DNF-920-K1000
Contents
1. b When working with lower sample concentrations total initial sample concentration lt 10 ng uL Dilute 1 000 bp DNA Ladder solution 50X with 0 1X TE buffer in sample well 1 uL 1 000 bp DNA Ladder 49 uL 0 1X TE buffer The highest level of sizing accuracy is obtained when the 1 000 bp DNA Ladder is diluted to a similar concentration range yielding similar peak height RFU values and with a similar diluent 1X or 0 1X TE to the samples being analyzed Sample Plate Preparation 1 Some suggested sample preparation guidelines are presented below It may be necessary to adjust the sample dilution and diluent concentration 1X TE or 0 1X TE depending upon initial sample concentration and sample matrix For best results the 1 000 bp DNA Ladder should be prepared with a similar concentration and diluent to the samples If total initial sample concentration is gt 10 ng uL e g PCR products a Using a clean 96 well sample plate pipette 22 uL of supplied 1X TE buffer solution to each well that is to contain sample or ladder 9 Rev DNF 920 2015JUL25 Advanced Analytical Technologies Inc b Pipette 2 uL of each DNA sample into the respective wells of the sample plate mix the contents of the well using the pipette by aspiration expulsion in the pipette tip c 1 000 bp DNA Ladder Pipette 2 uL of 1 000 bp DNA Ladder solution into the respective well s of the sample plate to contain ladder i e well 12 of each row analy2. by left clicking the Sample Tray dropdown or by clicking the appropriate sample plate tab alternate plate view and choosing the appropriate location 96 Capillary Systems Note that Sample 3 is typically assigned to the Capillary Storage Solution Left click a well of the desired sample plate row with the mouse The selected row will be highlighted in the plate map e g Row A in Figure 3 Enter the sample name if desired into the respective Sample ID cell by left clicking the cell and typing in the name Alternatively sample information can be imported from txt or csv file by selecting the Load from File option 11 Rev DNF 920 2015JUL25 Advanced Analytical Technologies Inc i i Fragment Analyzer 2555 User ID aati Database SysLog sdf a oe File Admin Utilities Help Operation Run Status Manually enter Sample ID data OR load from file option of save information by raAanmmMoaAa BRD Save Tray or Save Selected Row Trayname Enter Tray Name Here Select Row Run Selected Row Add to queue Edit method ead irom Fie ave Tray Save Selected Row Reset Row Reset tray rec Run Entire Tray Add to queue Edit method Capillary Array Condifoniag After entering data Add to queue Edit method select Add to queue Si 0 0KV Si 00uA 6 0 0 PSI LED Off Vent Closed Q Waste Closed Stage Bu
3. semi skirted PCR plate from Eppendorf 951020303 Please refer to Appendix C Fragment Analyzer Compatible Plates and Tubes in the Fragment Analyzer User Manual for a complete approved sample plate list The system has been designed to operate using these dimensions styles of PCR plates Plates with similar dimensions may be used but note that capillary damage may occur with the use of poor quality PCR plates IMPORTANT Contact AATI if a different vendor or style of PCR plate is to be used in order to verify compatibility The use of PCR plates with different dimensions to the above recommended plate could possibly damage the tips of the capillary array cartridge Refer to the Fragment Analyzer User Manual for a list of approved PCR sample plates 75 bp 15 000 bp Marker Preparation 1 Store the 75 bp and 15 000 bp Marker solution at 20 C upon arrival 2 Bring the 75 bp and 15 000 bp Marker solution to room temperature prior to use agitate solution to ensure it is properly mixed and centrifuge vial prior to dispensing 8 Rev DNF 920 2015JUL25 Advanced Analytical Technologies Inc 3 The Marker solution is supplied as a ready to use solution containing 0 5 ng uL of each fragment in a 1X TE buffer solution It is intended for use as an external standard marker plate Prepare the Marker solution plate by dispensing 30 UL well into Row A only 12 Capillary ot every well 96 Capillary of a separate sample plate
4. Cover the wells with 20 uL well of the supplied mineral oil to allow reuse for at least 30 injections The prepared Marker solution plate should be placed into Drawer M third from top of the Fragment Analyzer Ensure that the plate is loaded with well A1 toward the back left on the tray 1 000 bp DNA Ladder Preparation Store the 1 000 bp DNA Ladder solution at 20 C upon arrival Bring the 1 000 bp DNA Ladder solution to room temperature prior to use agitate solution to ensure it is properly mixed and centrifuge vial prior to dispensing The 1 000 bp DNA Ladder solution is supplied as a CONCENTRATE This enables the solution to be diluted with either 1X TE or 0 1X TE depending upon the available sample concentration and matrix see Sample Plate Preparation section below The solution contains 50 ng uL total DNA concentration in a 1X TE buffer solution It is intended for use as a sizing standard for calibration of DNA size For optimal sizing results the 1 000 bp DNA Ladder should be loaded in Well 12 of each row to be analyzed 12 capillary system ot Well H12 96 capillary system Prepare the working 1 000 bp DNA Ladder solution by diluting with either 1X TE buffer or 0 1X TE buffer Suggested dilutions are a When working with higher sample concentrations total initial sample concentration gt 10 ng L Dilute 1 000 bp DNA Ladder solution 12X with 1X TE buffer in sample well 2 uL 1 000 bp DNA Ladder 22 uL 1X TE buffer
5. SS NGS Fragment 1 6000bp mthds Tray Samp tray 1 D Gel 1 3 Method summary X Y Gi 0 0KV Si O0uA P 0 0 PSI LED OFF amp Vent Closed Waste Closed H Stage Buffer H Tray1H Figure 5 Main Screen after selection of samples to the run queue 15 Once an experiment has been loaded onto the queue the user can view or edit the method Administrator level only can edit a method by pressing the Method Summary field To remove the method from the queue press the X button to view the stepwise details of the method press the double down arrow icon 16 The user may add a Pause or Prime step into the queue by right clicking the mouse while over the queue and selecting Insert Pause or Insert Prime 17 The order of the experimental queue can be rearranged by dragging down individual entries Further information regarding the Method Queue operation is provided in the Fragment Analyzer User Manual 18 Once started the instrument will perform all the programmed experiments in the Method Queue uninterrupted unless a pause step is present Note that additional experiments can be programmed and added to the Method Queue at any time while the instrument is running if desired After completion of the last queued experiment the instrument stage will automatically move to the Store location 12 Capillary Systems Row H of the inlet buffer tray containing the Capillary Storage Solution 96 Capillary Systems Sample 3 lo
6. a plate seal on the sample plate and vortex the sample plate at 3000 rpm for 2 min Any suitable benchtop plate vortexer can be used Ensure that there is no well to well transfer of samples when vortexing The plate should be spun via a centrifuge after vortexing to ensure there are no trapped air bubbles in the wells C After adding 2 uL of sample to the 22 uL of diluent use a separate pipette tip set to a larger 20 uL volume and pipette each well up down to further mix D Use an electronic pipettor capable of mixing a 10 uL volume in the tip after dispensing the 2 uL sample volume Some models enable using the pipette tip for both adding and mixing 4 After mixing sample 1 000 bp DNA Ladder and diluent solution in each well centrifuge the plate to remove any ait bubbles Check the wells of the sample plate to ensure there are no ait bubbles trapped in the bottom of the wells The presence of trapped air bubbles can lead to injection failures 10 Rev DNF 920 2015JUL25 Advanced Analytical Technologies Inc Run the sample plate immediately once prepared or cover the sample plate with a cover film store at 4 C and use as soon as possible Alternatively to prevent evaporation place a mineral oil overlay on each sample 20 UL well To run the samples place the plate in one of the three sample plate trays Drawers 4 6 from the top of the Fragment Analyzer instrument Load or create the experimental method as described in t
7. arrival DO NOT FREEZE In a clean container e g 50 mL or 250 mL conical centrifuge tube add 20 mL of the 5X Capillary Conditioning Solution per 80 mL of deionized sub micron filtered water Agitate to mix The entire bottle can be mixed to 1X concentration and stored at room temperature if desired Once mixed place the 1X Capillary Conditioning Solution onto the instrument and insert the CONDITIONING fluid line Conditioning Solution pump position Ensure the fluid line is positioned at the bottom of the conical tube to avoid introducing air bubbles which can cause pressurization errors The 1X Capillary Conditioning Solution should be added to the system as use demands A typical 12 capillary experiment cycle consumes less than 4 mL a typical 96 capillary experiment consumes less than 35 mL When adding fresh 1X Capillary Conditioning Solution to the instrument update the solution levels in the Fragment Analyzer instrument control software From the Main Menu select Utilities Solution Levels A menu will be displayed to enter in the updated fluid levels Figure 1 7 Rev DNF 920 2015JUL25 Advanced Analytical Technologies Inc Instrument Preparation 1 Check the fluid level of the waste bottle and waste tray daily and empty as needed 2 Prepare a fresh 96 DeepWell 1mL Plate filled with 1 0 mL well of 1X dsDNA Inlet Buffer daily 12 Capillary System Row A only 96 Capillary System All Rows Do NOT overfill the w
8. 0 mL Part DNF 920 0500 Intercalating Dye 30 uL x 2 Part DNF 600 U030 5X dsDNA Inlet Buffer 300 mL dilute with sub micron filtered water prior to use Part DNF 455 0300 5X Capillary Conditioning Solution 100 mL dilute with sub micron filtered water prior to use Part DNF 475 0100 75 bp and 15 000 bp Markers 3 2 mL Part FS SMK920 0003 a 0 5 ng uL concentration each in 1X TE buffer 1 000 bp DNA Ladder 100 uL x 2 Part FS SLR920 U100 b 250 bp 10 000 bp 50 ng pL total DNA concentration in 1X TE buffer Mineral Oil 15 mL Part FS SMO15 Dilution Buffer 1X TE 125 mL Part DNF 495 0125 2 Rev DNF 920 2015JUL25 Advanced Analytical Technologies Inc Application The DNF 920 Reagent Kit from AATI is for the analysis of dsDNA fragments between 75 bp and 15 000 bp Sizing and relative quantification between samples can be obtained using this kit Example applications include PCR fragment sizing and restriction digest analysis Specifications Specifications Sample Volume Required Number of Samples per Run Total Electrophoresis Run Time DNA Sizing Range Separation Resolution DNA Sizing Accuracy DNA Sizing Precision DNA Fragment Concentration Range 2 uL adjustable depending upon sample concentration 12 Capillary 11 1 well DNA Ladder 96 Capillary 95 1 well DNA Ladder 50 minutes 33 55 Array 80 minutes 55 80 Array 75 bp 15 000 bp defined by lower upper marke
9. New Software Version Refer to red highlighted text for additional updates ADVANCED ANALYTICAL DNF 920 dsDNA Reagent Kit User Guide DNF 920 K0500 DNF 920 K1000 For use with the Fragment Analyzer Automated CE System Fragment Analyzer Software Version 1 0 2 PROSize 2 0 Software Version 1 3 Revised July 25 2015 Advanced Analytical Technologies Inc Ph 515 296 6600 2711 South Loop Drive Suite 4150 Fax 515 294 7141 Ames IA 50010 www aati us com Rev DNF 920 2015JUL25 Advanced Analytical Technologies Inc DNF 920 dsDNA Reagent Kit 75 bp 15 000 bp 500 Samples Part DNF 920 K0500 Kit Components 1 2i 3 dsDNA Gel 75 bp 15 000 bp 240 mL Part DNF 920 0240 Intercalating Dye 30 uL Part DNF 600 U030 5X dsDNA Inlet Buffer 125 mL dilute with sub micron filtered water prior to use Part DNF 455 0125 5X Capillary Conditioning Solution 50 mL dilute with sub micron filtered water prior to use Part DNF 475 0050 75 bp and 15 000 bp Markers 3 2 mL Part FS SMK920 0003 a 0 5 ng uL concentration each in 1X TE buffer 1 000 bp DNA Ladder 100 uL Part FS SLR920 U100 a 250 bp 10 000 bp 50 ng uL total DNA concentration in 1X TE buffer Mineral Oil 15 mL Part FS SMO15 Dilution Buffer 1X TE 60 mL Part DNF 495 0060 DNF 920 dsDNA Reagent Kit 75 bp 15 000 bp 1000 Samples Part DNF 920 K 1000 Kit Components 1 2 3 dsDNA Gel 75 bp 15 000 bp 50
10. Tray Name can be entered to identify the sample plate The Folder Prefix if entered will amend the folder name normally a time stamp of HH MM SS from the start of the CE run 7 To copy the experimental results to another directory location in addition to the default save directory C AATI Data check the Copy results box and select the desired Copy path directory by clicking the button and navigating the desired save directory 8 Any Notes can be entered regarding the experiment they will be saved and displayed in the final PDF report generated by the PROS7ze 2 0 software 9 Once all information has been entered press OK to add the method to the instrument queue press Cancel to abort adding the method 10 Repeat Steps 3 9 for any remaining sample rows to be analyzed 11 On 96 capillary systems or in 12 capillary systems if the entire 96 well sample tray is to be run using the same experimental method under the Run Entire Tray field press Add to queue A form similar to Figure 4 will be displayed for entering information and adding the run to the instrument queue for the entite 96 well sample tray 12 After a row or tray has been added to the queue the method s will be listed on the main screen under the Method Queue field Figure 5 13 Prior to starting the experiment verify all trays buffer storage waste marker sample etc have been loaded into their respective drawer locations 14 Press the Play ico
11. cation Viewing and Editing Experimental Methods 1 A User level operator can View the steps of the experimental method by pressing the View link on the Separation Setup screen or by pressing the Method Summary option once a method has been loaded onto the experimental queue User level operators cannot edit any steps of a queued separation method 14 Rev DNF 920 2015JUL25 Advanced Analytical Technologies Inc 2 Administrator level operators can Edit certain steps of the experimental method To open the method editor screen press the Edit link from the Separation Setup screen Figure 4 The method editor screen is displayed showing the steps of the method Figure 6 3 The preloaded optimized steps for the DNF 920 33 Figure 6 and DNF 920 55 Figure 7 methods are shown below The general steps of the method are as follows 1 2 3 4 5 6 7 Full Condition flushing method Automatically enabled Gel Selection Gel 1 Perform Prerun ENABLED 7 kV 30 sec Rinse DISABLED Marker Injection ENABLED Voltage Injection 3 kV 5 sec This step injects the 75 bp 15 000 bp marker plate Rinse DISABLED Sample Injection ENABLED Voltage Injection 3 kV 5 sec This step injects the prepared sample plate Separation ENABLED Voltage 7 kV 50 min This step performs the CE Separation E Separation Method Gel Selection Gel 1 X V Perform Prerun Voltage 70 kV Time 30 Sec E Rinse Tray Bu
12. ells of the inlet buffer plate 3 12 Capillary Systems In Row H of the same prepared buffer plate place 1 1 mL well of Capillary Storage Solution AATI GP 440 0100 Row H of the buffer plate is used for the Store location and the array moves to this position at the end of the experimental sequence 4 96 Capillary Systems In the Sample 3 drawer place a sample plate filled with 100 uL well of Capillary Storage Solution AATI GP 440 0100 Sample 3 is used for the Store location and the array moves to this position at the end of the experimental sequence IMPORTANT Ensure Row H of the buffer tray 12 capillary systems or Sample 3 96 capillary systems is always filled with Capillary Storage Solution and the capillary array is placed against Storage Solution when not in use to prevent the capillary tips from drying out and potentially plugging 5 Place the prepared inlet buffer plate into Drawer B top drawer of the Fragment Analyzer Ensure that the plate is loaded with well A1 toward the back left on the tray 6 Place an empty 96 DeepWell 1mL Plate into Drawer W second from top of the Fragment Analyzer This plate serves as the capillary waste tray and should be emptied daily Alternatively the supplied open reservoir waste plate may be used Marker Ladder Sample Preparation General Information 1 The recommended 96 well sample plate for use with the Fragment Analyzer system is a
13. ent size is below 5 000bp analyze using DNF 915 Reagent Kit DNF 915 35bp 5 000bp range to better resolve primer dimer species Sample peak s migrate after or co migrate with 15 000 bp Upper Marker DNA sample size out of range of assay Analyze samples with a Genomic DNA Analysis Kit DNF 487 0500 or DNF 488 0500 which contain no upper marker limit Poor resolution of ladder peaks Slower migration time than expected Capillary Array Vent Valve is partially plugged with gel Inspect and if necessary clean Capillary Array Vent Valve as described in the Fragment Analyzer Troubleshooting and Maintenance Guide Rev DNF 920 2015JUL25 19 Advanced Analytical Technologies Inc F No sample peak or 1 Air trapped at the 1 Check sample plate wells for trapped air Lower Marker peak bottom of sample plate bubbles Centrifuge plate observed for well or bubbles present individual sample in sample well 2 Insufficient sample 2 Verify proper volume of solution was added to volume A minimum of sample well 24 uL is required 3 Capillary is plugged 3 Check waste plate for liquid in the capillary well If no liquid is observed follow the steps outlined in Appendix G Capillary Array Cleaning of the Fragment Analyzer User Manual for unclogging a capillary array Technical Support Technical Support and Contact Information 1 For questions with Fragment A
14. ffer H Tray1H Figure 3 Main Screen showing selection of sample row and entering sample information After sample information for the row or plate has been entered under the Run Selected Row field press Add to queue The Separation Setup form will be displayed enabling the user to select the experimental method and enter additional information Figure 4 r a Separation Setup Method DNF 920 33 DNA 75 15000bp mthds gj Edit Get Trayname Enter Tray Name here Folder Prefix Enter folder prefix here Copy results Notes Enter any notes here Figure 4 Separation Setup form to select experimental Method and enter tray folder information 4 In the Separation Setup pop up form left click the dropdown and select the appropriate preloaded experimental Method file The available methods are sorted by kit number and are linked to the directory containing methods for the currently installed capillary array length e g 33cm or 55cm Select the following method a Select DNF 920 33 DNA 75 15000bp mthds when the 33 cm effective 55 cm total short capillary array is installed 12 Rev DNF 920 2015JUL25 Advanced Analytical Technologies Inc b Select DNF 920 55 DNA 75 15000bp mthds when the 55 cm effective 80 cm total long capillary array is installed 5 Select the appropriate Gel line being used for the experiment Gel 1 or Gel 2 using the dropdown 6 The
15. ffer Row A Dips 1 V Marker Injection Row A X Voltage Injection Voltage 3 00 kV Time 5 Sec D Vacuum Injection Pressure 2 0 PSI E Rinse Tray Buffer Row A Dips 1 V Sample Injection Voltage Injection Voltage 3 00 4 kV Time 52 Sec gt Vacuum Injection Pressure 2 0 PSI V Separation Voltage 70 kV Time 50 0 Min Figure 6 DNF 920 33 dsDNA Reagent Kit 75bp 15000 bp method 15 Rev DNF 920 2015JUL25 Advanced Analytical Technologies Inc 4 Figure 7 shows the preloaded method for the 55 cm effective 80 cm total length long array The Prerun and Separation voltage is set to 11 kV the Injection voltage to 7 5 kV and the Separation time to 80 min r Separation Method Gel Selection Gel 1 X V Perform Prerun Voltage 11 0 kV Time 305 Sec E Rinse Tray Buffer Row A Dips 1 V Marker Injection Row A X Voltage Injection Voltage 7 50S kV Time 5 Sec Vacuum Injection Pressure 2 0 PSI E Rinse Tray Buffer Row Dips 1 V Sample Injection Voltage Injection Voltage 750 kV Time 5f Sec Vacuum Injection Pressure 2 0 PSI V Separation Voltage 110 kV Time 80 0 Min Figure 7 DNF 920 55 dsDNA Reagent Kit 75bp 15000 bp method 5 An Administrator level user has the option to adjust the Gel Selection Prerun settings Rinse settings including Tray Row and Dips Marker Injection settings including Row Sample Injection settings and t
16. gure 1 r Solution Levels Check the fluid volumes before proceeding Ensure that the waste is empty and that the gel and conditioning solutions are full Record the solution volumes here Volume mL Solutions Gel 1 50 0 DNF 920 Gel 2 43 3 NaOH Conditioning Solution 47313 Waste 94 04 ok Cancel _ Figure 1 Solution Levels menu 8 When switching applications e g between kits prime the appropriate gel fluid line after loading fresh gel dye mixture From the Main Menu of the Fragment Analyzer instrament control software select Utilities Prime Select the desired fluid line s Conditioning Gel 1 or Gel 2 and press OK to purge the fluid line with fresh gel 6 Rev DNF 920 2015JUL25 Advanced Analytical Technologies Inc r ad Prime Fluid Selected Cycles 1 F Conditioning 3 I oe lee Ey Gel Empty Rate 300 us Figure 2 Prime menu Inlet Buffer Preparation Store the 5X dsDNA Inlet Buffer at 4 C upon arrival DO NOT FREEZE Bring the 5X dsDNA Inlet Buffer to room temperature prior to mixing and use In a clean container add 20 mL of the 5X dsDNA Inlet Buffer per 80 mL of deionized sub micron filtered water Agitate to mix The entire bottle can be mixed to 1X concentration and stored at 4 C if desired Capillary Conditioning Solution Preparation 1 Store the 5X Capillary Conditioning Solution at room temperature upon
17. he Separation settings For example if the marker solution is loaded into a row other than Row A on a 12 capillary instrument this can be adjusted prior to or while the method is loaded on the experimental queue 6 To apply any adjustments to the method being placed on the experimental queue press the OK button To exit the editor screen without applying any changes press the Cancel button IMPORTANT Any edits made to the experimental method from the Separation Setup or Method Summary screen will only apply to the currently loaded experiment in the queue No changes are made to the original separation method file 16 Rev DNF 920 2015JUL25 Advanced Analytical Technologies Inc Processing Experimental Data 1 When processing data the PROSize 2 0 software Version 1 3 and higher will automatically recognize the separation method performed and apply the appropriate matching configuration file from the C PROSize 2 0 Configurations directory a The DNF 920 33 separation method will be processed using the DNF 920 33 DNA 75 15000bp configuration file b The DNF 920 55 separation method will be processed using the DNF 920 55 DNA 75 15000bp configuration file NOTE If the preloaded PROS ze 2 0 software configuration files _DNF 920 33 DNA 75 15000bp and DNF 920 55 DNA 75 15000bp are not located in the C PROSize 2 0 Configurations directory contact AATI Technical Support to obtain these files T
18. he data is normalized to the lower marker set to 75 bp and upper marker set to 15 000 bp and calibrated to the 1 000 bp DNA Ladder run in parallel to the samples Figures 8 9 show examples of the 75 bp and 15 000 bp markers injected with the 1 000 bp DNA Ladder A total of 16 peaks should be observed The PROSize 2 0 configuration is set to the DNA mode in the Advanced Settings The Quantification settings should be set to Use Lower Marker for quantification with a Final Conc ng uL of 0 5 and a Dilution Factor of 12 2 uL sample 22 uL Diluent Marker Note if a pre dilution was performed prior to the experiment the Dilution Factor setting should be changed to reflect the estimated final sample concentration UPDATE June 25 2013 The Quantification settings should now be set to Use Lower Marker for quantification from the previous Use Upper Marker setting 4 For full information on processing data refer to the PROS7ze 2 0 User Manual Fragment Analyzer Shut Down Storage Instrument Shut Down Storage The instrument automatically places the capillary array in the Store position against Capillary Storage Solution 12 Capillary Systems Row H of the buffer tray 96 Capillary Systems Sample 3 after each experiment no further action is required If the instrument is to be idle for more than one day turn off power to the system to preserve lamp lifetime 17 Rev DNF 920 2015JUL25 Advanced Analytical Technologies Inc T
19. he following sections The CCD detection system of the Fragment Analyzer system provides a high dynamic range for detection An ideal injection range would yield peak heights from 100 20 000 RFUs Overloading of sample can decrease separation resolution and saturate the detector leading to mismatched lower upper marker peak heights and poor results It is important to optimize sample dilution and concentration and use experimental parameters to work with within the specified RFU range The highest level of sizing accuracy is obtained when the sample and DNA Ladder peak heights are of similar RFU peak heights TIP If the above methods yield peak heights consistently above 20 000 RFUs decrease the matker sample injection time or reduce the sample volume to 1 uL sample 23 uL 1X TE If low signals are encountered increase the marker sample injection time or alternatively add 4 uL of sample 20 uL of DI water in each well When making adjustments to the sample dilution the total volume should be maintained to at least 24 uL Whenever making adjustments to the sample dilution ensure the Dilution Factor of the PROSize 2 0 software is adjusted accordingly when processing the data Performing Experiments Running an Experiment 1 To set up an experiment from the Main Menu of the Fragment Analyzer instrument control software select the Operation tab Figure 3 Select the sample tray location to be analyzed 1 2 or 3
20. ial assay specific issues which may be encountered when using the DNF 920 Reagent Kit and suggested remedies For a full list of instrument specific troubleshooting information refer to the Troubleshooting and Maintenance Guide for the Fragment Analyzer system Issue Cause Corrective Action The peak signal is gt gt 20 000 RFU upper marker peak is low or not detected relative to lower marker No peak observed for DNA sample when expected Lower Upper Marker peaks observed Input DNA sample concentration is too high Sample concentration too low and out of range Sample was not added to 1X TE diluent or not mixed well Further dilute inout DNA sample concentration with 1X TE buffer and repeat experiment Reduce injection time and or injection voltage and repeat experiment Use the same injection voltage time settings for the Marker Plate and Sample Plate to maximize quantification accuracy Prepare more concentrated sample and repeat experiment e g 4 uL sample 20 uL DI water or repeat experiment using increased injection time and or injection voltage for Marker Sample Plate Verify sample was correctly added and mixed to sample well Sample peak s migrate before or co migrate with 75 bp Lower Marker Excess primer dimer species in sample Further dilute inout DNA sample concentration with 1X TE buffer to minimize primer dimer interference and repeat experiment If fragm
21. ing Solution 4 Rev DNF 920 2015JUL25 Advanced Analytical Technologies Inc Safety When working with chemicals always follow usual safety guidelines such as wearing a suitable lab coat disposable gloves and protective eyewear For more information about the specific reagents please refer to the appropriate material safety data sheets MSDSs that can be obtained from the product supplier Fragment Analyzer Start Up Instrument Preparation Gel Preparation 1 2 Store the dsDNA Separation Gel at 4 C upon arrival The Intercalating Dye is supplied as a 20 000X concentrate in DMSO and should be stored at 20 C NOTE For this assay the Intercalating Dye should be used at 2X normal concentration 1 10 000 dilution Bring the dsDNA Gel and Intercalating Dye to room temperature prior to mixing Mix appropriate volumes of Intercalating Dye and dsDNA Gel necessary for less than two weeks of operation Use the supplied 50 mL conical centrifuge tube to allow a small minimum working volume For larger volumes use a 250 mL conical centrifuge tube and remove the collar of the tube holder in the instrument reagent compartment For maximum accuracy it is recommended to dispense Separation Gel into a clean glass graduated cylinder for volume measurement and transfer to the working tube prior to adding Intercalating Dye NOTE Some loss of detection sensitivity will be observed over a two week period after the gel dye mi
22. n D to start the sequence loaded into the queue To Pause the queue after the currently running experiment is completed press the button To Clear the run queue of all loaded runs press the x button 13 Rev DNF 920 2015JUL25 Advanced Analytical Technologies Inc Fragment Analyzer 2555 User ID aati Database SysLog sdf n File Admin Utilities Help Operation un Status Sample Tray 1 Capillary Well Sample ID D1 SampD1 a 000000000000 D2 SampD2 s O000000000000 J a ln c O00000000000 n loi 2000000800008 EIOOOOOOOOOO0OO E ai sik F amp 000000000000 t To start running s O00000000000 p D7 SampD7 O00000 000000 s D8 SampD8 the queue press 123 4 5 6 7 8 9 10 11 12 9 D9 SampD9 Tray name Enter Tray Name Here 10 DIO SampD10 the Play button 11 D11 SampD11 12 D12 SampD12 Load from File Save Tray Save Selected Row Reset Row Reset tray Run Selected Row Method Queue Add to queue Edit method SEPARATION Method DNF 473 33 SS NGS Fragment 1 6000bp mthds Tray Samp tray 1 A Gel 1 2 Methodsummary X Y Run Entire Tray Add to queue Edit method SEPARATION Method DNF 47 agment 1 6000bp mthds y Samp tray 1 B Gel 1 x Capillary Array Conditioning SEPARATION Method DNF 47 ragment 1 6000bp mthds Tray Samp tray 1 C Gel 1 Methodsummary X Y Add to queue Edit method SEPARATION Method DNF 473 33
23. nalyzer operation or about the DNF 920 Reagent Kit contact AATI Technical Support by phone at 515 296 6600 or by email at support aati us com 20 Rev DNF 920 2015JUL25 Advanced Analytical Technologies Inc
24. r 75 bp 1 500 bp lt 5 1 500 bp 15 000 bp lt 10 5 or better 2 CV 0 5 ng L 50 ng uL input DNA adjustable by dilution of sample 1 Results using DNA Ladder or DNA Fragment standards initially prepared in 1X TE buffer Storage Conditions Store at 4 C Store at Room Temperature Store at 20 C DO NOT FREEZE DO NOT FREEZE dsDNA Gel Intercalating Dye Ca pulaty Coacidaning Solution 5X dsDNA Inlet Buffer 75 bp and 15 000 bp Markers Mineral Oil Dilution Buffer 1X TE 1 000 bp DNA Ladder Ensure all reagents are completely warmed to room temperature prior to use Rev DNF 920 2015JUL25 3 Advanced Analytical Technologies Inc Additional Material and Equipment Required Hardware Software and Reagents available from AATI 1 Hardware Fragment Analyzer 12 capillary or 96 capillary CE system with LED fluorescence detection e 12 Capillary Array Cartridge Fluorescence 33 cm effective 55 cm total length 50 um ID part A2300 1250 3355 OR e 12 Capillary Array Cartridge Fluorescence 55 cm effective 80 cm total length 50 um ID part A2300 1250 5580 OR e 96 Capillary Array Cartridge Fluorescence 33 cm effective 55 cm total length 50 um ID part A2300 9650 3355 OR e 6 Capillary Array Cartridge Fluorescence 55 cm effective 80 cm total length 50 um ID part A2300 9650 5580 Software Fragment Analyzer instrument control software Ver
25. sion 1 0 2 or higher e PROSize 2 0 data analysis software Version 1 3 or higher Reagents e Capillary Storage Solution 100 mL AATI GP 440 0100 Equipment Reagents to Be Supplied by User 1 96 well PCR sample plates Please refer to Appendix C Fragment Analyzer Compatible Plates and Tubes in the Frgzent Analyzer User Manual for a complete approved sample plate list Multichannel pipettor s and or liquid handling device capable of dispensing 1 100 uL volumes sample plates and 1000 uL volumes Inlet Buffer plate Pipette tips 96 well plate centrifuge for spinning down bubbles from sample plates Sub micron filtered DI water system for diluting the 5X dsDNA Inlet Buffer and 5X Capillary Conditioning Solutions Fisherbrand 96 DeepWell 1mL Plate Natural Polypropylene part 12 566 120 Inlet Buffer and Waste plate Reagent Reservoir 50 mL VWR 82026 355 or similar for use in pipetting Inlet Buffer plates sample trays Conical centrifuge tubes for prepared Separation Gel Dye mixture and or 1X Capillary Conditioning Solution a 250 mL for 96 Capillary instruments or larger volumes Corning 430776 available from Fisher 05 538 53 or VWR 21008 771 b 50 mL for 12 Capillary instruments or 50 mL volumes BD Falcon 352070 available from Fisher 14 432 22 or VWR 21008 940 Clean graduated cylinder for measurement of dsDNA Gel volume and dilution of 5X dsDNA Inlet Buffer and 5X Capillary Condition
26. xture has been prepared For best results it is recommended to prepare gel dye mixture daily It is not recommended to use gel dye mixture that is more than two weeks old The volume of dsDNA Gel required per run varies between 12 capillary and 96 capillary Fragment Analyzer systems The volumes required are summarized below For 12 capillary Fragment Analyzer systems oo Volume of Intercalating dye Volume of dsDNA Gel analyzed 12 1 0 uL 10 mL 24 1 5 pL 15 mL 36 20 ul 20 mL 48 2 5 uL 25 mL 96 4 5 uL 45 mL A 5 mL minimum volume should be initially added to the tube 5 Rev DNF 920 2015JUL25 Advanced Analytical Technologies Inc For 96 capillary Fragment Analyzer systems ORS EDs Volume of Intercalating dye Volume of dsDNA Gel analyzed 96 4 0 uL 40 mL 192 8 0 uL 80 mL 288 12 0 uL 120 mL 384 16 0 uL 160 mL 480 20 0 uL 200 mL 6 Place the prepared dsDNA Gel Intercalating Dye mixture onto the instrument and insert into the desired gel fluid line Gel 1 or Gel 2 pump position Ensure the fluid line is positioned at the bottom of the conical tube to avoid introducing air bubbles which can cause pressurization errors 7 When adding dsDNA Gel to the instrument update the solution levels in the Fragment Analyzer instrument control software From the Main Menu select Utilities Solution Levels A menu will be displayed to enter in the updated fluid levels Fi
27. ypical Separation Results 1 000 bp DNA Ladder 1 Figure 8 shows the typical expected results for the 1 000 bp DNA Ladder diluted 12X with 1X TE diluent co injected with the 75 bp lower marker and 15 000 bp upper marker using a 33 cm effective 55 cm total length capillary array A total of 16 peaks should be observed with the sizes annotated as in Figure 8 All fragments in the ladder should be resolved DNF 920 1000bp DNA Ladder 75 bp LM 15000 bp UM RFU gt Q T 2296 1 1 I J I I 1 00 10 00 00 15 00 00 20 00 00 25 00 00 30 00 00 35 00 00 40 00 00 45 00 00 50 00 Time HH MM SS Figure 8 Example result showing 1 000 bp DNA Ladder injected with 75 bp lower marker and 15 000 bp upper marker using the DNF 920 reagent kit Method DNF 920 33 short array Figure 9 shows the same separation performed on a 55 cm effective 80 cm total length capillary array DNF 920 1000bp DNA Ladder 3000 75 bp LM 6000 15000 bp UM RFU 2 Q T 377 5 7 T 7 1 1 7 T T 1 T 00 25 00 00 30 00 00 35 00 00 40 00 00 45 00 00 50 00 00 55 00 01 00 00 01 05 00 01 10 00 01 15 00 01 20 00 Time HH MM SS Figure 9 Example result showing 1 000 bp DNA Ladder injected with 75 bp lower marker and 15 000 bp upper marker using the DNF 920 reagent kit Method DNF 920 55 long array 18 Rev DNF 920 2015JUL25 Advanced Analytical Technologies Inc Troubleshooting The following table lists several potent
28. zed or well H12 for 96 capillary instrument mix the contents of the well using the pipette by aspiration expulsion in the pipette tip 3 If total initial sample concentration is lt 10 ng L e g restriction digests a Prepare a 0 1X TE solution by diluting the supplied 1X TE buffer 10X with deionized water b Using a clean 96 well sample plate pipette 20 uL of the 0 1X TE buffer solution to each well to contain sample Pipette 49 uL of the 0 1X TE buffer solution to any well s to contain 1 000 bp DNA Ladder c Pipette 4 uL of each DNA sample into the respective wells of the sample plate mix the contents of the well using the pipette by aspiration expulsion in the pipette tip d 1 000 bp DNA Ladder Pipette 1 uL of 1 000 bp DNA Ladder solution into the respective well s of the sample plate to contain ladder i e well 12 of each row analyzed or well H12 for 96 capillary instrument mix the contents of the well using the pipette by aspiration expulsion in the pipette tip Important Sample Mixing Information When mixing sample with diluent solution it is important to mix the contents of the well thoroughly to achieve the most accurate quantification It is highly suggested to perform one of the following methods to ensure complete mixing A When adding 2 uL of sample to the 22 uL of diluent swirl the pipette tip while pipetting up down to further mix B After adding 2 uL of sample to the 22 uL of diluent place
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