Home

Agilent 220 TapeStation System Troubleshooting Manual

Contents

1. Damage to the TapeStation instrument and impact system performance gt Ensure only Agilent 2200 TapeStation approved consumables are used Consumables required for the 2200 TapeStation e Loading tips 5067 5152 or 5067 5153 Optical Tube 8x Strip 401428 and Optical Cap 8x Strip 401425 or 96 well Sample Plates 5067 5150 and 96 well Plate Foil Seal 5067 5154 e Vortex mixer Additional Material Required Not Supplied e Volumetric pipettes e Centrifuge suitable for 8 way strips or 96 well plates Heating block or PCR machine for RNA and Protein assays Agilent 2200 TapeStation System Troubleshooting Manual 17 1 Essential Measurement Practices Manual Marker Assignment 18 The 2200 TapeStation software assigns the most likely upper and lower marker using their run position In the event that the incorrect markers are assigned manually correcting this is possible using the simple procedure below The following example corrects the lower marker in a DNA file The same method can be used to manually assign upper markers RNA and Genomic DNA assays contain only lower markers a S a 1 Turn Alignment off by pressing the Aligned button 2 Un aligning the gel image shows the run distance of each band uncorrected by known marker sizes In a fully functional file markers will migrate to a similar position and have a similar shape Check the gel image to identify which bands are the correct upper
2. Size bp 1 000 25 50 100 200 300 400 500 700 1 500 Figure 17 Example of missing sample peak 60 Agilent 2200 TapeStation System Troubleshooting Manual Table 8 Troubleshooting DNA Applications 5 DNA probable causes for unexpected migration profile Problem Likely Cause Solution Additional bands have appeared to those expected in the sample lane The upper and lower markers appear unusually intense in the gel image DNA lower markers are normalised to appear the same in the electropherogram which can result in sample peaks looking smaller than expected There may be a contaminant peak caused by dust or dirt on the ScreenTape Sample prepared too long before analysis Insufficient Mixing The sample used is too dilute Unalign the image using the Aligned button in the TapeStation Analysis Software Contaminant peaks can be unassigned by right clicking on either the gel image or electropherogram peak Peaks caused by contaminants present sharp strong bands which are often slanted or uneven in the gel image and are usually easily distinguishable from sample peaks To avoid evaporation or settling ensure that your run is started immediately after sample preparation If using a 96 well plate always cover with the recommended foil seal Ensure correct mixing by reading the mixing recommendations See Good Measurement Practices for DNA on pag
3. 1 16 Essential Measurement Practices m 2200 TapeStation Controller A 01 04 Ladder Needle Change Transport About Agilent Technologies iy Sample Descriptions j i Sample Well Description D1000 ScreenTape ID 00 S029 010103 99 138100 Expires 29 Oct 2013 Figure 3 Error Temperature out of Range If the quoted current temperature is above the specified range please move the system out of direct sunlight and away from any windows Check that any air conditioning is functioning If the quoted current temperature is below the specified range please allow the instrument to equilibrate to the ambient temperature and avoid using in a cooled area Transporting the TapeStation If transporting the TapeStation instrument always allow it to acclimatize to the ambient temperature of the operating environment before use Before transporting the TapeStation ensure transport mode has been enabled in the controller software using the drop down menu labelled Transport Always remove the Sample plate tip holder and ScreenTape from the instrument before transportation When transporting the TapeStation ensure the correct packaging is used Shipping boxes can be purchased using part number G2960 60100 Agilent 2200 TapeStation System Troubleshooting Manual Essential Measurement Practices 1 Consumables CAUTION Use of incorrect consumables
4. 2200 TapeStation Software ScreenTape Compatibility Matrix 22 This section gives an overview on the compatibility of the TapeStation Software The 2200 TapeStation Software can be downloaded from the Agilent Genomics website www agilent com genomics tapestation Important Ensure that the TapeStation instrument is not connected to the laptop during software installation or upgrade NOTE For information regarding software installation PC compatibility and known problems or limitations please consult our latest Readme document This can be found using the link above Agilent Technologies 21 2 2200 TapeStation Software ScreenTape Compatibility Matrix The following matrix details the minimum allowable TapeStation software version required to run each ScreenTape assay Table 1 ScreenTape Compatibility Matrix ScreenTape Product Number 5067 5371 5067 5365 5067 5582 5067 5584 5067 5576 5067 5579 P200 Genome pyO00 HS D1000 RNA HS RNA Name ScreenTape DNA ScreenTape ScreenTape ScreenTape ScreenTape P ScreenTape P P P P 2200 G2964AA A 01 01 A 01 03 A 01 04 A 01 04 A 01 04 A 01 04 TapeStation 2200 Nucleic Not G2965AA Acid A 01 03 A 01 04 A 01 04 A 01 04 A 01 04 L compatible 5 TapeStation x 2200 Not G2966AA ScreenPlex Lone A 01 03 A 01 04 A 01 04 A 01 04 A 01 04 E compatible 2 TapeStation A 5 Lab901 Seen Not Not Not Not Not Not E ST007 Single Load
5. Agilent 2200 TapeStation System Troubleshooting Manual Troubleshooting DNA Applications Molarity Molarity Molarity is determined from both size and quantity Errors in sizing and quantification will result in erroneous molarity calculations Always ensure that the good measurement practices for sizing and quantification have been followed to ensure accurate molarity values Agilent 2200 TapeStation System Troubleshooting Manual 5 49 5 50 Troubleshooting DNA Applications Genomic DNA Assay When using the Genomic DNA Assay it is important to note the following recommendations to ensure correct sizing Equilibrate reagents to room temperature Genomic DNA Reagents must be equilibrated to room temperature for 30 minutes before use Failure to do so can affect sizing results Cold reagents will overestimate the size of genomic DNA samples Table4 The effect of room temperature RT equilibrated Genomic DNA reagents as well as cold reagents on the sizing accuracy of the Genomic DNA ScreenTape assay Expected Reagents at RT Reagents at 4 C MW 17000 bp 18867 bp 24369 bp Accuracy 11 43 Always use fresh genomic DNA ladder Ladder must be prepared fresh for each run and run in the first available position e Run profile decreases as ladder warms up within the instrument e Sizing results will be affected by adjusted ladder run profiles No software ladder is available for genomic DNA A
6. File does not have Genomic DNA file where The Genomic DNA ScreenTape assay ladder assigned either no ladder has been always requires a ladder run or the ladder has been If a ladder has been run assign it using the unassigned Assign button in the main ribbon Otherwise prepare a new run with Genomic DNA Ladder in the first position Ladder run as sample For Genomic DNA files a Re assign this lane as a ladder using the ladder has been assigned Assign button in the main ribbon as a sample No DIN will be displayed Sample concentration The gDNA sample Either dilute or concentrate your sample outside functional concentration is outside until it is within the recommended range range for DIN the recommended for application as stated in the assay Quick functional range for DIN Guide then prepare a new run DIN values may not be present or reliable for this file Sample concentration The concentration of the Either dilute or concentrate your sample outside recommended sample is out of the until it is within the recommended range range concentration specified for for application as stated in the assay Quick the assay Concentration Guide then prepare a new run values are not reliable The original ladder for For gDNA comparison Where possible manually delete extra this lane had too many files The ladder of the peaks by right clicking on the affected peak peaks original file had too many and selecting delete peaks This may affect Please see Goo
7. suggested actions below If after completing the suggested actions the error message persists or instrument function does not return to normal please contact your local support representative Found new hardware A Found New Hardware pop up launched when the USB cable of the TapeStation was inserted into the laptop Probable cause Suggested actions 1 The USB cable has been inserted intoa new _ Re install software using the software Readme port Agilent 2200 TapeStation System Troubleshooting Manual Error Messages Failed to connect to the barcode scanner or camera Probable cause Suggested actions 1 The drivers have not been installed correctly Power cycle instrument leaving 60 seconds after power up before re launching controller Ifthe message persists try a different USB port on the laptop for the cable to connect to the TapeStation Camera error Freeze Image returned failure code 122 Probable cause Suggested actions 1 Camera connection error Power cycle instrument leaving 60 seconds after power up before re launching controller Agilent 2200 TapeStation System Troubleshooting Manual 9 99 9 Error Messages The instrument has failed to respond in a timely fashion Probable cause Suggested actions 1 Incorrect consumables have been used Check the correct consumables have been used and that the lids are removed from sample tubes before use Contact your Agilent support team with details o
8. Insufficient migration due to presence of salt Samples which are contaminated with genomic DNA contain a third peak which migrates in the region of the 18 and 28S Occasionally this can be mistaken for the 18 or 28S peak For correct sizing ensure that all peaks are annotated correctly Treat samples with DNAse and rerun to eliminate the genomic DNA peak Any issues with the run ladder lane will be annotated within the TapeStation Analysis Software sample table under Observations Manually insert or delete affected peaks or insert a software saved ladder to regain sizing information For best sizing results rerun to ensure a functioning ladder lane Ensure the correct marker peaks are picked up in the TapeStation Analysis Software See Manual Marker Assignment on page 18 Ensure that all sample peaks are within the recommended sizing range for the application Dilute your samples to ensure low levels of buffer salt True migration profiles can be seen by pressing the Aligned button in the TapeStation Analysis Software Please refer to the salt tolerance guidelines for your assay in the User Manual or appropriate Quick Guide Agilent 2200 TapeStation System Troubleshooting Manual 69 6 Troubleshooting RNA Applications Missing Marker Peaks Al neat j e f Z amp 2 E v Figure 24 Example of missing marker peaks in an RNA assay Table 11 RNA Possible causes of missing marker peaks In order
9. Run Properties This new curve will be used to fill in concentration data for all remaining sample peaks Samples used to construct the calibration curve show two concentration values e The manually entered value in red in brackets follows the value back calculated from the curve which is displayed in black For example 29 6 30 5 Manually entered values can be edited or deleted in the electropherogram view by clicking on the concentration field in the data table The calibration curve will then be corrected to use the updated or remaining values Agilent 2200 TapeStation System Troubleshooting Manual 81 7 Troubleshooting Protein Applications Incorrect Quantification results Table 16 Protein Possible causes for incorrect quantification results in order of probability Cause Solution Insufficient Mixing Sample concentration outside recommended range for application Incorrect Peak integration Incorrect heating procedure Incorrect protocol used Sample prepared too long before the run is performed Pipetting technique or pipette calibration Incorrect ScreenTape or reagent storage Ensure correct mixing see Mixing recommendations on page 10 Either dilute or concentrate the sample until it is within the recommended concentration range for application then prepare a new run Ensure that all sample peaks and markers are integrated correctly in the TapeStation Analysis Software by clicki
10. adapters a Ip Other devices fa 8CM2070240 fp Network Controller E Portable Devices PF Ports COM amp LPT ID Processors IP Security Devices i Sensors Sound video and game controllers pM System devices a Universal Serial Bus controllers 5180 Area Imager Generic USB Hub Generic USB Hub Generic USB Hub Honeywell Control Device Intel R 7 Series C216 Chipset Family USB Enhanced Host Controller 1 26 Intel R 7 Series C216 Chipset Family USB Enhanced Host Controller 1E2D Intel R USB 3 0 eXtensible Host Controller Intel R USB 3 0 Root Hub uEye Ul 224x Series USB Composite De Update Driver Software USB Mass Storage Disable USB Root Hub Uninstall USB Root Hub USB Serial Convert Scan for hardware changes Launches the Update Driver Softw Properties Make sure that all USB devices are enabled Disabled devices show up with an exclamation mark To enable disabled USB devices right click on the device and click Enable Agilent 2200 TapeStation System Troubleshooting Manual Instrument Communication 3 Barcode Reader For correct function the instrument must read barcodes both inside the instrument when idle and on the ScreenTape when ScreenTape is inserted Excess light can affect this procedure Ensure that the 2200 TapeStation instrument is placed away from direct light sources Excess light can interfere with barcode recognition and result in
11. compatible compatible compatible compatible compatible compatible DNA Lab901 TapeStation NO Not Not Not Not Not ST008 Single Load compatible compatible compatible compatible compatible compatible z Lab901 ini _ Not Not Not Not Not Not S ST009 ap compatible compatible compatible compatible compatible compatible 2 Single Load Lab901 Combined 7010 TaneStatian A 01 01 A 01 03 A 01 04 A 01 04 A 01 04 A 01 04 Lab901 ScreenPlex Not 1017 TapeStation compatible A 01 03 A 01 04 A 01 04 A 01 04 A 01 04 Lab901 Nucleic Acid Not ST019 TapeStation compatible A 01 03 A 01 04 A 01 04 A 01 04 A 01 04 22 Agilent 2200 TapeStation System Troubleshooting Manual Agilent 2200 TapeStation System Troubleshooting Manual 3 Instrument Communication Instrument and Laptop 24 USB Connection 25 Updating USB Drivers 26 Barcode Reader 33 Retrieving log files 34 This section gives an overview on possible issues caused by instrument communication Apg Agilent Technologies 23 3 Instrument Communication Instrument and Laptop Always power up the 2200 TapeStation System in this order a Laptop b Instrument c Controller e Always power down laptops and instruments at night e Always save run files to a local folder on the TapeStation laptop Always download anti virus software onto the 2200 TapeStation laptops 24 Agilent 2200 TapeStation System Troubleshooting Manual Instrument Communication 3 USB Connection 2200 TapeStati
12. documentation This section describes techniques for ensuring reliable quantification and sizing results using DNA assays on the 2200 TapeStation System and includes advice on the following topics Quantification Sizing e Molarity Genomic DNA Assay For more details please consult the Technical Overview using the link below www chem agilent com Library technicaloverviews Public 5991 5153EN pdf Agilent 2200 TapeStation System Troubleshooting Manual Troubleshooting DNA Applications 5 Quantification Sample mixing Sample and sample buffer must be vortex mixed according to protocol followed by centrifugation to remove any bubbles Insufficient mixing can cause discrepancies in quantification The effects of insufficient mixing were investigated using D1000 ScreenTape and reagents and results are presented in Figure 7 on page 41 and Figure 8 on page 42 as well as Table 3 on page 42 Poor mixing dramatically affects the reported sample concentration Following the recommended procedure is the best way to attain accurate quantification results Please see Mixing recommendations on page 10 or the appropriate assay Quick Guide for more details A B co Vortex Mixing Ibp Pipette Mixing a0 No Mixing 100 200 5 500 600 300 Size bp 100 Figure 7 The electropherogram A and the gel image B of the ScreenTape mixing tests In both panels the green tr
13. of probability Cause Solution Insufficient Mixing Ensure correct mixing see Mixing recommendations on page 10 Sample Either dilute or concentrate your sample until it is within the recommended range for the concentration application as seen in the appropriate assay Quick Guide then prepare a new run outside recommended range for application 70 Agilent 2200 TapeStation System Troubleshooting Manual Degraded RNA Ladder and or samples nt Figure 25 Example of degraded RNA resulting in missing peak identification A0 L a Troubleshooting RNA Applications 6 5000 4000 4 3000 2000 Sample Intensity FU 1000 cS Table 12 RNA Possible causes of degraded sample in order of probability Cause Solution Incorrect heating step RNAse contamination of the reagents RNAse contamination of the plasticware Ensure the correct heating recommendations for the assay are followed as described in the User Manual and appropriate Quick Guide Ensure Good Laboratory Practices are followed as detailed in the RNA section of the TapeStation user manual Prepare a new run using new reagents Ensure Good Laboratory Practices are followed as detailed in the RNA section of the TapeStation user manual Agilent 2200 TapeStation System Troubleshooting Manual 71 6 Troubleshooting RNA Applications Incorrect or missing RIN value Table 13 RNA Pos
14. over and under heating the samples can affect concentration values To avoid evaporation or settling ensure that the run is started immediately after sample preparation If using a 96 well plate always cover with the recommended foil seal Ensure correct pipetting technique and up to date pipette calibration Reverse pipetting technique is advised for RNA and High Sensitivity RNA assays Use the Aligned button in the TapeStation Analysis Software to detect the correct markers then assign them by right clicking see Manual Marker Assignment on page 18 Ensure that all sample peaks are within the recommended sizing range for the application Dilute your samples to ensure low levels of buffer salt True migration profiles can be seen by pressing the Aligned button in the TapeStation Analysis Software Please refer to the salt tolerance guidelines for your assay in the User Manual or appropriate Quick Guide 68 Agilent 2200 TapeStation System Troubleshooting Manual Troubleshooting RNA Applications 6 Incorrect Sizing Results Incorrect sizing results can have multiple causes If after checking each of these causes your results files continue to show sizing discrepancies please contact your local support representative Table 10 RNA Possible causes of incorrect sizing results in order of probability Cause Solution Genomic DNA contamination Issues with ladder lane Incorrect Marker peaks picked up
15. probability Cause Solution Only specific RNA peaks 16 18 23 28S and Lower marker peak will be annotated by the software Extremely degraded sample There are 2 bands present at the 18S peak position Genomic DNA contamination Manually add any important peaks by right clicking on either the gel or electropherogram and selecting add other peak The TapeStation Analysis Software can occasionally fail to recognize extremely degraded RNA sample peaks Prepare samples again ensuring Good Laboratory practices are followed There may have been insufficient sample denaturation prior to ScreenTape analysis The 18S peak may have partially reverted back to its original non denatured conformation Ensure the correct denaturation conditions during RNA sample preparation according to the assay sample preparation guide Samples should be kept on ice after preparation and run within 2 hours of denaturation Samples contaminated with genomic DNA contain a third peak which migrates in the region of the 18 and 288 Occasionally this peak can be mistaken for the 18 or 28S peak Ensure that all peaks are annotated correctly Treat samples with DNAse to eliminate the genomic DNA peak then prepare a new run Agilent 2200 TapeStation System Troubleshooting Manual 73 6 Troubleshooting RNA Applications Unexpected migration profile There are a number of issues which can affect migration and sample peak profile If th
16. value 72 Incorrect or Missing Peak Annotation 73 Unexpected migration profile 74 Troubleshooting Protein Applications 79 Buffer compatibility 80 Quantification of Protein Samples 81 Incorrect Quantification results 82 Incorrect Sizing Results 83 Unexpected Migration Profile 84 Instrument Maintenance 85 General Information 86 Changing the Needle 87 Preventative Maintenance Interval 91 ErrorMessages 93 Analysis Software Warning Messages 94 Communication Error Messages 98 Consumable Error Messages 103 10 ScreenTape Products Parts and Consumables 105 ScreenTape Products 106 Parts and Consumables 109 Agilent 2200 TapeStation System Troubleshooting Manual Agilent 2200 TapeStation System Troubleshooting Manual 1 Essential Measurement Practices Overview 6 Intended use of the 2200 TapeStation System 7 Performance Limitations of Use 8 Tools and Handling 9 Mixing recommendations 10 Reagents and Reagent Mixes 11 Samples 12 Screenlape 13 2200 TapeStation Instrument 15 Consumables 17 Consumables required for the 2200 TapeStation 17 Additional Material Required Not Supplied 17 Manual Marker Assignment 18 Apg Agilent Technologies Overview Overview This section lists all user relevant hints on handling the 2200 TapeStation instrument tools ScreenTape consumables and reagents For the latest information on 2200 TapeStation compatible assays visit the Agilent website at www agilent com genomics
17. 00 TapeStation Nucleic Acid System G2966AA 2200 TapeStation ScreenPlex System Agilent 2200 TapeStation System Troubleshooting Manual 87 Instrument Maintenance Needle change intervals e After 7680 lanes 3840 pierces in a Single loading system the controller software will inform the user that a needle change is pending The word Needle will appear in the bottom of the controller software inside a yellow box e After 8320 lanes 4160 pierces in a Single loading system a needle change is recommended The box around the word Needle will change from yellow to red e After 8960 lanes 4480 pierces in a Single loading system the needle has completed its lifetime and must be changed before the TapeStation will start leedle Change Transport Help TapeStationUser Agilent Technologies Sample Well Description i Sample Descriptions Please insert a ScreenTape Ready Figure 31 Controller software indicating a Needle change is recommended Agilent 2200 TapeStation System Troubleshooting Manual Instrument Maintenance 8 Parts required p n Description 1 G2960 60062 Needle cartridge for use in single pump systems For use with product numbers ST007 ST008 and ST009 OR 1 G2960 60063 Needle cartridge for use in dual pump systems For use with product numbers ST017 ST019 ST010 G2960A G2961A G2964AA G2965AA and G2966AA The needle replacement procedure as detailed below is performed during
18. 87 compatibility buffer 80 consumables 17 D degraded ladder RNA 71 degraded samples RNA 71 DNA incorrect sizing results 55 E equilibrate reagents 50 F flicking 48 fresh ladder 50 G genomic DNA 110 quantification 54 H handling 6 tools 9 l incorrect peak annotation RNA 73 instrument 15 24 integration 43 intended use 7 L lanes blank 36 laptop and instrument 24 laptop 24 LED status 15 limitations of use 8 logfiles 34 M marker peaks missing 5 7 marker manual assignment 18 matrix compatibility 22 migration profile protein 84 RNA 74 unexpected 58 3 issing marker peaks RNA 70 missing peak annotation RNA 73 missing marker peaks 57 ixing 41 recommendations 10 3 N needle change 87 0 operating temperature 15 overview handling 6 reagents 6 tape 6 tools 6 P peak 43 performance limitations 8 protein samples quantification 81 protein incorrect quantification 82 incorrect sizing results 83 unexpected migration profile 84 protocol 45 Q quantification Agilent 2200 TapeStation System Troubleshooting Manual incorrect 68 protein samples 81 protein 82 R reader barcode 33 reagent mixes 11 reagents 11 recommendations mixing 10 RINe incorrect 72 missing 72 RNA degraded ladder 71 degraded samples 71 incorrect peak annotation 73 missing marker peaks 70 missing peak annotation 73 unexpected migration profile 74 S sampl
19. Agilent 2200 TapeStation a System e e 0 20 lt e J Troubleshooting Manual f Agilent Technologies Notices Agilent Technologies Inc 2015 No part of this manual may be reproduced in any form or by any means including elec tronic storage and retrieval or translation into a foreign language without prior agree ment and written consent from Agilent Technologies Inc as governed by United States and international copyright laws Manual Part Number 62964 90110 Edition 01 2015 Printed in Germany Agilent Technologies Hewlett Packard Strasse 8 76337 Waldbronn Warranty The material contained in this docu ment is provided as is and is sub ject to being changed without notice in future editions Further to the max imum extent permitted by applicable law Agilent disclaims all warranties either express or implied with regard to this manual and any information contained herein including but not limited to the implied warranties of merchantability and fitness for a par ticular purpose Agilent shall not be liable for errors or for incidental or consequential damages in connection with the furnishing use or perfor mance of this document or of any information contained herein Should Agilent and the user have a separate written agreement with warranty terms covering the material in this document that conflict with these terms the warranty terms in the sep arate ag
20. Manual the specific assay quick guide and online www agilent com genomics tapestation e Protect the gel lanes of the ScreenTape from excessive force do not bend or flex the ScreenTape Store the ScreenTape in the provided packaging between 2 8 C e ScreenTape can be used straight from the fridge with no equilibration time e Handle ScreenTape carefully to avoid fingerprints or fibers which can affect imaging Ensure that ScreenTape is flicked gently before inserting into the instrument if there are any small bubbles present then this will move them to the top of the chamber see Figure 2 on page 14 The presence of small bubbles within the buffer chamber of the ScreenTape is normal These bubbles often occur at the gel buffer interface and need to be displaced prior to running Failure to remove bubbles from the gel buffer interface is detrimental to the performance of the ScreenTape Agilent 2200 TapeStation System Troubleshooting Manual 13 1 14 Essential Measurement Practices TAATAI EREIN E AAE A AR saifojouysay uap y z5 Figure 2 Flicking the ScreenTape consumable removes bubbles from the gel interface Partially used ScreenTape those that contain lanes run on previous occasions should be returned to the box and stored vertically between 2 8 C for a maximum of 2 weeks The laptop utilized for performing any previous use s of the ScreenTape must be utilized for all further re use ScreenTape
21. PI controllers IEEE 1394 Bus host controllers Z Imaging devices lt Keyboards E Memory technology driver A Mice and other pointing devices W Modems amp Monitors KP Network adapters lip Other devices Di 8CM20702A0 Dn Network Controller Y Ports COM amp LPT OF 5180 Area Imager C IP Communications Pc IEP ECP Printer Port LP7 Disable FP USB Serial Port CON Uninstall Update Driver Software 1B Processors IP Security Devices Scan for hardware changes Gi Sencor P ti Launches the Update Driver Softwar If the following pop up window appears choose Browse my computer for driver software and choose let me pick from a list on the next pop up window G Update Driver Software 5180 Area Imager COM12 Browse for driver software on your computer Search for driver software in this location E Program Files G86 Agilent 2200 TapeStation Controller Softwar v Browsen J Include subfolders gt Let me pick from a list of device drivers on my computer This list will show installed driver software compatible with the device and all driver software in the same category as the device Agilent 2200 TapeStation System Troubleshooting Manual Instrument Communication 3 Updating USB Drivers 3 Click on the 5180 Area Imager to mark it and click Next ee Do Update Driver Software 5180 Area Imager COM12 Select the device driver you want to install for this hardw
22. a non functioning instrument When inserting a ScreenTape into the TapeStation its orientation must be correct 1 Insert the ScreenTape with the label towards the front of the instrument and the barcode facing right N Agilent 2200 TapeStation System Troubleshooting Manual 33 3 Instrument Communication Retrieving log files 34 Log files can be retrieved using the TapeStation Analysis Software These are often useful for troubleshooting purposes Please ensure that when communicating with the Technical Support Channel both the log files and the analysis file in question are sent for technical troubleshooting 1 Open the TapeStation Analysis Software 2 In the file menu select Help then click on the export error logs button Export Error Logs Fy Export Error Logs from the Analysis and Controller applications to a zip file Agilent 2200 TapeStation System Troubleshooting Manual Agilent 2200 TapeStation System Troubleshooting Manual S ee 4 r Instrument Troubleshooting Blank Lanes 36 This section gives an overview on possible issues caused by the instrument Apg Agilent Technologies 35 4 Instrument Troubleshooting Blank Lanes 36 There are a number of reasons blank lanes can occur in a TapeStation Analysis file If after checking each of the causes listed below your results files continue to show blank lanes please contact your local support representative Sample I
23. ace shows the recommended protocol of vortex mixing using the IKA vortexer and adaptor at 2 000 rpm for 1 minute followed by brief centrifugation Blue shows results for pipette mixing only Red shows the effect of no mixing Images were taken from the Agilent TapeStation Analysis Software Agilent 2200 TapeStation System Troubleshooting Manual 41 5 42 Troubleshooting DNA Applications Good Measu ement Practices i re tor D J A 120 g 100 3 Ege SSS o c 60 Toog O 0Do OSE 3538 40 Es 25 20 0 Vortex mixing Pipette mixing No mixing then spin down only Figure 8 Chart of reported concentrations for the ScreenTape mixing tests using the D1000 Assay Concentrations are expressed as a percentage of the theoretical for the three mixing methods As above the green bar represents the recommended protocol of vortex mixing using the IKA vortexer and adaptor at 2 000 rpm for 1 minute followed by brief centrifugation blue pipette mixing only red no mixing Table3 ScreenTape Reagent mixing tests Quantification values obtained from the TapeStation Analysis Software and the Agilent D1000 ScreenTape Assay when using the correct protocol vortex mix followed by brief centrifugation and two incorrect mixing protocols pipette mixing only and no mixing as illustrated above Measured concentration Theoretical concentration ng pL ng pL Vortex mixing then 68 5 70 centrifugation P
24. ample or marker Follow the storage conditions specified for the assay peaks one or other condinons not both Sample concentration outside Either dilute or concentrate your sample until it is within the recommended range for recommended range for application as stated in the assay Quick application Guide then prepare a new run Sample Intensity FU Figure 20 Example of low signal intensity of the marker peaks D 800bp fragment 6 pg ul 2 s a Figure 21 Example of low signal intensity of the sample peaks Agilent 2200 TapeStation System Troubleshooting Manual 63 5 Troubleshooting DNA Applications Table8 DNA probable causes for unexpected migration profile Problem Likely Cause Solution Low signal intensity of sample and marker Incorrect reagent storage Follow the storage conditions specified for the assay peaks Both or of conditions Prepare a new run with fresh reagents ladder peaks A1 O A1 Ladder S S 2 2 2 ps A al Figure 22 Example of low signal intensity in the High Sensitivity D1000 Ladder 64 Agilent 2200 TapeStation System Troubleshooting Manual Troubleshooting DNA Applications 5 Table8 DNA probable causes for unexpected migration profile Problem Likely Cause Solution Incorrect migration Salt concentration samples have not reached the lower end of the gel lane Partial electr
25. and lower markers Agilent 2200 TapeStation System Troubleshooting Manual Essential Measurement Practices 1 Manual Marker Assignment Incorrectly Correctly assigned as the assigned as the Lower Marker Lower Marker Figure 4 Manual lower marker assignment in the TapeStation Analysis Software First identify the correct marker 3 To add a Peak assignment hover the mouse pointer above the unassigned peak and right click Then select Add Peak as shown in Figure 5 on page 20 Agilent 2200 TapeStation System Troubleshooting Manual 19 1 Essential Measurement Practices Manual Marker Assignment 4 Next assign the correct Marker peak by right clicking and selecting Assign as Upper Marker Assign as Lower Marker as shown in Figure 5 on page 20 Zoom Out Zoom Out Reset Zoom Reset Zoom f Snapshot this Graph Snapshot this Graph a Add Peak Delete Peak Delete Peak E Delete All Sample Peaks Delete All Sample Peaks Assign as Upper Marker Figure 5 Manual lower marker assignment in the TapeStation Analysis Software Assign the correct marker by right clicking 5 Turn the alignment back on by pressing the Aligned button again Check the updated gel and electropherogram images the data should now be correctly aligned 20 Agilent 2200 TapeStation System Troubleshooting Manual NOTE b oe ec 27 eee Agilent 2200 TapeStation System Troubleshooting Manual
26. are Select the manufacturer snd model of your hardware device and then click Next If you have a disk that contains the driver you want to install click Have Disk Vi Show compatible hardware Model ER This driver is digitally signed Have Disk T Geen 4 The Driver software will automatically update When complete the following window will appear KJ L Update Driver Software 5180 Area Imager COM12 Windows has successfully updated your driver software Windows has finished installing the driver software for this device 5180 Area Imager 5 Make sure that all USB devices are enabled Disabled devices show up with an exclamation mark To enable disabled USB devices right click on the device and click Enable Agilent 2200 TapeStation System Troubleshooting Manual 31 3 32 Instrument Communication Updating USB Drivers Updating Camera Driver software 1 To re install the camera on a protein capable 2200 TapeStation System G2964AA expand the Universal Serial Bus controller list and right click on the uEye UI 224x Series For a Nucleic Acid TapeStation G2965AA right click on the uEye UI 154x Series After identifying the camera driver identified as malfunctioning update the appropriate drivers as described in the Updating Barcode Reader and USB to Serial Convertor Drivers section above FE Device Menage File Action View Help ev S Bl on NS E Monitors KP Network
27. bservations Manually insert or delete affected peaks or insert a software saved ladder to regain sizing information Dilute your samples to ensure low levels of buffer salt True migration profiles can be seen by unaligning your gel image by pressing the Aligned button in the TapeStation Analysis Software Please refer to the P200 Buffer compatibility section and make the necessary adjustments to the sample buffer Store reagent kit according to assay Quick Guide Use stain solution within 1 week of preparation Agilent 2200 TapeStation System Troubleshooting Manual 83 7 Troubleshooting Protein Applications Unexpected Migration Profile Table 18 Protein Causes of unexpected migration profile Problem Likely Cause Solution Additional sharp bands are present at the top of the gel image The markers have appeared in the sample lane but the sample has not Higher than normal background noise Low signal intensity Incorrect migration samples have not reached the lower end of the gel lane The sample may be too concentrated for the application The sample used is below the concentration range recommended to the assay Insufficient Mixing Insufficient Mixing Incorrect ScreenTape and reagent storage conditions Insufficient Mixing Incorrect reagent storage conditions Salt concentration Dilute your sample until it is within the recommended concentration range for application then prepa
28. d Measurement Practices sizing for DNA on page 40 for advice on handling the Genomic DNA Ladder 96 Agilent 2200 TapeStation System Troubleshooting Manual Table 20 Analysis Software Warning Messages Error Messages 9 Affected Assay Observation Detailed description Corrective action RNA High Sensitivity RNA RIN edited RNA concentration outside recommended range for RIN Sample concentration outside recommended range User has manually changed the position of the 18S fragment RIN may have changed as a result RNA Sample concentration lies outside the functional range for RIN RIN values are not reliable The concentration of the sample is out of the concentration specified for the assay Concentration values are not reliable An edited RIN will be displayed To recover the original RIN assigned by the software use the Restore Default Settings button on the main ribbon Either dilute or concentrate your sample until it is within the recommended range for application as stated in the assay Quick Guide then prepare a new run Either dilute or concentrate your sample until it is within the recommended range for application as stated in the assay Quick Guide then prepare a new run Agilent 2200 TapeStation System Troubleshooting Manual 97 98 9 Error Messages Communication Error Messages Should you encounter an error message please work through the
29. e TapeStation Analysis Software ScreenTape types e D1000 High Sensitivity D1000 Genomic DNA RNA High Sensitivity RNA gt P200 TapeStation ScreenPlex Software ScreenTape types DS12 Agilent 2200 TapeStation System Troubleshooting Manual 103 9 Error Messages Pipette bin full One or more pipette tips may still be in the pipette tip bin from a previous run Empty the pipette tip bin before continuing Probable cause Suggested actions 1 The tip discard section has not been Remove all discarded tips and replace the tip emptied after the last run holder into the instrument Pipette pump affixed to the pump A pipette pump may be affixed to the pump Please insert an empty tip holder into the instrument close the lid then click OK to continue Probable cause Suggested actions 1 Itis likely that the TapeStation has lost Insert an EMPTY tip holder into the instrument power during a run and needs to discard then follow the on screen instructions The any tips which have been picked up before instrument will automatically discard any tips starting again into the tip bin Remember to empty the tip bin before starting the next run 104 Agilent 2200 TapeStation System Troubleshooting Manual Agilent 2200 TapeStation System Troubleshooting Manual e gn Pe e e e e 10 7 e ScreenTape Products Parts and 9 Consumables ScreenTape Products 106 Parts and Consumables 109 This section gives a
30. e 40 Concentrate your sample until it is within the recommended range for application then prepare a new run Figure 18 Example of intense upper and lower markers seen in the blue gel image Lower markers are normalised in electropherogram view and appear identical in signal Agilent 2200 TapeStation System Troubleshooting Manual 61 5 Troubleshooting DNA Applications Table 8 DNA probable causes for unexpected migration profile Problem Likely Cause Solution Insufficient Mixing Higher than normal background noise Incorrect ScreenTape or reagent storage Ensure correct mixing by reading the mixing recommendations See Good Measurement Practices for DNA on page 40 Follow the storage conditions specified for the assay Al B1 B1 Sheared DNA LI Ta 600 500 om m 400 m amp 300 z 200 amp 100 on ols Size s g s S 3 3 8 g 3 bp Figure 19 Example of high background noise 62 Agilent 2200 TapeStation System Troubleshooting Manual Troubleshooting DNA Applications 5 Table8 DNA probable causes for unexpected migration profile Problem Likely Cause Solution Insufficient Mixin Ensure correct mixing by reading the mixing recommendations g See Good Measurement Practices for DNA on page 40 Low signal intensity Incorrect reagent storage of s
31. e 41 samples 12 screentape 13 sizing results protein 83 software 21 system intended use 7 T tools handling 9 transport 16 U update usb drivers 26 usb drivers update 26 usb 25 update 26 Ww workflow 46 Agilent 2200 TapeStation System Troubleshooting Manual Index 111 www agilent com In This Book The manual describes the following Essential Measurement Practices e 2200 TapeStation Software e Instrument Communication Instrument Troubleshooting e Troubleshooting DNA Applications e Troubleshooting RNA Applications Troubleshooting Protein Applications e Instrument Maintenance e Error Messages Agilent Technologies 2015 Printed in Germany 01 2015 G2964 90110 fe Agilent Technologies
32. e issues below do not describe your analysis file please contact your support representative Table 15 RNA Possible causes of unexpected migration profile Problem Likely Cause Solution There are two bands evident in the 28S peak opposition There are two bands present at the 18 or The 28S peak may have partially reverted back to its original non denatured conformation The peak may have partially reverted back to its original There may have been insufficient sample denaturation prior to ScreenTape analysis Ensure the correct denaturation conditions during RNA sample preparation Samples should be kept on ice and run within 2 hours of denaturation There may have been insufficient sample denaturation prior to ScreenTape analysis Ensure the correct denaturation conditions during RNA sample 28S peak position non denatured preparation conformation Samples should be kept on ice and run within 2 hours of denaturation D1 01 8 Kia S 5000 4000 Fs 3000 2000 1000 0 ya Figure 26 Example of extra peaks being picked up at the 28S position 74 Agilent 2200 TapeStation System Troubleshooting Manual Troubleshooting RNA Applications 6 Table 15 RNA Possible causes of unexpected migration profile Problem Likely Cause Solution A split peak or additional Denaturation of the Ensure the correct denaturation conditions when preparing
33. enance service must be performed by a trained Agilent Field Service Engineer and can be scheduled by contacting your local Agilent Service representative Ladder NeedleChange Transport About Agilent Technologies Sample Descriptions Sample Well Description 0 ie 0 ie ie 0 ie 0 Please insert a ScreenTape Figure 32 Controller software indicating a Preventative Maintenance is due Agilent 2200 TapeStation System Troubleshooting Manual 91 8 Instrument Maintenance If both the Maintenance message and the Needle message are being displayed simultaneously then only a Preventative Maintenance should be performed The Preventative Maintenance procedure includes a Needle Change and following PM service both messages will disappear Ladder NeedleChange Transport About m Agilent Technologies Sample Descriptions Sample Well Description nm za Q5 0 O O Q QO 10 O O O OvO O O Figure 33 Controller software indicating a Preventative Maintenance is due Schedule Preventative Maintenance do not perform a Needle Change 92 Agilent 2200 TapeStation System Troubleshooting Manual Agilent 2200 TapeStation System Troubleshooting Manual Error Messages Analysis Software Warning Messages 94 Communication Error Messages 98 Found new hardware 98 Failed to connect to the barcode scanner orcamera 99 Camera error Freeze Image returned failure code 122 99 The i
34. ented in these modes will be different Electropherogram view is designed for use with discrete peaks and the default size reported is that of the highest point of the peak Region view calculates data over a whole smear or region for example NGS libraries and reports size as that of the center of the regions mass This gives the user an idea of the distribution of sizes within that sample Electropherogram View lt 3 MW 179bp Average MW 232 bp Figure 12 The sizing data obtained in Electropherogram and Region views of the Agilent TapeStation Analysis Software Agilent 2200 TapeStation System Troubleshooting Manual 47 5 48 Troubleshooting DNA Applications Identifying the correct markers The markers are used as internal references to determine the molecular weight size of the sample Incorrect identification will lead to miscalculations in reported sizing values Always ensure that the upper and lower markers have been identified correctly See Manual Marker Assignment on page 18 Flicking the ScreenTape The presence of small bubbles within the buffer chamber of the ScreenTape is normal e These bubbles often occur at the gel buffer interface and need to be displaced by flicking prior to running e Failure to remove bubbles from the gel buffer interface is detrimental to the performance of the ScreenTape See ScreenTape on page 13 for more details
35. es and Printers Default Programs Help and Support Agilent 2200 TapeStation System Troubleshooting Manual Instrument Communicatio 3 Updating USB Drivers 2 The Barcode reader 5180 Area Imager the USB to Serial convertor and the respective uEye UI camera should all be present in this list see red circles If one of the devices is not present or is highlighted as having issues with an exclamation mark a driver update is required To resolve this first click on File and choose Device Manager i gt Control Panel Hardware and Sound Devices and Printers Edt View Tools Help 5180 Ares Imager COM12 3 Make sure that all USB devices are enabled Disabled devices show up with an exclamation mark To enable disabled USB devices right click on the device and click Enable Agilent 2200 TapeStation System Troubleshooting Manual 29 3 30 Instrument Communication Updating USB Drivers Updating Barcode Reader and USB to Serial Convertor Drivers 1 To locate the Barcode reader and USB to Serial Drivers expand the Ports list Right click on the device identified as malfunctioning in the previous step and select Update Driver software The following instructions show updating the 5180 Area Imager barcode reader driver By Device File Action View Help e nO mn e PRs a Datasystem01 Batteries 7 Computer a Disk drives EX Display adapters xf DVD CD ROM drives Ca IDE ATA ATA
36. et A WARNING notice denotes a hazard It calls attention to an operating procedure practice or the like that if not correctly performed or adhered to could result in personal injury or death Do not proceed beyond a WARNING notice until the indi cated conditions are fully under stood and met Agilent 2200 TapeStation System Troubleshooting Manual Contents Contents 1 Essential Measurement Practices 5 Overview 6 Intended use of the 2200 TapeStation System 7 Performance Limitations of Use 8 Tools and Handling 9 Mixing recommendations 10 Reagents and Reagent Mixes 11 Samples 12 Screenlape 13 2200 TapeStation Instrument 15 Consumables 17 Manual Marker Assignment 18 2 2200 TapeStation Software 21 ScreenTape Compatibility Matrix 22 3 Instrument Communication 23 Instrument and Laptop 24 USB Connection 25 Updating USB Drivers 26 Barcode Reader 33 Retrieving log files 34 4 Instrument Troubleshooting 35 Blank Lanes 36 5 Troubleshooting DNA Applications 39 Good Measurement Practices for DNA 40 Incorrect Quantification Results 52 Genomic DNA Quantification 54 Incorrect Sizing Results 55 Missing Marker Peaks 57 Unexpected Migration Profile 58 Agilent 2200 TapeStation System Troubleshooting Manual Contents Troubleshooting RNA Applications 67 Incorrect Quantification Results 68 Incorrect Sizing Results 69 Missing Marker Peaks 70 Degraded RNA Ladder and or samples 71 Incorrect or missing RINe
37. f error and instrument log files The last stepper move of the Shuttle Z Axis X Axis finished xxx steps from the home flag Probable cause Suggested actions 1 Incorrect consumables have been used Check the correct consumables have been used and that the lids are removed from sample tubes before use Contact your Agilent support team with details of error and instrument log files 100 Agilent 2200 TapeStation System Troubleshooting Manual Error Messages 9 A hardware device associated with the last command is missing or malfunctioning Probable cause Suggested actions 1 The instrument temperature sensor is Contact your Agilent support team with details malfunctioning of error and instrument log files Failed to connect to the barcode reader Camera Probable cause Suggested actions 1 There could be a connection issue withthe Ensure no other USB devices are connected USB port e Power cycle the instrument by switching off waiting 2 minutes then switching back on again e If the error message remains connect the instrument using a different USB Port Agilent 2200 TapeStation System Troubleshooting Manual 101 9 Error Messages The tape just inserted was not recognized please re insert ensuring barcode is near the front of the instrument and facing right Probable cause Suggested actions 1 The idle barcode is not being read correctly Ensure that the ScreenTape is inserted correctly wit
38. gilent 2200 TapeStation System Troubleshooting Manual Troubleshooting DNA Applications 5 The effect of shaking the genomic DNA ladder vial e Shaking the Genomic DNA ladder vial can degrade the top fragment See Figure 13 on page 51 e This can result in inaccurate sizing results e Where the degradation has resulted in failure of the software to assign the top ladder fragment no DIN scores will be presented for the lanes in this file e Prior to addition with sample buffer the ladder vial should be handled carefully e Once mixed with sample buffer the ladder should be gently vortexed for 5 seconds as normal Del A AL Lasser w mo a ATLL i I 450 mmmn i 15 ii I 2000 Sa i GEE N Di ii 4000 mms toon SAAT Tem HW TOTAL 2000 as 2000 if 1500 ms 1 300 a 1 it yi Nit 1200 nn Lo AA A 2 et WL AE Whi I 00 n 00 mmm MEL APRA Wey a eae BE 0 ee ti om Figure 13 The effect of shaking the Genomic DNA Ladder A Genomic DNA Ladder has been vortex mixed for 5 seconds prior to analysis on the Genomic DNA ScreenTape assay B The Genomic DNA Ladder vial was shaken by manually inverting the tube 30 times Degradation of the top fragment 48 500 bp is clearly shown To minimize shaking during transit the Genomic DNA reagents are shipped frozen on dry ice Once received these should be kept at 2 8 C i
39. h the bar code is facing away from the sample block and towards the front of the instrument Ensure the TapeStation is facing away from any direct light sources which could interfere with barcode reading Power cycle the TapeStation by switching off waiting 2 minutes then switching back on again and restart the laptop If the problem persists contact Agilent support 2 The ScreenTape barcode is not being read e Ensure that the ScreenTape is inserted correctly correctly with the bar code is facing away from the sample block and towards the front of the instrument e Ensure the TapeStation is facing away from any direct light sources which could interfere with barcode reading e Power cycle the TapeStation by switching off waiting 2 minutes then switching back on again and restart the laptop If the problem persists contact Agilent support 102 Agilent 2200 TapeStation System Troubleshooting Manual Error Messages 9 Consumable Error Messages The licensed application of your system does not support the use of the tape just inserted A pop up window states The licensed application of your system does not support the use of the tape just inserted Probable cause Suggested actions 1 The wrong ScreenTape type has been Ensure the correct software and ScreenTape inserted for the analysis software mode type are used together Please see the note below Ensure that software versions installed are the most recent availabl
40. ion profile Problem Likely Cause Solution A bubble could be located at Always flick the ScreenTape before placing into the TapeStation the top of the ScreenTape See Good Measurement Practices for DNA on page 40 Sisntedm orsmeared The sample may not have Please follow the mixing recommendations for the assay been mixed correctly with the See Good Measurement Practices for DNA on page 40 and bands AAs s ii sample buffer Mixing recommendations on page 10 for more details The sample and sample buffer Always ensure that samples are run on the TapeStation may have started to evaporate immediately after preparation with sample buffer Figure 16 Effect of bubbles at the gel interface on TapeStation Analysis Software results Gel view Agilent 2200 TapeStation System Troubleshooting Manual 59 5 Troubleshooting DNA Applications Table8 DNA probable causes for unexpected migration profile Problem Likely Cause Solution Concentrate your sample until it is within the recommended range The markers have The sample used is too dilute ee for application then prepare a new run appeared in the sample lane but the Ensure correct mixing by reading the mixing recommendations Insufficient Mixin i sample has not g See Good Measurement Practices for DNA on page 40 D2 800bp fragment 6 pa ul C S Ka K m 1400 1200 4 5 1000 2 800 600 4 400 4 200 4 iy C i a ey ee
41. ipette mixing only 21 2 70 No mixing 6 5 70 Agilent 2200 TapeStation System Troubleshooting Manual Troubleshooting DNA Applications 5 Peak integration In DNA assays concentration values are calculated using the area of the sample peak compared to the known concentration of the upper marker The user must ensure that both marker and sample peaks are properly integrated by manually adjusting the peak when necessary Figure 9 on page 43 shows examples of correct upper marker peak integration In assays with no upper marker RNA High Sensitivity RNA Genomic DNA quantification is taken from the lower marker a eee A1 40 ng ul Covaris s2 6000 5000 4000 4 2 2000 2000 1000 lo oO i Is a Size 1S wr A R 2 al al ale s amp l wp ei i R rai i p i a E S i i A E Figure 9 Correct upper marker integration for the 2200 TapeStation DNA Assays Figure shows D1000 ScreenTape Assay Agilent 2200 TapeStation System Troubleshooting Manual 43 5 Troubleshooting DNA Applications Good Measurement Practices for DNA Figure 10 on page 44 demonstrates the effect of peak integration on the quantitative results Incorrect peak integration can significantly bias the determined DNA sample concentration Correct peak integration Incorrect peak integration Concentration 41 2 ng uL Concentration 14 9 ng uL Al 40 ng ul Covaris s2 Al 40 ng ul Co
42. ipetting procedure When filling the pipette tip push slightly past the first resistance Empty the pipette tip only to the first resistance This procedure ensures pipetting accuracy Agilent 2200 TapeStation System Troubleshooting Manual 9 1 Essential Measurement Practices Mixing recommendations 10 Use a vortexer which is designed for mixing 8 way tube strips or 96 well plates respectively e The IKA MS3 vortexer is recommended for use with the following applications D1000 ScreenTape Assay High Sensitivity D1000 ScreenTape Assay RNA ScreenTape Assay High Sensitivity RNA ScreenTape Assay e For these assays vortex mix samples with sample buffer using the default vortex setting 1 minute e If an IKA MS3 vortexer is not available please ensure thorough manual vortex mixing 10 seconds on maximum speed Figure 1 An IKA MS3 Vortexer is bundled with 2200 TapeStation instruments TapeStation instruments PN G2965AA or G2964AA are supplied with an optional IKA MS3 vortexer which includes a 96 well plate adaptor suitable for both 96 well PCR plates and 8 way strips It is recommended that all current TapeStation users purchase the IKA MS3 for best results Agilent Technologies will not sell these parts separately this vortexer can be obtained directly from IKA www ika com by quoting the part number 4674100 and Agilent Technologies This part number is supplied only to Agilent customers and may not be lis
43. mount 5067 5576 RNA ScreenTape 7 ScreenTape 5067 5577 RNA ScreenTape Sample Buffer 1 vial O 600 pL 5067 5578 RNA ScreenTape Ladder 1 vial D 10 pL Agilent 2200 TapeStation System Troubleshooting Manual 107 10 ScreenTape Products Parts and Consumables Kit Components P200 ScreenTape Assay Part Number Name Color Amount 5067 5371 P200 ScreenTape 7 ScreenTape 5067 5372 P200 Reagents e P200 5X Labeling Dye 70 pL P200 Labeling Buffer 350 pL P200 Reducing Sample Buffer O 550 uL P200 pH Buffer clear 1000 pL P200 Non Reducing Sample Buffer e 550 pL P200 Markers pre stained 270 uL P200 Ladder 40 pL e H 108 Agilent 2200 TapeStation System Troubleshooting Manual Parts and Consumables Table 21 Parts and Consumables ScreenTape Products Parts and Consumables 10 Product Contents Catalogue Number Filtered loading tips 10 pk 10 x 384 tips 5067 5152 Filtered loading tips 1 pk 1 x 384 tips 5067 5153 Optical Tube 8x Strip Box of 120 401428 Optical Cap 8x Strip Box of 120 401425 Sample block for tube strips 1 5067 5155 96 well sample plates 10 5067 5150 Sample block for 96 well 1 5067 5156 plate 96 well Plate Foil seal 1x 100 5067 5154 Loading Tip Holder 1 5067 5158 USB cable Power brick G2960 60064 G2960 20025 Agilent 2200 TapeStation System Troubleshooting Manual 109 Index Index B barcode reader 33 barcode 33 blank lanes 36 buffer compatibility 80 c change needle
44. mple tubes Do not re use sample strips from previous runs Re using samples can lead to poor results Always ensure that the positions selected in the controller software match the location of the sample strips within the instrument Always ensure that sample strip lids are removed before starting the run Failure to do so can result in damage to the instrument If the above causes have been ruled out there may be a hardware fault Please send all relevant data to your local support representative for investigation Agilent 2200 TapeStation System Troubleshooting Manual 37 4 Instrument Troubleshooting Blank Lanes 38 Agilent 2200 TapeStation System Troubleshooting Manual Agilent 2200 TapeStation System Troubleshooting Manual 5 Troubleshooting DNA Applications Good Measurement Practices for DNA 40 Quantification 41 Sizing 47 Molarity 49 Genomic DNA Assay 50 Incorrect Quantification Results 52 Genomic DNA Quantification 54 Incorrect Sizing Results 55 Missing Marker Peaks 57 Unexpected Migration Profile 58 This section gives an overview on how to avoid problems with DNA Applications ee Agilent Technologies 39 5 Troubleshooting DNA Applications Good Measurement Practices for DNA 40 For reliable results instructions regarding reagent preparation and instrument maintenance must be strictly followed Important technical details are described in the TapeStation User Manual and other supporting
45. n overview on ScreenTape Products Parts and Consumables ott Agilent Technologies 105 10 ScreenTape Products Parts and Consumables ScreenTape Products Kit Components High Sensitivity D1000 ScreenTape Assay Part Number Name Color Amount 5067 5584 High Sensitivity D1000 ScreenTape 7 ScreenTape 5067 5585 High Sensitivity D1000 Reagents 2 vials e High Sensitivity D1000 Ladder e 20 pL e High Sensitivity D1000 Sample Buffer eS 300 pL 5067 5587 High Sensitivity D1000 Ladder 1 vial 20 pL Kit Components D1000 ScreenTape Assay Part Number Name Color Amount 5067 5582 D1000 ScreenTape 7 ScreenTape 5067 5583 D1000 Reagents 2 vials D1000 Ladder 8 10 uL D1000 Sample Buffer 400 uL 5067 5586 D1000 Ladder 1 vial 10 pL Kit Components Genomic DNA ScreenTape Assay Part Number Name Color Amount 5067 5365 Genomic DNA ScreenTape 7 ScreenTape 5067 5366 Genomic DNA Reagents 2 vials Genomic DNA Ladder 75 pL Genomic DNA Sample Buffer O 1350 uL 106 Agilent 2200 TapeStation System Troubleshooting Manual ScreenTape Products Parts and Consumables 10 Kit Components High Sensitivity RNA ScreenTape Assay Part Number Name Color Amount 5067 5579 High Sensitivity RNA ScreenTape 7 ScreenTape 5067 5580 High Sensitivity RNA ScreenTape Sample 1 vial Buffer 250 pL 5067 5581 High Sensitivity RNA ScreenTape Ladder 1 vial 10 pL Kit Components RNA ScreenTape Assay Part Number Name Color A
46. n the refrigerator Agilent 2200 TapeStation System Troubleshooting Manual 51 5 Troubleshooting DNA Applications Incorrect Quantification Results Incorrect Quantification results can have multiple causes please read Good Measurement Practices for DNA on page 40 for further details If after reviewing this Technical overview and following the recommendations below your results files continue to show concentration discrepancies please contact your local support representative 52 Agilent 2200 TapeStation System Troubleshooting Manual Table 5 Troubleshooting DNA Applications 5 DNA Possible causes of incorrect quantification results in order of probability Cause Solution Insufficient Mixing Sample concentration outside recommended range for application Incorrect Peak integration Some sample has run concurrently with the Upper marker Incorrect protocol used Sample prepared too long before analysis Incorrect pipetting technique or pipette calibration Incorrect Marker peaks picked up Ensure correct mixing by reading the mixing recommendations See Good Measurement Practices for DNA on page 40 and Mixing recommendations on page 10 Either dilute or concentrate your sample until it is within the recommended range for application as stated in the assay Quick Guide then prepare a new run Ensure that all sample peaks and markers are integrated correctly in the TapeStation A
47. nalysis Software by clicking and dragging so that the whole peak is encompassed See Good Measurement Practices for DNA on page 40 Ensure your sample is within the recommended sizing range for the applications If using Sure Select Protocol ensure that all AMPure beads are removed by increasing the time your sample is on the magnetic plate See Good Measurement Practices for DNA on page 40 Ensure the correct sample protocol for the application is used Differences exist between standard and High Sensitivity assay protocols please consult the User Manual or appropriate Quick Guides for more detail See Good Measurement Practices for DNA on page 40 To avoid evaporation or settling ensure that your run is started immediately after your sample preparation If using a 96 well plate always cover with the recommended foil seal see Consumables on page 17 Follow guidelines in the essential measurement practices section Overview on page 6 Use the Aligned button in the TapeStation Analysis Software to detect the correct markers then assign them by right clicking See Manual Marker Assignment on page 18 Ensure that all sample peaks are within the recommended sizing range for the application Agilent 2200 TapeStation System Troubleshooting Manual 53 5 Troubleshooting DNA Applications Genomic DNA Quantification 54 Incorrect quantification in the Genomic DNA assay can occur due to
48. ng and dragging so that the whole peak is encompassed Ensure that the samples are heated denatured before running according to the assay instructions Over and under heating can affect concentration values Ensure the correct sample protocol for the application is followed To avoid evaporation or settling ensure that your run is started immediately after sample preparation If using a 96 well plate always cover with the approved foil seal Follow guidelines in the Essential Measurement Practices section see Essential Measurement Practices on page 5 Follow the storage conditions specified for the assay as stated in the User Manual and appropriate Quick Guide Use stain solution within 1 week of preparation 82 Agilent 2200 TapeStation System Troubleshooting Manual Incorrect Sizing Results Troubleshooting Protein Applications 7 Table 17 Protein Possible causes of incorrect sizing results in order of probability Cause Solution Incorrect sample buffer used P200 Reagent kits contain both reducing and non reducing buffer Issues with the ladder lane Incorrect migration samples have not reached the lower end of the gel lane Incorrect storage of ScreenTape or reagents Ensure the correct buffer is used Reducing or non reducing conditions will affect the size of fragments analyzed Any issues with the ladder lane will be annotated within the TapeStation Analysis Software sample table under O
49. ng your local Agilent Service representative During the PM the following components within the TapeStation system will be replaced e Fan filters Piercing Needles Electrophoresis Probes The instruments tip sensor will also be re adjusted all moving axis will be lubricated optics cleaned and the any foreign objects will be removed In addition to the Annual Preventative Maintenance service users with a high throughput of samples will also need to perform a Needle Change procedure every 8320 samples This simple procedure is explained in the Changing the needle section and can be performed by the end user with the aid of a disposable cartridge Agilent 2200 TapeStation System Troubleshooting Manual Changing the Needle Table 19 Overview of TapeStation Configuration Needle Cartridge Instrument Maintenance 8 It is important to know which TapeStation system you have before changing the needle s in order to purchase the correct needle cartridge Product Number TapeStation Configuration Pump Needle Cartridge Ordering Code ST007 TapeStation for ScreenPlex ST008 TapeStation for DNA Single G2960 60062 ST009 TapeStation for Nucleic acids ST017 TapeStation for ScreenPlex ST019 TapeStation for Nucleic acids Twin G2960 60063 ST010 TapeStation for Protein Combined TapeStation G2960A 2200 TapeStation System G2961A 2200 TapeStation Nucleic Acid System G2964AA 2200 TapeStation System Twin G2960 60063 G2965AA 22
50. nstrument has failed to respond in a timely fashion 100 The last stepper move of the Shuttle Z Axis X Axis finished xxx steps from the home flag 100 A hardware device associated with the last command is missing or malfunctioning 101 Failed to connect to the barcode reader Camera_ 101 The tape just inserted was not recognized please re insert ensuring barcode is near the front of the instrument and facing right 102 Consumable Error Messages 103 The licensed application of your system does not support the use of the tape just inserted 103 Pipette bin full 104 Pipette pump affixed to the pump 104 This section gives an overview on Error Messages Apg Agilent Technologies 93 9 Error Messages Analysis Software Warning Messages 94 Warning messages are displayed in the TapeStation Analysis Software within the sample table Each lane which has a warning is flagged by colored triangles containing an exclamation mark which are detailed in the Observations column Warning flags can be yellow or red e A yellow warning indicates an abnormal lane where the results may not be reliable A red warning indicates a significant problem with that lane where some data may be missing These warning messages can also be found in the TapeStation Analysis Software online help by clicking on the question mark anywhere within the software Agilent 2200 TapeStation System Troubleshooting Manual Table 20 Analy
51. ntensity FU Figure 6 Examples of blank lanes in the TapeStation Analysis Software Blank lanes can appear in either of the ways shown above As a white lane with no visible peaks or as a black grainy lane A black lane happens when there is very little sample signal causing any background noise to appear higher than normal In these cases the sample intensity scale shows very small values Agilent 2200 TapeStation System Troubleshooting Manual Table 2 Instrument Troubleshooting 4 Possible causes of blank lanes in order of probability Cause Solution Tips in the selected positions were missing from the tip rack Without tips at the correct positions sample cannot be picked up and loaded onto the ScreenTape A bubble in the sample tube prevented the sample from being picked up During normal instrument function tips pick up from the very bottom of the sample tubes The presence of bubbles here can result in no sample being picked up Insufficient sample present The samples were not selected correctly in the controller software The sample lids were not correctly removed before starting the run Hardware fault Always ensure that all 16 tips are loaded into the tip rack at the start of each run Always ensure that sample strips or 96 well plates are centrifuged after preparation to remove any bubbles present at the bottom of wells Always ensure a minimum of 3 uL of solution is present in the sa
52. oduct codes To run samples from a 96 well plate also use the 2200 TapeStation foil cover p n 5067 5154 to prevent the sample from leaving the plate during vortexing After vortexing use an appropriate centrifuge for either 96 well plates or 8 way strips to ensure that all of the samples are at the bottom of the tube before placing in the TapeStation Agilent 2200 TapeStation System Troubleshooting Manual 45 5 46 Troubleshooting DNA Applications TapeStation analysis in the Agilent Sure Select workflow The 2200 TapeStation system has been verified for use within the Agilent Sure Select protocol During the purification step however residual AMPure XP beads can give signal which runs with the upper marker See Figure 11 on page 46 Any signal under the upper marker causes a lower reported value of sample concentration e This artifact can be avoided by increasing the time for which the samples are incubated on the magnetic plate to 10 minutes thereby removing a higher percentage of the beads Sample intensity FU a A w 2 s s s Figure 11 Enlarged image of the upper marker showing additional signal from AMPure beads Agilent 2200 TapeStation System Troubleshooting Manual Troubleshooting DNA Applications 5 Sizing Peak maxima versus average molecular weight sizing Within the 2200 TapeStation analysis software sizing can be found in both electropherogram and region mode The sizing information pres
53. of these causes your results files continue to show concentration discrepancies please contact your local support representative Table9 RNA Possible causes of incorrect quantification in order of probability Cause Solution Sample Either dilute or concentrate your sample until it is within the recommended range for the concentration out with recommended range for application Incorrect Peak integration Some sample has run concurrently with the Lower marker Incorrect protocol used Insufficient Mixing Incorrect heating procedure Sample prepared too long before analysis Incorrect pipetting technique or pipette calibration Incorrect Marker peaks picked up Insufficient migration due to presence of salt application as seen in the appropriate assay Quick Guide then prepare a new run Ensure that all sample peaks and markers are integrated correctly in the TapeStation Analysis Software by clicking and dragging so that the whole peak is encompassed Ensure your samples are within the recommended sizing range for the applications Ensure the correct sample protocol for the application is used Differences exist between standard and High Sensitivity assay protocols please consult the User Manual or appropriate Quick Guides for more detail Ensure correct mixing see Mixing recommendations on page 10 Ensure that the samples are heat denatured according to the assay instructions before running Both
54. on instrument has three individual USB devices which are linked to the single USB connection on the back of the instrument The USB devices found within each instrument are 1 The Barcode reader The Barcode reader is generic across all instrument types and is identified in the list of Ports as 5180 Area Imager 2 The USB to serial converter The USB to serial converter is generic across all instrument types and is identified in the Universal Serial Bus controllers list as USB lt Serial Converter 3 The Camera The Camera type listed depends on the instrument Nucleic Acid 2200 TapeStation instruments G2965AA use a camera identified in the Universal Serial Bus controllers list as uEye UI 154x Series 2200 TapeStation instruments G2964AA use a camera identified in the Universal Serial Bus controllers list as uEye UI 224x Series Agilent 2200 TapeStation System Troubleshooting Manual 27 3 28 Instrument Communication Updating USB Drivers To update the USB Drivers please use the following instructions 1 Switch the instrument on and connect the USB cable Open the Windows Start menu and click on Devices and Printers M TapeStation Analysis TapeStation Controller al Notepad A Getting Started E F Paint Calculator gQ Snipping Tool Mi Sticky Notes gm XPS Viewer Windows Fax and Scan gt All Programs amp Admin Documents Pictures Music Computer Control Panel Devic
55. on instruments connect to bundled laptops via USB cable For instructions on connecting your 2200 TapeStation and laptop please consult the readme document Windows may display a Found New Hardware wizard once the software has loaded In this instance always perform the following steps 1 Select No not this time to prevent connecting to Windows Update and initiating a search for software 2 Inthe next window select Install the Software automatically 3 Ifa window appears indicating the software did not pass the windows logo testing click Continue Anyway A window will then appear indicating that the hardware has been successfully installed Agilent 2200 TapeStation System Troubleshooting Manual 25 3 Instrument Communication Updating USB Drivers This section describes the procedure to update relevant USB drivers and identifies the device names specific to the 2200 TapeStation System If a laptop fails to connect to the instrument or loses connection for any reason first check that the power and USB cables are attached to the instrument then if issues remain re install the USB drivers This process is the same as for any external USB device If a connection failure occurs a likely error message displayed in the controller software is as follows ERROR Failed to connect to the barcode scanner or camera 26 Agilent 2200 TapeStation System Troubleshooting Manual Instrument Communication 3 Each TapeStati
56. oncentrate your sample until it is within the recommended present atthe top of too concentrated for range for the application then prepare the sample again and rerun on the gel image the application ScreenTape If necessary use the High Sensitivity RNA ScreenTape assay 2 2 k i bA a Figure 28 Example of highly concentrated RNA sample presenting as additional bands presented with arrows above the 28S peak 76 Agilent 2200 TapeStation System Troubleshooting Manual Troubleshooting RNA Applications 6 Table 15 RNA Possible causes of unexpected migration profile Problem Likely Cause Solution The sample may be Concentrate your sample until it is within the recommended range for ka inoi ii tao ilute tor the application teh re ae the sample again and rerun on Sane e appeared in the application pp prep ple ag p sample lane but the sample has not Ensure correct mixing by reading the Mixing recommendations on Insufficient Mixing page 10 D T 5 2 S v pS E Ss A Figure 29 Example of missing sample peaks in an RNA run Higher than normal PE n Ensure correct mixing by reading the Mixing recommendations on Insufficient Mixing background noise page 10 oe 2 Ensure correct mixing by reading the Mixing recommendations on Insufficient Mixing g 9y g g page 10 Low signal intensity Incorrect reagent Follow the storage conditions specified for the assay as
57. ophoresis failure caused by bubbles at gel buffer interface AO L Al a pi 5 o S amp S W So Y 3 S gt 2 2 a E S a 3 8 o Unalign the image using the Aligned button in the TapeStation Analysis Software This will show the true position of all peaks present High salt concentrations can cause short running within the gel lane which can cause incorrect identification of Lower marker peaks Please refer to the salt tolerance guidelines for the assay Ensure ScreenTape is flicked to remove any bubbles prior to use Figure 23 Example of shortened migration caused by high salt concentration Agilent 2200 TapeStation System Troubleshooting Manual 65 5 Troubleshooting DNA Applications Unexpected Migration Profile 66 Agilent 2200 TapeStation System Troubleshooting Manual Agilent 2200 TapeStation System Troubleshooting Manual 6 Troubleshooting RNA Applications Incorrect Quantification Results 68 Incorrect Sizing Results 69 Missing Marker Peaks 70 Degraded RNA Ladder and or samples 71 Incorrect or missing RINe value 72 Incorrect or Missing Peak Annotation 73 Unexpected migration profile 74 This section gives an overview on how to avoid problems with RNA Applications ee Agilent Technologies 67 6 Troubleshooting RNA Applications Incorrect Quantification Results Incorrect quantification results can have multiple causes If after checking each
58. or DNA on page 40 and Mixing recommendations on page 10 Sample concentration Either dilute or concentrate your sample until it is within the recommended range for outside recommended range application as stated in the assay Quick Guide then prepare a new run for application Blank lane See Blank lane section Insufficient migration To check migration true distance unalign the gel image using the Aligned button See Unexpected Migration Profile on page 58 Agilent 2200 TapeStation System Troubleshooting Manual 57 5 Troubleshooting DNA Applications Unexpected Migration Profile There are a number of issues which can affect migration and sample peak profile If the issues below do not describe your analysis file please contact your support representative Table8 DNA probable causes for unexpected migration profile Problem Likely Cause Solution Ensure the correct marker peaks are picked up in the TapeStation Analysis Software The gel image looks Incorrect Marker peaks picked See ScreenTape on page 13 and Manual Marker distorted up Assignment on page 18 Ensure that all sample peaks are within the recommended sizing range for the application Incorrectly assigned as the Lower Marker Figure 15 Identifying the correct lower marker 58 Agilent 2200 TapeStation System Troubleshooting Manual Troubleshooting DNA Applications 5 Table8 DNA probable causes for unexpected migrat
59. ration ranges Using samples outside the stated ranges can affect performance For salt tolerance guidelines please refer to the apporpriate Assay Quick Guide Please ensure that sample buffers are below the maximum recommendation Allow all samples to equilibrate to room temperature for 30 minutes Mix and spin down prior to use Pipette carefully Always pipette reagents against the side of the sample tube Ensure that no residual material is left on the outside of the tip When adding sample buffer to sample please ensure that they are mixed correctly by following assay instructions Improper mixing can lead to quantification errors see Mixing recommendations on page 10 Once mixed briefly centrifuge to collect the contents at the base of tubes Used sample strips and tips should be disposed of in accordance to local safety regulations For successful loading the sample solution must be placed at the bottom of the tube or well without any air bubbles The 2200 TapeStation will load a sample from a minimum of 3 uL onto ScreenTape Lids on sample tubes Failure to remove lids can cause damage to the 2200 TapeStation and impact performance gt Ensure lids have been removed from the sample tubes before starting the run 12 Agilent 2200 TapeStation System Troubleshooting Manual Essential Measurement Practices 1 ScreenTape Details and specifications for each assay are available in the 2200 TapeStation User
60. re a new run Concentrate your sample until it is within the recommended concentration range for application then prepare a new run Ensure correct mixing see Mixing recommendations on page 10 Please follow the mixing recommendations for the assay Follow the storage conditions specified for the assay Ensure correct mixing see Mixing recommendations on page 10 Follow the storage conditions specified for the assay Dilute your samples to ensure low levels of buffer salt True migration profiles can be seen by unaligning your gel image by pressing the Aligned button in the TapeStation Analysis Software Please refer to the P200 Buffer compatibility section and make the necessary adjustments to the sample buffer 84 Agilent 2200 TapeStation System Troubleshooting Manual Agilent 2200 TapeStation System Troubleshooting Manual e0 ee 3 r Instrument Maintenance General Information 86 Changing the Needle 87 Preventative Maintenance Interval 91 This section gives an overview on the importance of Preventive Maintenance gg Agilent Technologies 85 Instrument Maintenance General Information 86 For continuous reliable operation the TapeStation system requires a defined set of Preventative Maintenance PM operations to be performed every 16000 samples or on an annual basis This maintenance must be performed by a trained Agilent Field Service Engineer and can be scheduled by contacti
61. reement shall control Technology Licenses The hardware and or software described in this document are furnished under a license and may be used or copied only in accor dance with the terms of such license Restricted Rights Legend If software is for use in the performance of a U S Government prime contract or subcon tract Software is delivered and licensed as Commercial computer software as defined in DFAR 252 227 7014 June 1995 or as a commercial item as defined in FAR 2 101 a or as Restricted computer soft ware as defined in FAR 52 227 19 June 1987 or any equivalent agency regulation or contract clause Use duplication or dis closure of Software is subject to Agilent Technologies standard commercial license terms and non DOD Departments and Agencies of the U S Government will receive no greater than Restricted Rights as defined in FAR 52 227 19 c 1 2 June 1987 U S Government users will receive no greater than Limited Rights as defined in FAR 52 227 14 June 1987 or DFAR 252 227 7015 b 2 November 1995 as applicable in any technical data Safety Notices CAUTION A CAUTION notice denotes a hazard It calls attention to an operating procedure practice or the like that if not correctly per formed or adhered to could result in damage to the product or loss of important data Do not proceed beyond a CAUTION notice until the indicated condi tions are fully understood and m
62. residual sample remaining at the top of the gel This signal can be viewed in the TapeStation Analysis Software using the Scale to Molecular Weight button Any signal seen which is annotated well should be included in the analyzed region Samples designated well are too large to have migrated onto the gel please be aware that an accurate concentration for the entire sample cannot be generated Agilent 2200 TapeStation System Troubleshooting Manual Troubleshooting DNA Applications 5 Incorrect Sizing Results For best sizing precision and accuracy the user should run the appropriate ladder with the samples Incorrect sizing results can have multiple causes please read the Good Measurement Practices for DNA on page 40 If after reviewing this Technical overview and following the recommendations below your results files continue to show sizing discrepancies please contact your local support representative Table6 DNA Possible causes of incorrect sizing results in order of probability Cause Solution Incorrect Marker peaks picked up Incorrect Analysis software mode Issues with the ladder lane Insufficient migration due to presence of salt Ensure the correct marker peaks are picked up in the TapeStation Analysis Software See Manual Marker Assignment on page 18 Ensure that all sample peaks are within the recommended sizing range for the application Reported sizing can differ between electrophe
63. rogram and region modes Select the correct mode for your samples See Good Measurement Practices for DNA on page 40 for more details Any issues with the run ladder lane will be annotated within the TapeStation Analysis Software sample table under Observations Manually insert or delete affected peaks or insert a software saved ladder to regain sizing information if available Dilute your samples to ensure low levels of buffer salt True migration profiles can be seen by unaligning your gel image by pressing the Aligned button in the TapeStation Analysis Software Please refer to the salt tolerance guidelines for your assay in the User Manual or appropriate Quick Guide Agilent 2200 TapeStation System Troubleshooting Manual 55 5 Troubleshooting DNA Applications Genomic DNA Sizing Incorrect or inaccurate sizing can occur using the Genomic DNA ScreenTape assay when there has been an issue with the ladder lane For more details please see the Good Measurement Practices for DNA on page 40 56 Agilent 2200 TapeStation System Troubleshooting Manual Troubleshooting DNA Applications 5 Missing Marker Peaks 2 a a 2 5 2 G E DA A Figure 14 Example of missing marker peak Table 7 DNA Possible causes of missing marker peaks in order of probability Cause Solution Insufficient Mixing Ensure correct mixing by reading the mixing recommendations See Good Measurement Practices f
64. run data is stored locally on the instrument laptop Changing or updating the laptop can cause this information to be lost resulting in partially used ScreenTape lanes being reused Used ScreenTape sample strips and tips should be disposed of in accordance with local safety regulations Agilent 2200 TapeStation System Troubleshooting Manual Essential Measurement Practices 1 2200 TapeStation Instrument LED Status When powered up and idle the instrument will have a blue LED visible on the front of the case When running normally this blue LED will flash slowly e A quickly flashing LED indicates that the instrument has encountered an issue Operating Temperature e Ensure that environmental conditions for the chosen assay are met e Optimal Instrument operating temperature 20 C 68 F e Assay specific operating temperatures 12 37 C 54 99 F for D1000 ScreenTape 17 37 C 63 99 for High Sensitivity D1000 ScreenTape 15 30 C 59 86 F for Genomic DNA ScreenTape 14 30 C 57 86 F for RNA and High Sensitivity RNA ScreenTape 10 33 C 50 91 F for P200 ScreenTape Instrument operating temperature may be higher than ambient lab temperature especially after prolonged use If the instrument is out of the recommended temperature range for the ScreenTape inserted the following error message will appear in the controller software Agilent 2200 TapeStation System Troubleshooting Manual 15
65. sible causes for missing or incorrect RIN results in order of probability Cause Solution There are 2 bands present at the 18S peak position Extremely degraded sample Sample concentration outside recommended range for application Genomic DNA contamination There may have been insufficient sample denaturation prior to ScreenTape analysis The 18S peak may have partially reverted back to its original non denatured conformation Ensure the correct denaturation conditions during RNA sample preparation according to the assay Quick Guide Samples should be kept on ice after preparation and run within 2 hours of denaturation The TapeStation Analysis Software can occasionally fail to recognize extremely degraded RNA sample peaks Either dilute or concentrate your sample until it is within the recommended range for the application as seen in the appropriate assay Quick Guide then prepare a new run Samples contaminated with genomic DNA contain a third peak which migrates in the region of the 18 and 28S Occasionally this peak can be mistaken for the 18 or 28S peak Ensure that all peaks are annotated correctly Treat samples with DNAse to eliminate the genomic DNA peak then prepare a new run 72 Agilent 2200 TapeStation System Troubleshooting Manual Troubleshooting RNA Applications 6 Incorrect or Missing Peak Annotation Table 14 RNA Possible causes of incorrect or missing peak annotation in order of
66. sing Marker Peaks on page 70 for RNA Applications as appropriate Manually assign the upper and or lower marker See Manual Marker Assignment on page 18 for details If possible manually add extra peaks by right clicking on the affected peak and selecting add For gDNA assay prepare a new run with Genomic DNA Ladder in the first position Please see Good Measurement Practices for DNA on page 40 for advice on handling the Genomic DNA Ladder Where possible manually delete extra peaks by right clicking on the affected peak and selecting delete For Genomic DNA files please see Good Measurement Practices for DNA on page 40 for advice on handling the Genomic DNA Ladder Agilent 2200 TapeStation System Troubleshooting Manual 95 9 Error Messages Table 20 Analysis Software Warning Messages Affected Assay Observation Detailed description Corrective action Genomic DNA DIN edited Marker The User has manually A DIN will be displayed To recover the position changed changed the position of a original DIN value assigned by the marker peak software use the Restore Default Settings DIN may have changed as button on the main ribbon a result DIN edited Ladder User has manually A DIN will be displayed To recover the sizing changed changed the position of a original DIN assigned by the software use ladder peak the Restore Default Settings button on the DIN may have changed as_ main ribbon a result
67. sis Software Warning Messages Error Messages 9 Affected Assay Observation Detailed description Corrective action Caution Expired ScreenTape All Assays Caution Expired ScreenTape used after two weeks of first use Marker s not detected Markers outside standard running position Issue with ladder peak detection too few peaks detected Issue with ladder peak detection too many peaks detected ScreenTape was used after its expiration date ScreenTape was used after two weeks of the first usage The lower and or upper marker has not been identified by the software The image is unaligned and no sizing information provided The lower and or upper marker is running outside the expected detection window for the assay This may affect sizing though the image will not be unaligned Less than the expected number of ladder peaks have been identified This may affect sizing For gDNA samples no DIN will be displayed in this file More than the expected number of ladder peaks have been identified This may affect sizing For Genomic DNA files DIN results in this file may not be reliable Re run with in date ScreenTape and consumables Re run with in date ScreenTape and consumables Manually assign the upper and or lower marker See Manual Marker Assignment on page 18 for details If markers are not present see Missing Marker Peaks on page 57 for DNA or Mis
68. stated in the user storage conditions manual and appropriate Quick Guide Agilent 2200 TapeStation System Troubleshooting Manual 77 6 Troubleshooting RNA Applications Table 15 RNA Possible causes of unexpected migration profile Problem Likely Cause Solution Insufficient migration Dilute your samples to ensure low levels of buffer salt due to presence of True migration profiles can be seen by unaligning your gel image as Incorrect migration salt described in Manual Marker Assignment on page 18 samples have not Partial reached the lower electrophoresis end of the gel lane failure caused by bubbles at gel buffer interface Ensure Screentape is flicked to remove any bubbles prior to use see ScreenTape on page 13 AO L Al B1 D1 E1 F1 G1 H1 A2 B2 i q M RIN RIN RIN RIN RIN RIN RIN ea R Figure 30 Example of short migration caused by high salt concentration Lanes A1 B2 show the same sample in buffers of decreasing salt concentrations allowing migration further through the gel lane 78 Agilent 2200 TapeStation System Troubleshooting Manual Agilent 2200 TapeStation System Troubleshooting Manual 7 Troubleshooting Protein Applications Buffer compatibility 80 Quantification of Protein Samples 81 Incorrect Quantification results 82 Incorrect Sizing Results 83 Unexpected Migration Profile 84 This section gives an overview on how to avoid problems with Protein Applica
69. tapestation 6 Agilent 2200 TapeStation System Troubleshooting Manual Essential Measurement Practices 1 Intended use of the 2200 TapeStation System The 2200 TapeStation system carries out electrophoretic separation of Nucleic Acids and Proteins The system detects e Fluorescently stained double stranded DNA including genomic DNA e Fluorescently stained total RNA Eukaryotic and Prokaryotic Fluorescently labelled proteins Agilent 2200 TapeStation System Troubleshooting Manual 7 1 Essential Measurement Practices Performance Limitations of Use The 2200 TapeStation System can analyze a maximum of 16 samples on a single ScreenTape device more samples can be run using a 96 well plate and multiple ScreenTape The user is responsible for establishing performance characteristics necessary for upstream and downstream applications Appropriate controls must be included in any upstream application requiring analysis on the 2200 TapeStation System 8 Agilent 2200 TapeStation System Troubleshooting Manual Essential Measurement Practices 1 Tools and Handling Always follow the GLP rules established in the laboratory e Always wear gloves to prevent contamination e When pipetting sample use pipette tips that are of adequate size Pipette tips that are too large will lead to poor quantitation accuracy Change pipette tips between steps to avoid cross contamination e For RNA and High Sensitivity RNA assays use reverse p
70. ted on standard price lists For all other assays ensure thorough mixing at each mixing step following the recommendations in the assay Quick Guide and ensuring movement of the liquid within the sample tubes Agilent 2200 TapeStation System Troubleshooting Manual Essential Measurement Practices 1 Reagents and Reagent Mixes e Handle and store all reagents according to the instructions given in the appropriate Assay Quick Guide e Keep all reagents at the directed temperature when not in use Reagents left at room temperature for a long period of time may decompose leading to poor measurement results e Allow all reagents to equilibrate to room temperature for 30 minutes Mix and spin down prior to use e When pipetting sample buffer ensure that excess buffer droplets are removed from the tip before transfer to the sample tubes Care must be taken due to viscosity of Sample Buffers e When adding sample buffer to samples please ensure that they are mixed correctly See Mixing recommendations on page 10 and appropriate Assay Quick Guide e When pipetting small volumes ensure that no sample remains within the tip Special care must be taken with High Sensitivity assays which use small volumes of sample and sample buffer Agilent 2200 TapeStation System Troubleshooting Manual 11 1 Essential Measurement Practices Samples CAUTION Refer to the appropriate Assay Quick Guide for the recommended sizing and concent
71. the annual Preventative Maintenance procedure Customers with high throughput may require additional needle changes between PM services and should use the table above to order the correct parts for their instrument New needle cartridges can be ordered at any time from Agilent Technologies by contacting your local sales agent For details on the correct needle cartridge for your TapeStation model refer to Table 19 on page 87 Agilent 2200 TapeStation System Troubleshooting Manual 89 8 Instrument Maintenance Changing the needle cartridge 1 Remove the sample plate and tip holder 2 Remove the foil tab from the top of the needle cartridge Care must be taken to keep the needle cartridge level after removing the foil tab 3 Insert the needle cartridge into the tip holder space using the label for orientation The cartridge should be placed so that the label faces to the right and the printed arrow points to the front of the TapeStation Needle cartridge s Rear TapeStation Front 4 Close the lid 5 Go to Needle Change on the Controller software toolbar and select Run 90 Agilent 2200 TapeStation System Troubleshooting Manual Instrument Maintenance 8 Preventative Maintenance Interval After 16000 samples the controller software will inform the user that Preventative Maintenance is required The word Maintenance will appear in the bottom of the controller software inside a yellow box The Preventative Maint
72. the ladder unexpected band is RNA Ladder may not Ladder and samples should be kept on ice and run within 2 hours of evident in the RNA have been sufficient denaturation Ladder Samples contaminated with genomic DNA contain a third peak which migrates in the region of the 18 and 28S Genomic DNA Occasionally this peak can be mistaken for the 18 or 28S peak contamination Ensure that all peaks are annotated correctly Additional bands are Treat samples with DNAse to eliminate the genomic DNA peak then present in the region prepare a new run of the 18 and 28S peak Unalign the image using the Aligned button in the TapeStation Analysis There may bea Software Contaminant peaks can be unassigned by right clicking on either contaminant peak the gel image or electropherogram peak caused by dust or dirt Peaks caused by contaminants present sharp strong bands which are on the ScreenTape often slanted or uneven in the gel image and usually easily distinguishable from sample peaks G1 G1 200ng RNA 75ng gDNA 2 S 1 Ss S o f Sample Intensity FU 1043 N p o Figure 27 Example of genomic DNA contamination of RNA sample Agilent 2200 TapeStation System Troubleshooting Manual 75 6 Troubleshooting RNA Applications Table 15 RNA Possible causes of unexpected migration profile Problem Likely Cause Solution Additional bands are The sample may be Dilute or c
73. tions Apg Agilent Technologies 79 7 Troubleshooting Protein Applications Buffer compatibility Please refer to the technical overview Understanding the effects of proteins and buffers on staining denaturation and electrophoresis when analyzing proteins with Agilent P200 ScreenTape www chem agilent com library technicaloverviews Public 5990 9603EN pdf 80 Agilent 2200 TapeStation System Troubleshooting Manual Troubleshooting Protein Applications 7 Quantification of Protein Samples Quantification of protein samples in the TapeStation Analysis Software is currently customized by the user This means that when a file first loads no concentration values are displayed In order to see this information a calibration curve must be constructed manually by running samples of known concentrations It is possible to manually enter concentrations for known sample peaks in the electropherogram tab by following these instructions 1 Right click on the sample peak with known concentration 2 Select Assign Concentration This will automatically take you to the concentration field for that peak in the peak data table Manually type in the concentration and press enter 4 Repeat this process with as many known peaks as possible The more values entered the better the calibration curve constructed will be A calibration curve will now have been created using the manually entered data which can be viewed in the file menu under
74. varis s2 e e g 3000 Sample Intensity FU z 2 a Figure 10 Example of correct A and incorrect B sample peak integration and their effect on reported sample concentration 44 Agilent 2200 TapeStation System Troubleshooting Manual Troubleshooting DNA Applications 5 Use the correct protocol Each ScreenTape type is designed for use with its corresponding Reagent kit Ensure that sample and sample buffer volumes from the correct protocol are followed Ensure that the reagents are used with the corresponding ScreenTape type It is important to choose the correct assay based on the concentration of the sample using sample concentrations outside the specified quantitative ranges will lead to inaccurate quantification For correct volumes and quantitative ranges please refer to the User Manual or the appropriate assay Quick Guide Use the correct tools for the job Use calibrated pipettes which are sufficient for the volume required Ensure correct pipetting technique so that volumes are precise and that the concentrations can be calculated correctly Use a vortexer designed for mixing 8 way tube strips or 96 well plates respectively For more details please see Mixing recommendations on page 10 Use only the correct Agilent supplied consumables including loading tips 8 way strips and 96 well plates See Parts and Consumables on page 109 for pr

Download Pdf Manuals

Related Search

Related Contents

Wiley Beginning Microsoft Visual C# 2008  05_ミーリングチャック ミルエースMLY  Pioneer DVR-108 User's Manual  MANUAL DO UTILIZADOR  G-scan取扱説明書（いすゞ編）第21版【2014.03.15】  Fujitsu ESPRIMO E910  Installati  digisolutions  manuel d`utilisation des friteuses à gaz de série pro  

Copyright © All rights reserved. Failed to retrieve file