex 21 System 200 reactions DC8902 PowerPlex 18D System 200 reactions DC1802 800 reactions DC1808 PowerPlex ESX 16 System 100 reactions DC6711 400 reactions DC6710 PowerPlex ESX 17 System 100 reactions DC6721 400 reactions DC6720 PowerPlex ESI 16 System 100 reactions DC6771 400 reactions DC6770 PowerPlex ESI 17 Pro System 100 reactions DC7781 400 reactions DC7780 Not for Medical Diagnostic Use Accessory Components Product Size Cat Nuclease Free Water 50ml P1193 Water Amplification Grade 6 250ul 5 x 1 25041 DW0991 PowerPlex 5 Dye Matrix Standards 310 50ul each dye DG4600 Not for Medical Diagnostic Use Promega Corporation gt 2800 Woods Hollow Road gt Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TBD024 Revised 7 12 Page 21 This product or portions thereof is manufactured and sold under license from GE Healthcare under patents Austria Pat No E236994 Australia Pat No 692230 Belgium Pat No 0743987 Swit
12. at this information is present for each row that contains a sample name Select OK f Run your plate as described in the instrument user s manual g Upon completion of the run check the status of the spectral calibration in the Event Log window For the Applied Biosystems 3130xl Genetic Analyzers we recommend that a minimum of 12 of the 16 capillaries pass calibration For the Applied Biosystems 3130 Genetic Analyzers we recommend that a minimum of three of four capillaries pass calibration If fewer than the recommended numbers of capillaries pass repeat the spectral calibration 6 Troubleshooting For questions not addressed here please contact your local Promega Branch Office or Distributor Contact information available at www promega com E mail genetic promega com 6 A Applied Biosystems 3500 and 3500xL Genetic Analyzers Symptoms Causes and Comments Fewer than the recommended Matrix standards were too dilute Matrix number of capillaries passed the samples that are too dilute will result in low spectral calibration peak heights which may result in spectral calibration failure or bleedthrough or oversubtraction in other dye colors Decrease the dilution of each fragment in Step 2 of Section 3 A Matrix standards were too concentrated Matrix samples that are too concentrated may result in spectral calibration failure or bleedthrough or oversubtraction in other dye colors Increase the dilution of each fragme
13. e Version 3 0 Applied Biosystems 3130 and 3130x Genetic Analyzers 14 A Matix Gam ple Preparatiounen ii a isiu 14 Bix Ais tunen Prepad igi aniser iN E 16 6 Troubleshoot ene ne eee erent e Perr erro ener ert ete eee rreere a reereererere 19 A Applied Biosystems 3500 and 3500xL Genetic Analyzers s s 19 B ABI PRISM 3100 and 3100 Avant and Applied Biosystems 3190 and o DON Genee ANALY ZENS aiioe 20 Pao Related Produ S nnna 21 Promega Corporation gt 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TBD024 Revised 7 12 Page 1 1 Description Proper generation of a spectral calibration file is critical to evaluate multicolor systems with the ABI PRISM 3100 and 3100 Avant and Applied Biosystems 3130 3130xl 3500 and 3500xL Genetic Analyzers The PowerPlex 5 Dye Matrix Standards 3100 3130 consists of DNA fragments labeled with five different fluorescent dyes one tube contains a DNA fragment labeled with fluorescein one tube contains a DNA fragment labeled with JOE one tube contains a DNA fragment labeled with TMR ET one tube contains a DNA fragment labeled with CXR ET and one tube contains a DNA fragment labeled with CCS These matrix fragments are mixed and used on the ABI PRISM 3100 or 3100 Avant or Applied Biosystems 3130 3130x1 3500 or 3500xL Genetic Analyzer to perform a spectra
14. have found that the use of fresh polymer and new capillary array results in an optimal spectral 1 Set the oven temperature to 60 C then select the Start Pre Heat icon at least 30 minutes prior to the first injection to preheat the oven Figure 1 Common Operations gt gt EE Quick Start Create Create Plate Edit Existing Run New Plate from Template Plate Run Results Quick View e v Gauges POP4 Polymer ABC Anode CBC Cathode 36cm 24 cap Array 192 3 4 3 4 64 Titep Wilp i i gi eT e oe aT eis er i ar iji 4 gt 1 ay 4 A ak P 6 r gt 38 a 4 4 E I4 IN 4384 0 a 0 pone on te 48 Samples Remaining 7 Days Remaining 7 Days Remaining 66 Injections Performed 6 Injections Remaining 46 Injections Remaining 46 Injections Remaining Instrument 3500 Instrument State Idle View AA Details Pre Heat the Oven Laser On Oven On Set Temperature to EP Off ocne Khe Oven Temperature C 60 0 i Detection Cell Temperature C 49 96 60 a CC Start Pre Heat Instrument Door Close v Consumables Information Consumable C Polymer POP4 48 Samples Remaining 5 06 Jan 2011 06 1006009 4393715 Anode Buffer ABC 7 Days Remaining 0 18 Mar 2011 07 1006014 4393927 Cathode Buffer CBC 7 Days Remaining 0 23 Mar 2011 07 1007021 4408256 Capillary Array 36cm 24 cap 94 Injections Remaining 51 03 Feb 2011 02 08 4404687 Serial M309K0803 v Maintenance Notifications
15. he run check the status of the spectral calibration in the Event Log window For the ABI PRISM 3100 and Applied Biosystems 3130x Genetic Analyzers we recommend that a minimum of 12 of the 16 capillaries pass calibration For the ABI PRISM 3100 Avant and Applied Biosystems 3130 Genetic Analyzers we recommend that a minimum of three of four capillaries pass calibration If fewer than the recommended numbers of capillaries pass repeat the spectral calibration Promega Corporation gt 2800 Woods Hollow Road gt Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TBD024 Revised 7 12 Page 13 5 Instrument Preparation and Spectral Calibration Using POP 7 Polymer and Data Collection Software Version 3 0 Applied Biosystems 3130 and 3130x Genetic Analyzers Materials to Be Supplied by the User e 95 C dry heating block water bath or thermal cycler e crushed ice or an ice water bath e centrifuge compatible with 96 well plates e 3130 or 3130xl capillary array 36cm e performance optimized polymer 7 POP 7 for the 3130 or 3130 1 e 10X genetic analyzer buffer with EDTA e MicroAmp optical 96 well plate e aerosol resistant pipette tips e Hi Di formamide Applied Biosystems Cat 4311320 Q The quality of formamide is critical Use Hi Di formamide Freeze formamide in aliquots at 20 C Multiple freeze thaw cycles or long te
16. in USA 800 356 9526 Part TBD024 Page 8 Madison WI 53711 5399 USA www promega com Printed in USA Revised 7 12 9327TA 4 Instrument Preparation and Spectral Calibration Using POP 4 Polymer and Data Collection Software Version 2 0 or Version 3 0 ABI PRISM 3100 and 3100 Avant and Applied Biosystems 3130 and 3130x Genetic Analyzers Materials to Be Supplied by the User e 95 C dry heating block water bath or thermal cycler e crushed ice or an ice water bath e centrifuge compatible with 96 well plates e 3100 or 3130 capillary array 36cm e performance optimized polymer 4 POP 4 for the 3100 or 3130 e 10X genetic analyzer buffer with EDTA e MicroAmp optical 96 well plate e aerosol resistant pipette tips e Hi Di formamide Applied Biosystems Cat 4311320 The quality of formamide is critical Use Hi Di formamide Freeze formamide in aliquots at 20 C Multiple freeze thaw cycles or long term storage at 4 C may cause breakdown of formamide Poor quality formamide may contain ions that compete with DNA during injection which results in lower peak heights and reduced sensitivity A longer injection time may not increase the signal Formamide is an irritant and a teratogen avoid inhalation and contact with skin Read the warning label and take appropriate precautions when handling this substance Always wear gloves and safety glasses when working with formamide 4 A Matrix Sample P
17. l calibration on a specified dye set The spectral calibration should be performed on dye set G5 Once generated this file is applied during sample detection to calculate the spectral overlap between the five different dyes and separate the raw fluorescent signals into individual dye signals The PowerPlex 5 Dye Matrix Standards 3100 3130 was developed for use with the 5 dye PowerPlex Systems A matrix should be generated for each individual instrument Protocols to operate the ABI PRISM 3100 or 3100 Avant or Applied Biosystems 3130 3130x1 3500 or 3500xL Genetic Analyzer should be obtained from the manufacturer 2 Product Components and Storage Conditions Product Size Cat PowerPlex 5 Dye Matrix Standards 3100 3130 25ul each dye DG4700 Not for Medical Diagnostic Use Includes 25ul Fluorescein Matrix 5 Dye 3100 3130 e 25ul JOE Matrix 5 Dye 3100 3130 25ul TMR ET Matrix 5 Dye 3100 3130 25ul CXR ET Matrix 5 Dye 3100 3130 25ul CC5 Matrix 5 Dye 3100 3130 e 125ml Nuclease Free Water Storage Conditions Store all components at 30 C to 10 C in a nonfrost free freezer The matrix standards fragments are light sensitive and must be stored in the dark We strongly recommend that the matrix standards be stored with post amplification reagents and used separately with different pipettes tube racks etc Do not store the diluted matrix standard fragments Promega Corporation gt 2800 Woods Hollow
18. l dilution of concentrated fragments Before combining the matrix standards dilute the individual matrix standards 1 10 in Nuclease Free Water as described below Vortex for 5 10 seconds to mix Place on ice Component Fluorescein JOE TMR ET CXR ET COG Concentrated Dye 5ul 5ul 5ul 5ul 5ul Nuclease Free Water 45ul 45ul 45ul 45ul 45 ul 3 Fragment mix using 1 10 dilutions of matrix standards After the initial dilution in Step 2 combine the 1 10 dilutions as directed below Vortex for 5 10 seconds to mix Component Volume Hi Di formamide A75ul Fluorescein from initial dilution 5 0ul JOE from initial dilution 5 0ul TMR ET from initial dilution 5 0ul CXR ET from initial dilution 5 0ul CC5 from initial dilution 5 0ul 4 On the ABI PRISM 3100 and Applied Biosystems 3130x Genetic Analyzers 16 wells are used for spectral calibration on 16 capillaries wells A1 through H2 of a 96 well plate Load 251 of fragment mix prepared in Step 3 into each of the 16 wells Briefly centrifuge the plate to remove bubbles On the ABI PRISM 3100 Avant and Applied Biosystems 3130 Genetic Analyzers four wells are used for spectral calibration on four capillaries wells A1 through D1 in a 96 well plate Load 25ul of fragment mix into each of the four wells Briefly centrifuge the plate to remove any bubbles 5 Denature samples at 95 C for 3 minutes then immediately chill on crushed ice or in an ice water bath for 3 minutes Denature samples just p
19. ns Please visit our Web site for more information All prices and specifications are subject to change without prior notice Product claims are subject to change Please contact Promega Technical Services or access the Promega online catalog for the most up to date information on Promega products Promega Corporation gt 2800 Woods Hollow Road gt Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TBD024 Printed in USA Page 22 Revised 7 12
20. nt in Step 2 of Section 3 A Samples were not denatured completely Incomplete denaturation can cause extra peaks Heat denature samples for the recommended time and cool on crushed ice or in an ice water bath immediately prior to loading the capillary Do not cool samples in a thermal cycler or 20 C freezer Promega Corporation gt 2800 Woods Hollow Road gt Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TBD024 Revised 7 12 Page 19 6 B ABI PRISM 3100 and 3100 Avant and Applied Biosystems 3130 and 3130x Genetic Analyzers Symptoms Causes and Comments Fewer than the recommended Peak heights for the matrix standards were too number of capillaries passed the low Increase the injection voltage or time If spectral calibration the matrix peak heights are below 1 000RFU decrease the dilution of each fragment in Step 2 of Section 4 A or 5 A and repeat the spectral calibration Peak heights for the matrix standards were too high Decrease the injection voltage or time Matrix sample peak heights that are too high may result in spectral calibration failure If matrix sample peak heights are too high gt 5 000RFU increase the dilution of each fragment in Step 2 of Section 4 A or 5 A and repeat the spectral calibration For best spectral calibration results use a fresh bottle of polymer fresh buffer and water and a capillary arra
21. or the chemistry e Select the spectral module you created in the previous step in the Run Module drop down list Finally select Edit Parameters and make the following modifications e Change the lower condition bound to 4 0 and change the upper condition bound to 12 0 e Confirm that the Minimum Quality Score Q value is 0 95 Select OK in the Edit Parameters window and select OK in the Protocol Editor Note The condition number C value obtained when generating a spectral calibration will vary with the instrument After obtaining a spectral calibration that performs acceptably the condition bounds range in the previous step may be narrowed to more critically evaluate C values for subsequent spectral calibrations d In the Plate Manager create a new plate record as described in the instrument user s manual In the dialog box that appears select Spectral Calibration in the Application drop down list and select 96 well as the plate type Add entries in the owner and operator windows name the plate and select OK e In the spectral calibration plate editor dialog box place sample names in the appropriate cells In the Instrument Protocol column select the protocol you created in Step 2 c Ensure that this information is present for each row that contains a sample name Select OK f Run your plate as described in the instrument user s manual g Upon completion of t
22. rected below Vortex for 5 10 seconds to mix Component Volume Hi Di formamide 665ul Fluorescein from initial dilution 7 0ul JOE from initial dilution 7 0ul TMR ET from initial dilution 7 Oul CXR ET from initial dilution 7 Oul CC5 from initial dilution 7 Oul 4 On the Applied Biosystems 3500xL Genetic Analyzer only wells A1 to H3 of the 96 well plate are used for spectral calibration Load 25 of fragment mix prepared in Step 3 into each of the 24 wells After placing the septa on the plate briefly centrifuge the plate to remove bubbles On the Applied Biosystems 3500 Genetic Analyzer only wells A1 to H1 of the 96 well plate are used for spectral calibration Load 25 of fragment mix prepared in Step 3 into each of the 8 wells After placing the septa on the plate briefly centrifuge the plate to remove bubbles 5 Denature samples at 95 C for 3 minutes then immediately chill on crushed ice or in an ice water bath for 3 minutes Denature samples just prior to loading the instrument 6 Place the plate in the 3500 series 96 well standard plate base and cover with the plate retainer Place the plate assembly in Position A on the autosampler with the labels facing you Promega Corporation gt 2800 Woods Hollow Road gt Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TBD024 Printed in USA Page 4 Revised 7 12 Promega 3 B Instrument Preparation We
23. reparation There may be instrument to instrument variation in the sensitivity of detection The dilutions described here may need to be optimized in individual laboratories depending on the sensitivity of each ABI PRISM 3100 or 3100 Avant or Applied Biosystems 3130 or 3130x Genetic Analyzer The optimal dilution may differ for each dye fragment You also may need to adjust injection time or voltage in Section 4 B to achieve a passing spectral calibration Peak heights in the range of 1 000 4 000RFU are ideal Peak heights above 750RFU are required The same plate of matrix standards can be re injected up to four times To re inject the same matrix standards plate add an injection by selecting Plate Manager then Edit Select Edit again in the top left corner of the window then select Add sample run Injection time and voltage may require optimization to obtain peak heights above 750RFU and below the saturation point 1 Thaw the matrix standards on ice Mix each matrix standard by vortexing for 5 10 seconds prior to use Do not centrifuge the matrix standards as this may cause the DNA to be concentrated at the bottom of the tube Place on ice Promega Corporation gt 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TBD024 Revised 7 12 Page 9 4 A Matrix Sample Preparation continued 2 Initia
24. rior to loading the instrument Promega Corporation gt 2800 Woods Hollow Road gt Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TBD024 Printed in USA Page 10 Revised 7 12 4 B Instrument Preparation We have found that the use of fresh polymer and new capillary array results in an optimal spectral Representative raw data are shown in Figure 5 Another example of a passing spectral calibration in the Spectral Viewer is shown in Figure 6 o 20 4400 600 s00 1000 1200 1400 1600 1800 2000 2200 2400 Intensity ys Scan Number a a a ee U p g ee ee es JAS NU WO OA TAT T 1000 1200 1400 1600 1800 2000 2200 intensity ys Scan Number 1000 1200 1400 1600 1800 2000 2200 Intensity ys Scan Number ee a ee ee ee ee eee eee ee ee ees ee 0 ee U ee es ees Gey Ss ee ee ee ee ee 2 0 200 400 600 800 1000 1200 1400 1600 1800 2000 2200 2400 intensity ys Scan Number 8283TA Figure 5 Representative data for the PowerPlex 5 Dye Matrix Standards 3100 3130 on the Applied Biosystems 3130 Genetic Analyzer using POP 4 polymer and Data Collection Software Version 3 0 Figure shows the CC5 CXR ET TMR ET JOE and fluorescein peaks in the raw data profile from each of the four capillaries in the Capillaries Viewer Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800
25. rm storage at 4 C may cause breakdown of formamide Poor quality formamide may contain ions that compete with DNA during injection which results in lower peak heights and reduced sensitivity A longer injection time may not increase the signal O Formamide is an irritant and a teratogen avoid inhalation and contact with skin Read the warning label and take appropriate precautions when handling this substance Always wear gloves and safety glasses when working with formamide 5 A Matrix Sample Preparation There may be instrument to instrument variation in the sensitivity of detection The dilutions described here may need to be optimized in individual laboratories depending on the sensitivity of each Applied Biosystems 3130 or 3130xl Genetic Analyzer The optimal dilution may differ for each dye fragment You also may need to adjust injection time or voltage in Section 5 B to achieve a passing spectral calibration Peak heights in the range of 1 000 4 000RFU are ideal Peak heights above 750RFU are required The same plate of matrix standards can be re injected up to four times To re inject the same matrix standards plate add an injection by selecting Plate Manager then Edit Select Edit again in the top left corner of the window then select Add sample run Injection time and voltage may require optimization to obtain peak heights above 750RFU and below the saturation point 1 Thaw the matrix standards on ice
26. romega 5 dye set created in Step 2b i e Promega G5 from the Dye Set drop down menu Figure 3 d Select Start Run Library Maintenance Tools v Manage v Preferences Help v Log Out Print hi Calibration Settings Current Instrument Consumables Polymer Type POP4 Capillary Length 36cm y Sama Status Ready Chemistry Standard Matrix Standard Set Promega G5 Number of Wells 96 96 FastTube 384 Plate Position A OB 0 7 Allow Borrowing Capillary Run Data Capillary 3 4 R B 18 5 Run 1 __ zm i aa bE Run 2 ea a E Run3 as Overall ial a ae se ae fa Passed Failed i Borrowed Not Calibrated Quality Value Condition Status Message v Intensity vs Scan Number 2208880 4000 8000 12000 16000 20000 24000 28000 32000 0 50000 40000 30000 20000 10000 Intensity vs Scan Number gt Intensity vs Pixel Number 3 Calibration Run 2800 Woods Phone 608 274 4330 Hollow Road Madison WI 53711 5399 USA Fax 608 277 2516 www promega com Part TBD024 Page 7 Promega 3 B Instrument Preparation continued 3 If fewer than the recommended number of capillaries pass the spectral calibration run will be repeated automatically up to three times Upon completion of the spectral calibration check the quality of the spectral in the Capillary Run Data displa
27. s Dye Set Template G5 Template Z lt L QC Protocols Sequencing Analysis Protocols Arrange Dyes MicroSeqiD Protocols Dye Selecion Reduced Selection Fragment Analysis Protocols Calibration Peak Order 5 Parameters The parameters will be used for instruments configured with 36cm capillary array and polymer POP4 Matrix Condition Number Upper Limit BS Bi Before Scan 5000 Locate Start Point After Scan 500 _ i Limit Scans To 2500 Sensitivity 04 GS Minimum Quality Score 0 95 a NFA EPA Figure 2 Create New Dye Set Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TBD024 Printed in USA Page 6 Revised 7 12 9325TA Dashboard Edit v XX Maintenance lt Catibrate Spatial D Performance Check Sequencing Install Standard Fragment Install Standard HID Install Standard y Maintenance Wizards P Planned Maintenance Notifications Log Service Log Schedule aa D Figure Promega Corporation Toll Free in USA 800 356 9526 Printed in USA Revised 7 12 O Promega To perform the spectral calibration with the Promega 5 dye chemistry go to the Maintenance tab select Spectral and under the Calibration Run tab choose the appropriate fields Choose Matrix Standard from the Chemistry Standard drop down menu and the new P
28. s when working with formamide For additional information on performing spectral calibration refer to the Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 3 A Matrix Sample Preparation There may be instrument to instrument variation in the sensitivity of detection The dilutions described here may need to be optimized in individual laboratories depending on the sensitivity of each Applied Biosystems 3500 or 3500xL Genetic Analyzer 1 Thaw the matrix standards on ice Mix each matrix standard by vortexing each tube for 5 10 seconds prior to use Do not centrifuge the matrix standards as this may cause the DNA to be concentrated at the bottom of the tube Place on ice 2 Initial dilution of concentrated fragments Before combining the matrix standards dilute the individual matrix standards 1 10 in Nuclease Free Water as described below Vortex for 5 10 seconds to mix Place on ice Component Fluorescein JOE TMR ET CXR ET COS Concentrated Dye 5ul 5ul 5ul 5ul 5ul Nuclease Free Water 45ul 45ul 45ul 45ul 45ul Promega Corporation gt 2800 Woods Hollow Road gt Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TBD024 Revised 7 12 Page 3 3 A Matrix Sample Preparation continued 3 Fragment mix using 1 10 dilutions of matrix standards After the initial dilution in Step 2 combine the 1 10 dilution of each matrix standard as di
29. using POP 7 polymer and Data Collection Software Version 3 0 Figure shows the CC5 CXR ET TMR ET JOE and fluorescein peaks in the raw data profile from each of the four capillaries in the Capillaries Viewer 10608TA Promega Corporation gt 2800 Woods Hollow Road gt Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TBD024 Printed in USA Page 16 Revised 7 12 50 100 150 200 250 _lntensmy vs Pixel Number 0 1000 2000 3000 4000 5000 Intensity vs Stan Number Copley Dsta Tus Apr 03 11 4036 COT 2912 A01 e Dye Set Gay fea Fes oe FE fe Fe ee ye Set 35 a Actie Callbrstion for Dye Set GS Matix used tor Contlory 1 1 Tus Apr 03 11 40 36 COT 2012 Condtion 11 355294 Q Vaus 0959642 Ug of Cabraore tor Dye Set OS Tue pr 03 11 40 35 COT 2012 v override Scects_ Rename Figure 8 Representative data for the PowerPlex 5 Dye Matrix Standards 3100 3130 on the Applied Biosystems 3130 Genetic Analyzer using POP 7 polymer and Data Collection Software Version 3 0 Figure shows the CC5 orange CXR ET red TMR ET yellow JOE green and fluorescein blue peaks in the Spectral Viewer from one of the four capillaries Promega Corporation gt 2800 Woods Hollow Road gt Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TBD024 Revised
30. y Figure 4 and choose either Accept or Reject Note Refer to the 3500 Series Data Collection Software Version 1 0 HID User Manual for the criteria recommended by Applied Biosystems when accepting or rejecting a spectral calibration Dashboard Edit v Library Maintenance Tools Manage v Preferences Help LogOu x Maintenance iP Export Spectral Calibration Results g E View Spectral Calibration Report Qy Prit Calibration Run g CA Spectral Report H Calibrate v Calibration Information aa Dye Set Chemistry Standard Calibration Date v Capillary Array Serial Number pati f Matrix Standard 13 May 2010 01 46 06 PM M309E 1005 F Matrix Standard 13 May 2010 11 30 35 AM M309E 1005 No D Performance Check 65 Matrix Standard 12 May 2010 12 47 49 PM M309E 1005 No 2 Sequencing Install Standard 7 Capillary Run Data Fragment Install Standard HID Install Standard py Maintenance Wizards M Planned Maintenance Notifications Log E Passed BB Failed Borrowed Not Calibrated Service Log Capillary L Run 1 Quality Value 0 991481 Condition 9 895967 Status Passed Message q 0 991 c 9 896 Schedule v Intensity vs Scan Number een aA 0 400 800 1200 1600 2000 2400 2800 S Ys ERK 3000 DN 2000 1000 Intensity vs Scan Number Figure 4 The Capillary Run Data display Hollow Road Fax 608 277 2516 2800 Woods Phone 608 274 4330 Promega Corporation Toll Free
31. y with fewer than 100 injections All capillaries failed spectral Monitoring fragment migration in the calibration Capillaries Viewer during the spectral calibration run can provide information that will be useful for troubleshooting purposes Re inject the spectral calibration plate and monitor the Capillaries Viewer during the run Note any unusual peak formations or extremely high or low peak heights Based on information obtained while watching the Capillary Viewer it may be necessary to adjust the run conditions Samples were degraded due to improper storage Store matrix standards at 30 C to 10 C and protected from light Do not store in the freezer door and do not store in a frost free freezer The CXR ET labeled matrix fragment came off too early Decrease the data delay time and re inject the matrix standards Multicolor peaks were detected prior to the CXR ET peak Increase the data delay time and re inject the matrix standards Promega Corporation gt 2800 Woods Hollow Road gt Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TBD024 Printed in USA Page 20 Revised 7 12 Promega Symptoms Causes and Comments Spectral failure due to improper Confirm that the parameters in the run module C value are set correctly between 4 0 and 12 0 see Sections 4 B and 5 B Clean the instrument and install a new array and fresh polymer A dr
32. zerland Pat Nos 0743987 and 0851867 Germany Pat Nos 19581489 69530286 8 and 0851867 EP Pat Nos 0743987 and 0851867 Spain Pat Nos 2197193 and 2173310 France Pat Nos 0743987 and 0851867 United Kingdom Pat Nos 0743987 and 0851867 Italy Pat Nos 0743987 and 0851867 Liechtenstein Pat Nos 0743987 and 0851867 Netherlands Pat Nos 0743987 and 0851867 Sweden Pat Nos 0743987 and 0851867 U S Pat Nos 5 654 419 5 688 648 5 869 255 6 177 247 5 707 804 6 028 190 6 544 744 7 015 000 and 5 728 528 Canada Pat No 2231475 Japan Pat No 3066984 and other pending and foreign patent applications End User Terms and Conditions Acceptance These terms and conditions shall govern the purchase use transfer and acceptance of the products described in the purchase order quotation or invoice which products are sold and distributed by Promega to the buyer transferee of such products the End User The transfer sale of products to the End User is expressly conditional upon End User s acceptance of these terms and conditions Restrictions on Use End Users are specifically not authorized to and are forbidden from reselling transferring or distributing any products either as a stand alone product or as a component of another product The right to use the products does not in and of itself include or carry any right of the End User to any GE Healthcare Bio Sciences Corp s technology or intellectual property other than expressly provided
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