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Agilent 2100 Bioanalyzer Software Maintenance Guide PDF

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Summary of Content for Agilent 2100 Bioanalyzer Software Maintenance Guide PDF

Agilent Technologies

Agilent 2100 Bioanalyzer System

Maintenance and Troubleshooting Guide

Notices Agilent Technologies, Inc. 2011-2020 No part of this manual may be reproduced in any form or by any means (including electronic storage and retrieval or transla- tion into a foreign language) without prior agreement and written consent from Agi- lent Technologies, Inc. as governed by United States and international copyright laws.

Document Number SD-UF0000099 Rev. C.00

Edition 02/2020 Published 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 agreement 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 accordance 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 sub- contract, 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 software as defined in FAR 52.227-19 (June 1987) or any equivalent agency regu- lation or contract clause. Use, duplication or disclosure of Software is subject to Agi- lent 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 met.

WARNING

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 understood and met.

For Research Use Only

Not for use in diagnostic procedures

Maintenance and Troubleshooting

In this Book

In this Book

This manual provides maintenance and troubleshooting information for the Agilent 2100 Bioanalyzer system. It includes essential measurement practices, troubleshooting hints for hardware, software and applications, maintenance procedures and a list of spare parts and accessories.

This manual is based on the 2100 Expert Software revision B.02.08. Other software revisions may have an impact on results.

Maintenance and Troubleshooting 3

Contents

Contents

1 Essential Measurement Practices 7 Overview 8 Tools and Handling 9 Chip Priming Station 10 Reagents and Reagent Mixes 11 Samples 12 Chips 13 Agilent 2100 Bioanalyzer System 14

2 Troubleshooting the Instrument Communication 15 Verify the Instrument Communication 16 Troubleshooting Communication Issues 18 Changing COM Port Settings 20 USB to Serial Adapter 21 How to create a Support Package 22

3 Troubleshooting the 2100 Expert Software 25 Run Installation Qualification Test 26

4 Hardware Diagnostics 29 Overview 30 Diagnostic Test Procedure 32

5 Troubleshooting the DNA Application 35 Overview 36 Symptoms (DNA) 37 Symptoms (High Sensitivity DNA) 62

6 Troubleshooting the RNA Application 67 Overview 68 Symptoms (RNA) 69

4 Maintenance and Troubleshooting

Contents

7 Troubleshooting the Protein Application 89 Overview 90 Symptoms (Protein) 91 Symptoms (High Sensitivity Protein) 114

8 Maintenance of the Electrode Cartridge 117 Overview 118 DNA and Protein Assays 119 RNA Nano Assay 121 RNA Pico or Small RNA Assay 125 How to Clean the Pin Set of the Electrode Cartridge 128

9 Maintenance of the Chip Priming Station 133 Overview 134 Replacing the Syringe 135 Cleaning the Syringe Adapter 136 Replacing the Syringe Adapter 138 Replacing the Gasket 139 Checking the Chip Priming Station for Proper Performance - Seal Test 141

10 Maintenance of the Agilent 2100 Bioanalyzer instrument 143 Overview 144 Cleaning the Lens 145 Changing the Fuses 146

11 Maintenance of the Vortexer 151 Changing the Adapter 152

12 Spare Parts and Accessories 153 Overview 154

Maintenance and Troubleshooting 5

Contents

6 Maintenance and Troubleshooting

Maintenance and Troubleshooting

1 Essential Measurement Practices Overview 8 Tools and Handling 9 Chip Priming Station 10 Reagents and Reagent Mixes 11

Gel and Gel-Dye Mix 11 Samples 12 Chips 13 Agilent 2100 Bioanalyzer System 14

7Agilent Technologies

1 Essential Measurement Practices Overview

Overview

This section lists all user relevant hints on handling tools, chips, reagents and the Agilent 2100 Bioanalyzer system. For the latest information on assay-related hints, go to the Agilent web site at:

www.agilent.com/genomics/bioanalyzer

8 Maintenance and Troubleshooting

Essential Measurement Practices 1 Tools and Handling

Tools and Handling

Always follow the GLP-rules established in the laboratory.

Always wear gloves when handling chips to prevent contamination.

When pipetting sample, use non-filter 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.

Always insert the pipette tip to the bottom of the well when dispensing liquid. Placing the tip at the edge of the well leads to bubbles and poor results. Holding the pipette at a slight angle will ensure proper dispensing of the liquid.

Use a new syringe and electrode cleaner with each new kit.

Maintenance and Troubleshooting 9

1 Essential Measurement Practices Chip Priming Station

Chip Priming Station

Refer to the appropriate Kit Guide for the correct position of the syringe clip and base plate.

Replace the syringe with each new kit.

Check the performance of the chip priming station by applying the seal test on a monthly basis. For details see Maintenance of the Chip Priming Station on page 133. If necessary, replace the gasket and/or adapter.

10 Maintenance and Troubleshooting

Essential Measurement Practices 1 Reagents and Reagent Mixes

Reagents and Reagent Mixes

Handle and store all reagents according to the instructions given in the specific Kit Guide.

Keep all reagents and reagent mixes (for example, the gel-dye mix) refrigerated at 4C when not in use for more than 1 hour. Reagents left at room temperature may decompose, leading to poor measurement results.

Allow all reagents and samples to equilibrate to room temperature for 30 minutes. Mix and spin down prior to use.

Gel and Gel-Dye Mix Use gel-dye mix within the specified time frame stated in the instructions

from the kit guide. Otherwise, it may decompose and lead to poor measurement results.

Protect dye and gel-dye mixes from light. Dye decomposes when exposed to light.

Maintenance and Troubleshooting 11

1 Essential Measurement Practices Samples

Samples

Refer to the assay specific Kit Guides for maximum allowed sample and salt concentration.

For RNA assays: Heat denature all RNA samples and RNA ladder for 2 minutes at 70C before use.

For protein assays: Use 0.5 mL tubes for denaturation. Using larger tubes will lead to poor results.

12 Maintenance and Troubleshooting

Essential Measurement Practices 1 Chips

Chips

Prepared chips must be used within 5 minutes. Reagents may evaporate, leading to poor results.

For DNA and RNA assays, vortex chips for 1 minute. Inappropriate and insufficient vortexing will lead to poor results. Use only the IKA vortexer for chip vortexing. Replace the chip adapter if it is worn out. For the MS-2 vortexers with 3 mounting screws, the replacement part number is 5065-9966. For MS-3 vortexers with 4 mounting screws, replacement adapters may be purchased directly from IKA (www.ika.de) with part number 3428300.

Do not touch the wells of the chip. The chip could get contaminated resulting in poor measurement results.

Do not leave any wells of the chip empty. The assay will not run properly.

For DNA and RNA assays: Add 1 L of sample buffer to each unused sample well so the total liquid volume in each well is at least 6 L.

For protein assays: pipette a sample or ladder replicate in any empty sample well.

Do not touch the underside of the chip.

Maintenance and Troubleshooting 13

1 Essential Measurement Practices Agilent 2100 Bioanalyzer System

Agilent 2100 Bioanalyzer System

Do not touch the 2100 Bioanalyzer instrument during a run and never place it on a vibrating surface or near air-circulating instruments (for example, temperature cyclers).

Do not force the chip to fit in the 2100 Bioanalyzer instrument. The electrode cartridge may be damaged when the lid is closed.

Clean electrodes on a daily basis using the electrode cleaner. For more details, see Maintenance of the Electrode Cartridge on page 117.

Thorougly clean electrodes on a monthly basis using a toothbrush and distilled water. For more details, see How to Clean the Pin Set of the Electrode Cartridge on page 128.

Clean the focusing lens once a month (or after any liquid spill) using isopropanol. For more details, see Cleaning the Lens on page 145.

14 Maintenance and Troubleshooting

Maintenance and Troubleshooting

2 Troubleshooting the Instrument Communication Verify the Instrument Communication 16 Troubleshooting Communication Issues 18

Overview 18 Troubleshooting Communication Issue Flow Chart 19

Changing COM Port Settings 20 USB to Serial Adapter 21 How to create a Support Package 22

15Agilent Technologies

2 Troubleshooting the Instrument Communication Verify the Instrument Communication

Verify the Instrument Communication

To check whether your PC communicates with the Agilent 2100 Bioanalyzer instrument:

1 Start the instrument. The power switch is located at the rear where the power cable plugs in.

The status indicator lamp will light green if power is present and all instument self-tests have been passed successfully.

2 Start the 2100 Expert Software.

3 Select the instrument tab in the Instrument context.

4 In the tree view, highlight the appropriate instrument.

The connection to the selected instrument is established.

5 Open and close the lid the icon in the Instrument context should change from closed to open, see Table 1 on page 16.

NOTE A green status indicator does not indicate that the instrument is communicating with the PC, the lamp is green even if the instrument is not connected.

Table 1 2100 Bioanalyzer instrument icons

Switched off or not connected to PC.

Online and lid closed.

Online and lid open.

16 Maintenance and Troubleshooting

Troubleshooting the Instrument Communication 2 Verify the Instrument Communication

If the instrument is connected successfully, additional hardware information (serial number, cartridge type,...) is displayed on top of the screen, see Figure on page 17.

If the icon does not change, the instrument is not connected successfully. For solutions, see Overview on page 18.

Maintenance and Troubleshooting 17

2 Troubleshooting the Instrument Communication Troubleshooting Communication Issues

Troubleshooting Communication Issues

Overview Check if license keys have been registered with the software. Go to Help >

Registration > Add Licenses. Ensure that 2 licenses have been entered: the instrument control license and the electrophoresis license.

Check the COM port settings in the 2100 Expert Software, see Changing COM Port Settings on page 20.

Check whether the status indicator is red. If it is red, turn off power to the 2100 Bioanalyzer instrument and turn on again. If the problem persists, contact Agilent Technologies at www.agilent.com/genomics/contact.

Check whether the status indicator is on. If it is off and the fan is not running, replace the fuses as described under Changing the Fuses on page 146. A set of spare fuses comes with the instrument. If the status indicator is off and the fan is running, contact Agilent Technologies at www.agilent.com/genomics/contact.

Check that the RS232 communication cable is connected as described in the Installation and Safety Guide.

Check if another hardware device is connected to your computer.

Replace the RS232 cable.

Reinstall the 2100 Expert Software.

If the 2100 Bioanalyzer instrument still will not communicate, contact Agilent Technologies at www.agilent.com/genomics/contact.

18 Maintenance and Troubleshooting

Troubleshooting the Instrument Communication 2 Troubleshooting Communication Issues

Troubleshooting Communication Issue Flow Chart

1 www.agilent.com/genomics/contact 2 see How to create a Support Package on page 22 3 see USB to Serial Adapter on page 21

Maintenance and Troubleshooting 19

2 Troubleshooting the Instrument Communication Changing COM Port Settings

Changing COM Port Settings

The 2100 Bioanalyzer instrument communicates via a serial RS-232 cable with the PC. The number of COM ports available depends on the type of PC used. Laptop PCs have only one COM port. The 2100 Expert Software allows adjustment of the COM port.

To change the COM port settings:

1 Select the Instrument tab in the Instrument context. In the tree view, highlight the appropriate instrument.

2 Under COM Port choose a different port number from the drop down list.

3 Check the icon of the 2100 Bioanalyzer instrument on the screen. If it is no longer dimmed, communication between the 2100 Bioanalyzer instrument and PC is working properly. In addition, hardware information is displayed, see Figure on page 17

4 If you have a PC connected to your instrument and the icon is still dimmed, repeat step 2, choosing a different COM port each time, until it is not dimmed anymore. If the 2100 Bioanalyzer instrument still will not communicate create a support package (How to create a Support Package on page 22) and contact Agilent Technologies at www.agilent.com/genomics/contact.

NOTE The demo port refers to demo assays that do not require PC-instrument communication. For more information on demo assays, please refer to the Online Help or Users Guide.

20 Maintenance and Troubleshooting

Troubleshooting the Instrument Communication 2 USB to Serial Adapter

USB to Serial Adapter

Agilent strongly recommends to connect the 2100 Bioanalyzer instrument directly to a serial port on the PC. However, if the PC does not offer this option, connect the 2100 Bioanalyzer instrument to USB drive utilizing an Agilent USB-Serial Adapter cable. This cable includes a controller component, which requires the installation of a driver allowing the USB to emulate a serial port.

For Expert software B.02.08 and greater It is recommended to use the new Agilent USB-Serial Adapter cable (Part number 5188-8031, black cable) for 2100 Expert Software version B.02.08 and greater.

If the PC is connected to the internet, the correct driver will install automatically when the USB-Serial Adapter cable is plugged into the USB port of your PC.

For off-line systems, please install the driver from the Agilent 2100 Expert Software CD by clicking on the executable file found in SupportDriver/88-8031CDM20824.exe. If a CD is not available, download the driver.

Install the driver (http://www.genomics.agilent.com/article.jsp?pageId=2353 ) prior to connecting the adapter and the 2100 Bioanalyzer instrument and proceed as described below.

Close the 2100 Expert Software.

Execute the installation program of the driver and follow the instructions. Reboot the operating system.

Physically connect the 2100 Bioanalyzer instrument and the laptop by using the USB-Serial Adapter cable and the standard Serial RS-232 cable.

Maintenance and Troubleshooting 21

2 Troubleshooting the Instrument Communication How to create a Support Package

How to create a Support Package

The Support Package collects files and puts them into a ZIP file named similarly to Expert_06122015_164231. The ZIP file is saved automatically on your desktop and has to be send out manually (via email) to Agilent support.

In order to create a Support Package, in the 2100 Expert software, open Help > Create support package. In the pop-up window, a list of tasks is presented. Make sure all tasks are selected, then click collect to proceed.

22 Maintenance and Troubleshooting

Troubleshooting the Instrument Communication 2 How to create a Support Package

NOTE Alternatively:

In the Windows Start menu, open All Programs > Agilent 2100 Bioanalyzer > Utilities > Create support package. In the pop-up window, a list of tasks is presented.

Make sure all tasks are selected, then click collect to proceed.

Maintenance and Troubleshooting 23

2 Troubleshooting the Instrument Communication How to create a Support Package

24 Maintenance and Troubleshooting

Maintenance and Troubleshooting

3 Troubleshooting the 2100 Expert Software Run Installation Qualification Test 26

25Agilent Technologies

3 Troubleshooting the 2100 Expert Software Run Installation Qualification Test

Run Installation Qualification Test

If it is suspected that the 2100 Expert Software is not working properly, check for corrupted or missing files.

1 Start the 2100 Expert Software and select the Validation (Verification) context.

2 In the tree view, select New Validation (Verification) > Installation Validation (Verification) > Software > [My PC Name] > Installation Qualification Test.

3 Under Available Tests select the checkbox of the Installation Qualification Test. This test verifies that files and configurations have been installed to their appropriate locations and display correct attributes.

4 Start the software test tool by clicking Start.

5 The Save As dialog box appears. Define the name and location of the verification file.

6 When the test is finished, the Verification Run Complete message appears.

26 Maintenance and Troubleshooting

Troubleshooting the 2100 Expert Software 3 Run Installation Qualification Test

7 The result of the installation qualification test depends on whether the software installation is complete and no files are corrupted. To review the results, switch to the Results tab:

8 If the test passes and the 2100 Bioanalyzer system still does not function properly, see Verify the Instrument Communication on page 16 and Overview on page 30 for further troubleshooting procedures. Finally, to check the application, see Overview on page 36, Overview on page 68 , or Overview on page 90.

9 If the test fails, reinstall the 2100 Expert Software using the software UFD media that is supplied with the system.

10 If the test continues to fail, contact Agilent Technologies at https://www.agilent.com/en/contact-us/page.

Maintenance and Troubleshooting 27

3 Troubleshooting the 2100 Expert Software Run Installation Qualification Test

28 Maintenance and Troubleshooting

Maintenance and Troubleshooting

4 Hardware Diagnostics Overview 30 Diagnostic Test Procedure 32

29Agilent Technologies

4 Hardware Diagnostics Overview

Overview

Several tests are provided for the 2100 Bioanalyzer system to check the functionality of the hardware. These tests should be performed on a regular basis, or if incorrect measurements or error messages occur. Table 2 on page 30 provides an overview of the available test chips.

Table 2 2100 Bioanalyzer System Test Chips - Electrophoresis Mode

Test chip type Comment Quant.

Autofocus test chip Values for fluorescence and offset are printed on the chip; can be used multiple times.

1

Electrode/Diode test chip Can be used multiple times. 1

30 Maintenance and Troubleshooting

Hardware Diagnostics 4 Overview

Table 3 on page 31 shows a complete list of hardware diagnostic tests that can be run with the electrode cartridge.

Table 3 Diagnostic tests for electrophoresis mode

Test Description

Electronic test Verifies proper functioning of all electronic boards.

Fan test Checks that the fan is running.

Lid sensor test Checks for the devices sensing open or closed lid, and for laser and LED off when lid is closed.

Temperature test Checks that the temperature ramp up speed of the heater plate is within specifications.

Stepper motor test Checks for proper movement of the stepper motor.

Electrode/Diode test Checks photodiode and current versus voltage performance of the 2100 Bioanalyzer instrument. Electrode/Diode test chip required.

High voltage stability test Checks the accuracy and stability of all 16 high voltage power supplies. Unused chip (DNA, RNA or protein) required.

High voltage accuracy test Checks high voltage controller. Unused chip (DNA, RNA or protein) required.

High voltage accuracy-on load test (only for G2939A, G2938B and C instruments)

Checks channel-reference diode in transmission direction. Unused chip (DNA, RNA or protein) required.

Short circuit test Checks for instrument leak currents using an empty chip. Note: the limits of this test specify an ambient temperature of 25C and relative humidity less than or equal to 50%. Higher temperatures of relative humidity could result in a leak current. Unused chip (DNA, RNA or protein) required.

Optics test Checks for proper alignment of internal optics and proper function of the laser. Electrode/Diode test chip required.

Autofocus test Checks focusing capability of optical system. Autofocus test chip required. Input values are located on top of the chip.

Laser stability test Measures red laser signal stability. Autofocus test chip required.

Maintenance and Troubleshooting 31

4 Hardware Diagnostics Diagnostic Test Procedure

Diagnostic Test Procedure

For details on the test procedure, please refer to the documentation included with the test chip kits.

1 Start the 2100 Expert Software.

2 Access the hardware diagnostic tests by selecting the Diagnostics tab in the Instrument context of the 2100 Expert Software.

3 In case two 2100 Bioanalyzer instruments are connected to the PC, highlight the appropriate instrument in the tree view.

4 Select any of the hardware tests from the list given or choose Select All to run all tests.

5 Select Start and follow the instructions as given by the 2100 Expert Software.

6 At the end of the procedure, all tests must have passed.

NOTE Diagnostic tests cannot be run while the 2100 Expert Software is performing a chip run.

NOTE Tests can only be performed if the instrument is online. In the offline mode, the test entries are dimmed.

32 Maintenance and Troubleshooting

Hardware Diagnostics 4 Diagnostic Test Procedure

7 If there are failures, repeat the failed tests.

8 If failures persist, contact Agilent Technologies at www.agilent.com/genomics/contact.

The results of diagnostic tests are stored as .xdy files in Agilent\2100 bioanalyzer\2100 expert\diagnosis. If tests fail, send the .xdy files to Agilent Technical Support.

Maintenance and Troubleshooting 33

4 Hardware Diagnostics Diagnostic Test Procedure

34 Maintenance and Troubleshooting

Maintenance and Troubleshooting

5 Troubleshooting the DNA Application Overview 36 Symptoms (DNA) 37

Residual Gel in Spin Filter after Centrifugation 38 Too High Quantitation Results 38 Too Low Quantitation Results 39 Wrong Sizing Result 40 Run Aborted 41 Chip Not Detected 42 Additional Sample or Ladder Peaks 43 Spikes 44 Low Signal Intensity 45 Missing Peaks 47 Missing Upper Marker 48 Broad Peaks 49 Baseline Dips 50 Baseline Noise 51 Baseline Jumps 52 Wavy Baseline 53 Late Migration 54 Peak Tailing 56 Unexpected Run Time 57 Error Message: No data received since 5 seconds 61

Symptoms (High Sensitivity DNA) 62 Artefact Peaks 63 Split Peaks 64 Baseline Negative Dips 65

35Agilent Technologies

5 Troubleshooting the DNA Application Overview

Overview

Error messages appearing on the screen describe a problem that has occurred with either the hardware or the software.

Additional information regarding the nature of a problem may be found in the Run Log for the data file. Select the Log Book tab in the Data and Assay context. The Run Log lists all the actions and errors that occurred during the run.

In rare cases, results generated by the 2100 Bioanalyzer system might not be as expected. To help find the reason for the discrepancy, see Symptoms (DNA) on page 37.

For most observations, there will be at least one corresponding example, depicting a typical electropherogram, gel-like image or result table. Once the observation that resembles the outcome of the experiment has been identified, a set of assigned causes will be listed by priority.

The causes are grouped into three levels:

Most probable cause

Probable cause

Least probable cause

A list of solutions to help fix the problems are assigned to the causes. For successful troubleshooting, go through all the solution hints listed by probability.

36 Maintenance and Troubleshooting

Troubleshooting the DNA Application 5 Symptoms (DNA)

Symptoms (DNA)

Click to go straight to the troubleshooting hints.

Residual Gel in Spin Filter after Centrifugation on page 38

Too High Quantitation Results on page 38

Too Low Quantitation Results on page 39

Wrong Sizing Result on page 40

Run Aborted on page 41

Chip Not Detected on page 42

Additional Sample or Ladder Peaks on page 43

Spikes on page 44

Low Signal Intensity on page 45

Missing Peaks on page 47

Missing Upper Marker on page 48

Broad Peaks on page 49

Baseline Dips on page 50

Baseline Noise on page 51

Baseline Jumps on page 52

Wavy Baseline on page 53

Late Migration on page 54

Peak Tailing on page 56

Unexpected Run Time on page 57

Error Message: No data received since 5 seconds on page 61

Maintenance and Troubleshooting 37

5 Troubleshooting the DNA Application Symptoms (DNA)

Residual Gel in Spin Filter after Centrifugation

Too High Quantitation Results

Back to Symptoms (DNA) on page 37

Most probable causes Solution

Gel was filtered at insufficient g-value. Refer to the Kit Guide for proper centrifuge settings.

Cooled centrifuge was used for gel filtration.

Repeat centrifugation step at room temperature.

Gel was too cool or viscous. Reagents must be equilibrated at room temperature for 30 minutes prior to use.

Most probable causes Solution

Pipetting error during preparation of reagent mixes.

Check dilution procedure and check calibration of pipette.

Chip pipetting error. Prepare new chip. Always insert the pipette tip to the bottom of the well when dispensing the liquid. Use appropriate pipette and tips.

Insufficient vortexing of chip. Vortex chip for 1 minute. Only use the IKA vortexer. Adjust the speed to the set-point.

Probable causes Solution

Dye concentration too low (marker disappears).

Use dye concentration according to the Kit Guide. Let the dye warm up to room temperature for 30 minutes before preparing the gel-dye mix. Protect dye from light during this time.

Low or missing upper marker. Check Missing Upper Marker on page 48.

Least probable causes Solution

Loaded chip kept for too long before run.

Prepared chips must be used within 5 minutes.

38 Maintenance and Troubleshooting

Troubleshooting the DNA Application 5 Symptoms (DNA)

Too Low Quantitation Results

Back to Symptoms (DNA) on page 37

Most probable causes Solution

Pipetting error during preparation of reagent mixes.

Check dilution procedure and calibration of pipette.

Chip pipetting error. Prepare new chip. Always insert the pipette tip to the bottom of the well when dispensing the liquid. Use appropriate pipette and tips.

Insufficient vortexing of chip. Vortex chip for 1 minute. Only use the IKA vortexer. Adjust the speed to the set-point.

Probable causes Solution

Loaded chip kept for too long before run.

Prepared chips must be used within 5 minutes.

Dye concentration too high. Use dye concentration according to the Kit Guide. Let the dye warm up to room temperature for 30 minutes before preparing the gel-dye mix.

Least probable causes Solution

Sample concentration too high. Use sample concentration according to the Kit Guide.

Maintenance and Troubleshooting 39

5 Troubleshooting the DNA Application Symptoms (DNA)

Wrong Sizing Result

Back to Symptoms (DNA) on page 37

Most probable causes Solution

DNA ladder degraded. Check expiration date of reagents.

Chip not properly primed. Clogged chip priming station or wrong priming station settings.

Prepare a new chip. Check chip priming station as described in Checking the Chip Priming Station for Proper Performance - Seal Test on page 141. Clean/replace syringe, gasket, and plastic adapter, if necessary. Check if clip and base plate of priming station are in the correct position (see Kit Guide).

Chip contaminated. Wear powder-free gloves only. Do not touch the underside of the chip. Do not touch the wells of the chip. Clean the electrodes. Load the chip immediately after taking it out of its sealed bag.

Markers called incorrectly. Manually assign lower marker. Follow instructions for Manual Marker Assignment on page 58

Probable causes Solution

Loaded chip kept for too long before run.

Prepared chips must be used within 5 minutes.

No ladder in ladder well. Prepare a new chip.

Least probable causes Solution

Vibration of 2100 Bioanalyzer instrument.

Do not touch 2100 Bioanalyzer instrument during a run. Remove vibration devices, such as vortexers and vacuum pumps, from bench.

Changes of ambient temperature of more than 5C during the run.

Place 2100 Bioanalyzer instrument in thermally stable environment.

High voltage power supply defective. Check high voltage power supply using the Overview on page 30. If the power supply is defective, contact Agilent Technologies at www.agilent.com/genomics/contact.

40 Maintenance and Troubleshooting

Troubleshooting the DNA Application 5 Symptoms (DNA)

Run Aborted

NOTE In the logbook, an error will appear: Run aborted on port x. Instrument error occurred on port x, Unusual high or low voltage or current was detected during the start phase of the on-chip analysis. The marked wells will indicate the wells on the chip that caused the problem.

Most probable causes Solution Insufficient volume in well(s). Check Kit Guide for the amount of liquid to be pipetted. Ensure all wells contain sufficient

ladder, samples or buffer. Air bubble at the bottom of the well, obstructing access to microchannels.

Always insert the pipette tip to the bottom of the well when dispensing the liquid. Remove large air bubbles with a pipette tip (small bubbles on top of the well will not affect the assay).

Dirty electrodes. Clean electrodes according to instructions in How to Clean the Pin Set of the Electrode Cartridge on page 128.

Chip not properly primed. Clogged chip priming station or wrong priming station settings.

Prepare a new chip. Check chip priming station as described in Checking the Chip Priming Station for Proper Performance - Seal Test on page 141. Clean/replace syringe, gasket, and plastic adapter, if necessary. Check if clip and base plate of priming station are in the correct position (see Kit Guide).

Probable causes Solution Chip prepared with cold reagents.

Prepare a new chip. Allow all reagents and reagent mixes to warm up to room temperature for 30 minutes prior to use. Store chips at room temperature.

Least probable causes Solution High voltage power supply defective.

Check high voltage power supply using the Overview on page 30. If the power supply is defective, contact Agilent Technologies at www.agilent.com/genomics/contact.

Maintenance and Troubleshooting 41

5 Troubleshooting the DNA Application Symptoms (DNA)

Chip Not Detected

Back to Symptoms (DNA) on page 37

Most probable causes Solution

No communication between instrument and PC.

Check instrument communication as described in Verify the Instrument Communication on page 16.

Insufficient volume in well(s). Check Kit Guide for the amount of liquid to be pipetted. Ensure all wells contain ladder, samples or buffer.

Chip not properly primed. Air bubble in chip.

Prepare a new chip. Check chip priming station as described in Checking the Chip Priming Station for Proper Performance - Seal Test on page 141. Clean/replace syringe, gasket, and plastic adapter, if necessary. Check if clip and base plate of priming station are in the correct position (see Kit Guide).

Probable causes Solution

Expired reagents. Prepare new chip with fresh reagents.

Least probable causes Solution

High voltage power supply defective. Check high voltage power supply using the Overview on page 30. If the power supply is defective, contact Agilent Technologies at www.agilent.com/genomics/contact.

42 Maintenance and Troubleshooting

Troubleshooting the DNA Application 5 Symptoms (DNA)

Additional Sample or Ladder Peaks

Back to Symptoms (DNA) on page 37

Most probable causes Solution Chip or gel-dye mix contaminated with particles.

Prepare new chip with new gel-dye mix: Wear powder-free gloves only. Do not touch the underside of the chip. Do not touch the wells of the chip. Clean the electrodes. Load the chip immediately after taking it out of its sealed bag.

Sample degraded or contaminated.

Always wear gloves when handling chips and samples.

Probable causes Solution Chip prepared with cold reagents.

Prepare a new chip. Allow all reagents and reagent mixes to warm up to room temperature for 30 minutes before use.

Chip not properly primed. Clogged chip priming station or wrong priming station settings.

Prepare a new chip. Check chip priming station, see Checking the Chip Priming Station for Proper Performance - Seal Test on page 141. Clean/replace syringe, gasket, and plastic adapter, if necessary. Check if clip and base plate of priming station are in the correct position (see Kit Guide).

Vibration of 2100 Bioanalyzer instrument.

Do not touch 2100 Bioanalyzer instrument during a run. Remove vibration devices, such as vortexers and vacuum pumps, from bench.

Dye agglomerates present in the gel-dye mix.

Use dye concentration according to the Kit Guide. Let the dye warm up to room temperature for 30 minutes before preparing the gel-dye mix. Protect the dye from light during this time. After centrifugation, the gel-dye mix should be taken up from the top of the tube.

Least probable causes Solution DNA ladder degraded. Check expiration date of reagents. Use fresh DNA ladder.

Maintenance and Troubleshooting 43

5 Troubleshooting the DNA Application Symptoms (DNA)

Spikes

Back to Symptoms (DNA) on page 37

Most probable causes Solution

Vibration of 2100 Bioanalyzer instrument. Do not touch 2100 Bioanalyzer instrument during a run. Remove vibration devices, such as vortexers and vacuum pumps, from bench.

Chip or gel-dye mix contaminated with particles. Prepare new chip with new gel-dye mix: Wear powder-free gloves only. Do not touch the underside of the chip. Do not touch the wells of the chip. Clean the electrodes. Load the chip immediately after taking it out of its sealed bag.

Dye agglomerates present in the gel-dye mix. Use dye concentration according to the Kit Guide. Let the dye warm up to room temperature for 30 minutes before preparing the gel-dye mix. Protect the dye from light during this time. After centrifugation, the gel-dye mix should be taken up from the top of the tube.

Probable causes Solution

Chip prepared with cold reagents. Prepare a new chip. Allow all reagents and reagent mixes to warm up to room temperature for 30 minutes before use.

44 Maintenance and Troubleshooting

Troubleshooting the DNA Application 5 Symptoms (DNA)

Low Signal Intensity

Most probable causes Solution

Dye concentration too low. Use dye concentration according to the Kit Guide. Let the dye warm up to room temperature for 30 minutes before preparing the gel-dye mix. Protect the dye from light during this time.

Pipetting error during preparation of reagent mixes. Check dilution procedure and calibration of pipette.

Chip pipetting error. Pipette new chip. Always insert the pipette tip to the bottom of the well when dispensing the liquid. Holding the pipette at a slight angle will ensure proper dispensing of the liquid. Use appropriate pipette and tips.

Probable causes Solution

Fingerprint on focusing lens or on the backside of the chip. Clean lens as described in Cleaning the Lens on page 145. Do not touch the underside of the chip.

Insufficient vortexing of chip. Vortex chip for 1 minute. Only use IKA vortexer for chip vortexing. Adjust speed to set-point.

Maintenance and Troubleshooting 45

5 Troubleshooting the DNA Application Symptoms (DNA)

Back to Symptoms (DNA) on page 37

Least probable causes Solution

Chip contaminated. Wear powder-free gloves only. Do not touch the underside of the chip. Do not touch the wells of the chip. Clean the electrodes. Load the chip immediately after taking it out of its sealed bag.

Vibration of 2100 Bioanalyzer instrument. Do not touch 2100 Bioanalyzer instrument during a run. Remove vibration devices, such as vortexers and vacuum pumps, from bench.

Autofocus failure. Check autofocus using the Hardware Diagnostics on page 29. If autofocus fails, contact Agilent Technologies at www.agilent.com/genomics/contact.

46 Maintenance and Troubleshooting

Troubleshooting the DNA Application 5 Symptoms (DNA)

Missing Peaks

Back to Symptoms (DNA) on page 37

Most probable causes Solution Sample salt concentration is too high. Refer to the maximum sample buffer salt limits as specified in the Kit Guide.

Dilute samples with deionized DNase free water, if necessary. Chip not properly primed. Clogged chip priming station or wrong priming station settings.

Prepare a new chip. Check chip priming station as described in Checking the Chip Priming Station for Proper Performance - Seal Test on page 141. Clean/replace syringe, gasket and plastic adapter, if necessary. Check if clip and base plate of priming station are in the correct position (see Kit Guide).

Leak currents due to contaminated electrodes.

Clean electrodes with analysis-grade water and a toothbrush, see How to Clean the Pin Set of the Electrode Cartridge on page 128.

Least probable causes Solution Laser broken. Perform Optics, Autofocus, and Laser Stability tests as described in Hardware

Diagnostics on page 29. If tests fail, contact Agilent Technologies at www.agilent.com/genomics/contact.

Autofocus failure or high voltage power supply defective

Check autofocus and high voltage power supply by means of the Hardware Diagnostics on page 29. If a diagnostic test fails, contact Agilent Technologies at www.agilent.com/genomics/contact.

Maintenance and Troubleshooting 47

5 Troubleshooting the DNA Application Symptoms (DNA)

Missing Upper Marker

Back to Symptoms (DNA) on page 37

Most probable causes Solution

Alignment of upper marker not set properly.

Manually assign upper marker. Follow instructions for Manual Marker Assignment on page 58

Upper marker digested by restriction enzymes.

Inactivate restriction enzymes by adding EDTA or heat according to the manufacturer's instructions.

Probable causes Solution

Sample salt concentration is too high. Refer to the maximum sample buffer salt limits as specified in the Kit Guide. Dilute samples with deionized DNase free water if necessary.

48 Maintenance and Troubleshooting

Troubleshooting the DNA Application 5 Symptoms (DNA)

Broad Peaks

Back to Symptoms (DNA) on page 37

Most probable causes Solution

Leak currents due to contaminated electrodes.

Clean electrodes with analysis-grade water and a toothbrush, see How to Clean the Pin Set of the Electrode Cartridge on page 128.

Chip not properly primed. Clogged chip priming station or wrong priming station settings.

Prepare a new chip. Check the priming station as described in Checking the Chip Priming Station for Proper Performance - Seal Test on page 141. Clean/replace syringe, gasket and plastic adapter, if necessary. Check if clip and base plate of priming station are in the correct position (see Kit Guide).

Dye concentration too high. Use dye concentration according to the Kit Guide. Let the dye warm up to room temperature for 30 minutes before preparing the gel-dye mix.

Probable causes Solution

Genomic DNA or cDNA contamination. Check DNA preparation procedure.

Maintenance and Troubleshooting 49

5 Troubleshooting the DNA Application Symptoms (DNA)

Baseline Dips

Back to Symptoms (DNA) on page 37

Most probable causes Solution

Sample concentration is too high. Use sample concentration according to the Kit Guide.

Sample impurities: e.g. genomic DNA, ss DNA, etc.

Check DNA-isolation protocol. If possible, clean up samples.

Probable causes Solution

Sample salt concentration is too high. Refer to the maximum sample buffer salt limits as specified in the Kit Guide. Dilute samples with deionized DNase free water, if necessary.

Dye concentration is too low. Use dye concentration according to the Kit Guide. Let the dye warm up to room temperature for 30 minutes before preparing the gel-dye mix. Protect the dye from light during this time.

Least probable causes Solution

Autofocus failure. Check autofocus by means of the Overview on page 30. If the diagnostic test fails, contact Agilent Technologies at www.agilent.com/genomics/contact.

50 Maintenance and Troubleshooting

Troubleshooting the DNA Application 5 Symptoms (DNA)

Baseline Noise

Back to Symptoms (DNA) on page 37

Most probable causes Solution

Chip contaminated. Wear powder-free gloves only. Do not touch the underside of the chip. Do not touch the wells of the chip. Clean the electrodes. Load the chip immediately after taking it out of its sealed bag.

Probable causes Solution

Fingerprint on focusing lens or on the backside of the chip.

Clean lens as described in Cleaning the Lens on page 145. Do not touch the underside of the chip.

Least probable causes Solution

Autofocus failure or high voltage power supply defective.

Check autofocus and high voltage power supply by means of the Hardware Diagnostics on page 29. If tests fail, contact Agilent Technologies at www.agilent.com/genomics/contact.

Maintenance and Troubleshooting 51

5 Troubleshooting the DNA Application Symptoms (DNA)

Baseline Jumps

Back to Symptoms (DNA) on page 37

Most probable causes Solution

Vibration of 2100 Bioanalyzer instrument.

Remove vibration devices, such as vortexers and vacuum pumps, from bench.

Instrument lid was touched during the run.

Do not touch the 2100 Bioanalyzer instrument during a run.

Least probable causes Solution

Laser defective. Check laser using the Hardware Diagnostics on page 29. If the diagnostic test fails, contact Agilent Technologies at www.agilent.com/genomics/contact.

52 Maintenance and Troubleshooting

Troubleshooting the DNA Application 5 Symptoms (DNA)

Wavy Baseline

Back to Symptoms (DNA) on page 37

Most probable causes Solution Leak currents due to contaminated electrodes.

Clean electrodes as described in How to Clean the Pin Set of the Electrode Cartridge on page 128.

Leak currents due to liquid spillage on top of the chip (detergents in sample buffer lower surface tension in the wells of the chip).

Prepare a new chip. Lower vortexing speed or mix samples manually.

Dye concentration too low. Use dye concentration according to the Kit Guide. Let the dye warm up to room temperature for 30 minutes before preparing the gel-dye mix.

Probable causes Solution Chip not properly primed. Clogged chip priming station or wrong priming station settings.

Prepare a new chip. Check the priming station as described in Checking the Chip Priming Station for Proper Performance - Seal Test on page 141. Clean/replace syringe, gasket and plastic adapter, if necessary. Check if clip and base plate of priming station are in the correct position (see Kit Guide).

Leak currents due to wet cartridge. Use only 350 L of water in the cleaning chip. Ensure the humidity in the room is below 70% at 15 27 C (59 81 F).

Least probable causes Solution Changes in ambient temperature of more than 5C during the run.

Place the 2100 Bioanalyzer instrument in a thermally stable environment.

High voltage power supply defective. Check high voltage power supply using the Overview on page 30. If the power supply is defective, contact Agilent Technologies at www.agilent.com/genomics/contact.

Maintenance and Troubleshooting 53

5 Troubleshooting the DNA Application Symptoms (DNA)

Late Migration

Most probable causes Solution

Chip not properly primed. Clogged chip priming station or wrong priming station settings.

Prepare a new chip. Check chip priming station as described in Checking the Chip Priming Station for Proper Performance - Seal Test on page 141. Clean/replace syringe, gasket and plastic adapter, if necessary. Check if clip and base plate of priming station are in the correct position (see Kit Guide).

Leak currents due to contaminated electrodes.

Clean electrodes as described in How to Clean the Pin Set of the Electrode Cartridge on page 128.

Leak currents due to liquid spillage on top of the chip (detergents in sample buffer lower surface tension in the wells of the chip).

Prepare a new chip. Lower vortexing speed or mix samples manually.

Probable causes Solution

Loss of gel separation properties. Gel or gel-dye mix expired or stored incorrectly. Check Kit Guide for proper storage of gel and gel-dye mix. Use gel-dye mix within indicated time. Do not use expired reagents.

Dye concentration too high Use dye concentration according to the Kit Guide. Let the dye warm up to room temperature for 30 minutes before preparing the gel-dye mix.

Vortex speed too high. Vortex chip for 1 minute. Only use the IKA vortexer. Ensure speed is adjusted to the setpoint.

54 Maintenance and Troubleshooting

Troubleshooting the DNA Application 5 Symptoms (DNA)

Back to Symptoms (DNA) on page 37

Genomic DNA or high molecular weight DNA contamination.

Check DNA isolation protocol.

Least probable causes Solution

Vortex adapter not connected tightly. Press vortex adapter tightly on mount (vortex adapter must not rock). Replace vortex adapter as described in Maintenance of the Vortexer on page 151.

Changes in ambient temperature of more than 5C during the run.

Place the 2100 Bioanalyzer instrument in a thermally stable environment.

Maintenance and Troubleshooting 55

5 Troubleshooting the DNA Application Symptoms (DNA)

Peak Tailing

Back to Symptoms (DNA) on page 37

Most probable causes Solution

Sample salt concentration is too high. Refer to the maximum sample buffer salt limits as specified in the Kit Guide. Dilute samples with deionized DNase free water, if necessary.

56 Maintenance and Troubleshooting

Troubleshooting the DNA Application 5 Symptoms (DNA)

Unexpected Run Time

Unexpected Run Time

Back to Symptoms (DNA) on page 37

Most probable causes Solution

Lower and/or upper markers are called incorrectly.

Turn off alignment and check which bands are the correct lower and upper markers. For more details, see Manual Marker Assignment on page 58.

Maintenance and Troubleshooting 57

5 Troubleshooting the DNA Application Symptoms (DNA)

Manual Marker Assignment

1 Turn alignment off . Check the gel-like image to identify which bands are the correct lower and upper markers.

2 Turn the alignment back on . Check the electropherogram of each sample. Go to the Peak Table tab to adjust the markers.

58 Maintenance and Troubleshooting

Troubleshooting the DNA Application 5 Symptoms (DNA)

3 To adjust the lower marker, right click on the correct peak, and choose Manually Set Lower Marker.

4 To adjust the upper marker, right click on the correct peak, and choose Manually Set Upper Marker.

Maintenance and Troubleshooting 59

5 Troubleshooting the DNA Application Symptoms (DNA)

The data is properly aligned after the markers are called correctly.

60 Maintenance and Troubleshooting

Troubleshooting the DNA Application 5 Symptoms (DNA)

Error Message: No data received since 5 seconds

Most probable causes Solution

Disrupted communication between instrument and computer.

Please refer to Troubleshooting the Instrument Communication on page 15 for troubleshooting instrument communication issue. Ensure the Agilent USB-Serial Adapter cable, black cable (5188-8031) for 2100 Expert Software version B.02.08 and greater is used to connect the 2100 Bioanalyzer instrument to the computer through a USB port. See USB to Serial Adapter on page 21.

Maintenance and Troubleshooting 61

5 Troubleshooting the DNA Application Symptoms (High Sensitivity DNA)

Symptoms (High Sensitivity DNA)

Click to go straight to the troubleshooting hints.

Artefact Peaks on page 63

Split Peaks on page 64

Baseline Negative Dips on page 65

NOTE Follow protocols appropriate for the Next Generation Sequencing or targeted-enrichment system used. Additional inquiries should be directed to the manufacturer of those products.

62 Maintenance and Troubleshooting

Troubleshooting the DNA Application 5 Symptoms (High Sensitivity DNA)

Artefact Peaks

Back to Symptoms (High Sensitivity DNA) on page 62

Most probable causes Solution

Samples are in water. Refer to the Kit Guide for sample buffer specifications. For optimal results, samples should be dissolved in 10 mM Tris and 1 mM EDTA.

Probable causes Solution

Chip, gel-dye mix, or samples are contaminated with particles.

Wear powder-free gloves only. Do not touch the wells of the chip. Clean the electrodes. Load the chip immediately after taking it out of its sealed bag.

Maintenance and Troubleshooting 63

5 Troubleshooting the DNA Application Symptoms (High Sensitivity DNA)

Split Peaks

Back to Symptoms (High Sensitivity DNA) on page 62

Most probable causes Solution

Sample concentration is too high. Prepare a new chip. Follow guidelines in the Kit Guide for the amount of sample to be loaded.

64 Maintenance and Troubleshooting

Troubleshooting the DNA Application 5 Symptoms (High Sensitivity DNA)

Baseline Negative Dips

Most probable causes Solution

Residual RNase ZAP and/or SDS contamination on the electrode pins.

Only use RNaseZAP when decontaminating the pins according to How to Clean the Pin Set of the Electrode Cartridge on page 128. Clean the pin set thoroughly with water after running Protein or RNA assays. Change the water in the electrode cleaner Chip and/or the electrode cleaner Chip itself regularly.

Maintenance and Troubleshooting 65

5 Troubleshooting the DNA Application Symptoms (High Sensitivity DNA)

66 Maintenance and Troubleshooting

Maintenance and Troubleshooting

6 Troubleshooting the RNA Application Overview 68 Symptoms (RNA) 69

Residual Gel in Spin Filter after Centrifugation 70 Too High Quantitation Results 70 Too Low Quantitation Results 71 Chip Not Detected 72 Run Aborted 73 Additional Sample or Ladder Peaks 74 Additional Saturating Bands 75 Degraded RNA Ladder and/or Samples 76 Spikes 77 Low Signal Intensity 78 Baseline Noise 79 Broad Peaks 80 Missing Peaks 81 Missing RNA Fragment 82 Wavy Baseline 83 Late Migration 84 Error Message: No data received since 5 seconds 85 RNA Integrity Number (RIN): N/A 86

67Agilent Technologies

6 Troubleshooting the RNA Application Overview

Overview

Error messages appearing on the screen describe a problem that has occurred with either the hardware or the software.

Additional information regarding the nature of a problem may be found in the run log for the data file. Select the Log Book tab in the Data and Assay context. The Run Log lists all the actions and errors that occurred during the run.

In rare cases, results generated by the 2100 Bioanalyzer system might not be as expected. To help find the reason for the discrepancy, see Symptoms (RNA) on page 69.

For most observations there will be at least one corresponding example depicting a typical electropherogram, gel-like image or result table. Once the observation that resembles the outcome of the experiment has been identified, a set of assigned causes will be listed by priority.

The causes are grouped into three levels:

Most probable cause

Probable cause

Least probable cause

A list of solutions to help fix the problems are assigned to the causes. For successful troubleshooting, go through all the solution hints listed by probability.

68 Maintenance and Troubleshooting

Troubleshooting the RNA Application 6 Symptoms (RNA)

Symptoms (RNA)

Click to go straight to the troubleshooting hints.

Residual Gel in Spin Filter after Centrifugation on page 70

Too High Quantitation Results on page 70

Too Low Quantitation Results on page 71

Chip Not Detected on page 72

Run Aborted on page 73

Additional Sample or Ladder Peaks on page 74

Additional Saturating Bands on page 75

Degraded RNA Ladder and/or Samples on page 76

Spikes on page 77

Low Signal Intensity on page 78

Baseline Noise on page 79

Broad Peaks on page 80

Missing Peaks on page 81

Missing RNA Fragment on page 82

Wavy Baseline on page 83

Late Migration on page 84

Error Message: No data received since 5 seconds on page 85

RNA Integrity Number (RIN): N/A on page 86

Maintenance and Troubleshooting 69

6 Troubleshooting the RNA Application Symptoms (RNA)

Residual Gel in Spin Filter after Centrifugation

Too High Quantitation Results

Back to Symptoms (RNA) on page 69

Most probable causes Solution

Gel was filtered at insufficient g-value. Refer to the Kit Guide for proper centrifuge settings.

Cooled centrifuge was used for gel filtration.

Repeat centrifugation step at room temperature.

Gel was too cool or viscous. Reagents must be equilibrated at room temperature for 30 minutes prior to use.

Most probable causes Solution

Pipetting error during preparation of ladder or samples.

Check dilution procedure and calibration of pipettes.

Chip pipetting error. Prepare new chip. Always insert the pipette tip to the bottom of the well when dispensing the liquid. Use appropriate pipette and tips.

Insufficient vortexing of chip. Vortex chip for 1 minute. Only use the IKA vortexer. Adjust speed to set-point.

Probable causes Solution

RNA ladder degraded. Prepare a new chip using a new ladder aliquot. Always wear gloves when handling chips and RNA samples to avoid contamination. Follow decontamination procedure, see How to Clean the Pin Set of the Electrode Cartridge on page 128.

Electrodes contaminated with RNases. Clean electrodes with RNaseZAP. Follow decontamination procedure, see How to Clean the Pin Set of the Electrode Cartridge on page 128

Dye concentration too low. Use dye concentration according to the Kit Guide. Let the dye warm up to room temperature for 30 min before preparing the gel-dye mix. Protect the dye from light during this time.

RNA ladder not denatured. Heat denature the RNA ladder as described in the Kit Guide.

70 Maintenance and Troubleshooting

Troubleshooting the RNA Application 6 Symptoms (RNA)

Too Low Quantitation Results

Back to Symptoms (RNA) on page 69.

Most probable causes Solution

Reference measurement (e.g. UV absorption) was elevated due to contaminants in sample.

Purify sample prior to measurement.

Pipetting error during preparation of ladder, samples, or reagent mixes.

Use appropriate calibrated pipette and tips. Check dilution procedure. Always insert the pipette tip to the bottom of the well when dispensing the liquid.

Insufficient vortexing of chip. Vortex chip for 1 minute. Only use the IKA vortexer. Adjust the speed to the set-point.

Probable causes Solution

RNA ladder not denatured. Heat denature the RNA ladder as described in the Kit Guide.

Sample concentration too high. Use the sample concentration recommended by the Kit Guide.

Least probable causes Solution

Dye concentration too high. Use dye concentration according to the Kit Guide. Let the dye warm up to room temperature for 30 minutes before preparing the gel-dye mix. Protect the dye from light during this time.

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6 Troubleshooting the RNA Application Symptoms (RNA)

Chip Not Detected

Back to Symptoms (RNA) on page 69

Most probable causes Solution

Insufficient volume in well(s) or chip is empty.

Check Kit Guide for the amount of liquid to be pipetted. Ensure all wells contain ladder, samples or buffer.

Chip not properly primed. Air bubble in chip.

Prepare a new chip. Check chip priming station as described in Checking the Chip Priming Station for Proper Performance - Seal Test on page 141. Clean/replace syringe, gasket and plastic adapter, if necessary. Check if clip and base plate of priming station are in the correct position (see Kit Guide).

Probable causes Solution

No communication between the instrument and PC.

Test the PC-instrument communication as described in Verify the Instrument Communication on page 16.

Least probable causes Solution

High voltage power supply defective. Check high voltage power supply using the Overview on page 30. If the power supply is defective, contact Agilent Technologies at www.agilent.com/genomics/contact.

72 Maintenance and Troubleshooting

Troubleshooting the RNA Application 6 Symptoms (RNA)

Run Aborted

NOTE In the logbook, an error will appear: Run aborted on port x. Instrument error occurred on port x, Unusual high or low voltage or current was detected during the start phase of the on-chip analysis. The marked wells will indicate the wells on the chip that caused the problem.

Most probable causes Solution Insufficient volume in well(s). Check Kit Guide for the amount of liquid to be pipetted. Ensure all wells contain

sufficient ladder, samples or buffer. Air bubble at the bottom of the well, obstructing access to microchannels.

Always insert the pipette tip to the bottom of the well when dispensing the liquid. Remove large air bubbles with a pipette tip (small bubbles on top of the well will not affect the assay).

Dirty electrodes. Clean electrodes according to instructions in How to Clean the Pin Set of the Electrode Cartridge on page 128.

Chip not properly primed. Clogged chip priming station or wrong priming station settings.

Prepare a new chip. Check chip priming station as described in Checking the Chip Priming Station for Proper Performance - Seal Test on page 141. Clean/replace syringe, gasket, and plastic adapter, if necessary. Check if clip and base plate of priming station are in the correct position (see Kit Guide).

Probable causes Solution Chip prepared with cold reagents. Prepare a new chip. Allow all reagents and reagent mixes to warm up to room

temperature for 30 minutes prior to use. Store chips at room temperature.

Least probable causes Solution High voltage power supply defective. Check high voltage power supply using the Overview on page 30. If the power

supply is defective, contact Agilent Technologies at www.agilent.com/genomics/contact.

Maintenance and Troubleshooting 73

6 Troubleshooting the RNA Application Symptoms (RNA)

Additional Sample or Ladder Peaks

Back to Symptoms (RNA) on page 69

Most probable causes Solution Gel-dye mix expired. Use prepared gel-dye mix within one day. RNA ladder or sample not denatured properly. Heat ladder or samples at 70C for 2 minutes. Particles in tubes. For reagent preparation, use tubes that are supplied with the kit. Do not use

autoclaved tubes. Chip or gel-dye mix contaminated with particles.

Wear powder-free gloves only. Do not touch the wells of the chip. Clean the electrodes. Load the chip immediately after taking it out of its sealed bag.

Probable causes Solution Dye agglomerates present in the gel-dye mix. Use dye concentration according to the Kit Guide.

Let the dye warm up to room temperature for 30 minutes and vortex for 10 seconds before preparing the gel-dye mix. Protect the dye from light during this time. After centrifugation, the freshly prepared gel-dye mix should be taken up from the top of the tube.

Vibration of 2100 Bioanalyzer instrument Do not touch the 2100 Bioanalyzer instrument during a run. Remove vibration devices, such as vortexers and vacuum pumps, from the bench.

Chip preparation with cold reagents or chips. Prepare a new chip. Allow all reagents and reagent mixes to warm up to room temperature for 30 minutes before use. Store chips at room temperature.

Least probable causes Solution RNA ladder or sample degraded. Always wear gloves when handling chips or RNA samples to prevent them from

getting contaminated. Follow decontamination procedure, see How to Clean the Pin Set of the Electrode Cartridge on page 128.

74 Maintenance and Troubleshooting

Troubleshooting the RNA Application 6 Symptoms (RNA)

Additional Saturating Bands

Back to Symptoms (RNA) on page 69

NOTE Only present in RNA 6000 Pico or Small RNA assays.

Most probable causes Solution

Residual RNaseZAP on electrode pins. A dedicated electrode cassette for the use of RNA 6000 Pico and Small RNA assays. This cassette should only be washed with water in the electrode cleaner chip before and after each run. Only use RNaseZAP when decontaminating the pins according to How to Clean the Pin Set of the Electrode Cartridge on page 128.

Maintenance and Troubleshooting 75

6 Troubleshooting the RNA Application Symptoms (RNA)

Degraded RNA Ladder and/or Samples

Back to Symptoms (RNA) on page 69

Most probable causes Solution

RNase contamination of the pin set. Decontaminate pin set. Follow decontamination procedure, see How to Clean the Pin Set of the Electrode Cartridge on page 128. Decontaminate pipettes and work space.

RNase contamination of chips and/or reagents.

Prepare a new chip and fresh reagents. Wear powder-free gloves when preparing the chip. Decontaminate pipettes and work space.

76 Maintenance and Troubleshooting

Troubleshooting the RNA Application 6 Symptoms (RNA)

Spikes

Back to Symptoms (RNA) on page 69

Most probable causes Solution Vibration of 2100 Bioanalyzer instrument.

Do not touch the 2100 Bioanalyzer instrument during a run. Remove vibration devices, such as vortexers and vacuum pumps, from the bench.

Particles in tubes. For reagent preparation, use tubes that are supplied with the kit. Do not use autoclaved tubes.

Chip or gel-dye mix contaminated with particles.

Prepare new chip with new gel-dye mix. Wear powder-free gloves only. Do not touch the underside of the chip. Do not touch the wells of the chip. Clean the electrodes. Load the chip immediately after taking it out of its sealed bag.

Dye agglomerates present in the gel-dye mix.

Use dye concentration according to the Kit Guide. Let the dye warm up to room temperature for 30 minutes and vortex for 10 seconds before preparing the gel-dye mix. Protect the dye from light during this time. After centrifugation, the freshly prepared gel-dye mix should be taken up from the top of the tube.

Probable causes Solution Chip prepared with cold reagents. Prepare a new chip. Allow all reagents and reagent mixes to warm up to room

temperature for 30 minutes before use.

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6 Troubleshooting the RNA Application Symptoms (RNA)

Low Signal Intensity

Back to Symptoms (RNA) on page 69

Most probable causes Solution Gel-dye mix expired. Use prepared gel-dye mix within one day. Dye concentration too low. Use dye concentration according to the Kit Guide.

Let the dye warm up to room temperature for 30 minutes before preparing the gel-dye mix. Protect the dye from light during this time.

Pipetting error during preparation of reagent mixes.

Check dilution procedure and calibration of pipette.

Chip pipetting error. Prepare new chip. Always insert the pipette tip to the bottom of the well when dispensing the liquid. Use appropriate pipette and tips.

Probable causes Solution Fingerprint on focusing lens or on the backside of the chip.

Clean lens as described in Cleaning the Lens on page 145. Do not touch the underside of the chip

Insufficient vortexing of chip. Vortex chip for 1 minute. Only use IKA vortexer. Adjust speed to set-point . Least probable causes Solution Autofocus or laser failure. Check autofocus and laser using the Overview on page 30. If laser stability

diagnostic tests fail, contact Agilent Technologies at www.agilent.com/genomics/contact.

78 Maintenance and Troubleshooting

Troubleshooting the RNA Application 6 Symptoms (RNA)

Baseline Noise

Back to Symptoms (RNA) on page 69

Most probable causes Solution

Fingerprint on focusing lens or on the backside of the chip.

Clean lens as described in Cleaning the Lens on page 145. Do not touch the underside of the chip.

Chip contaminated with particles. Wear powder-free gloves only. Do not touch the underside of the chip. Do not touch the wells of the chip. Clean the electrodes. Load the chip immediately after taking it out of its sealed bag.

Vibration of 2100 Bioanalyzer instrument.

Do not touch the 2100 Bioanalyzer instrument during a run. Remove vibration devices, such as vortexers and vacuum pumps, from the bench.

Probable causes Solution

Dye concentration too low. Use dye concentration according to the Kit Guide. Let the dye warm up to room temperature for 30 minutes before preparing the gel-dye mix. Protect the dye from light during this time.

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6 Troubleshooting the RNA Application Symptoms (RNA)

Broad Peaks

Back to Symptoms (RNA) on page 69.

Most probable causes Solution

Sample contaminated with genomic DNA.

Check RNA isolation protocol. To remove genomic DNA, perform DNase treatment.

Leak currents due to contaminated pin set.

Clean the pin set of the electrode cartridge. Follow cleaning procedure, see How to Clean the Pin Set of the Electrode Cartridge on page 128

Probable causes Solution

Chip not properly primed. Clogged chip priming station or wrong priming station settings.

Prepare a new chip. Check the priming station as described in Checking the Chip Priming Station for Proper Performance - Seal Test on page 141. Clean/replace syringe, gasket and plastic adapter, if necessary. Check if clip and base plate of priming station are in the correct position (see Kit Guide).

Least probable causes Solution

Dye concentration too high. Use dye concentration according to the Kit Guide. Let the dye warm up to room temperature for 30 minutes before preparing the gel-dye mix. Protect the dye from light during this time.

80 Maintenance and Troubleshooting

Troubleshooting the RNA Application 6 Symptoms (RNA)

Missing Peaks

Back to Symptoms (RNA) on page 69.

Most probable causes Solution Sample salt concentration is too high.

Refer to the maximum sample buffer salt limits as specified in the Kit Guide. Dilute samples with deionized RNase free water, if necessary.

Chip not properly primed. Clogged chip priming station or wrong priming station settings.

Prepare a new chip. Check chip priming station as described in Checking the Chip Priming Station for Proper Performance - Seal Test on page 141. Clean/replace syringe, gasket and plastic adapter, if necessary. Check if clip and base plate of priming station are in the correct position (see Kit Guide).

Leak currents due to contaminated electrodes.

Clean electrodes as described in How to Clean the Pin Set of the Electrode Cartridge on page 128.

Leak currents due to liquid spillage on top of the chip (detergents in sample buffer lower surface tension in the wells of the chip).

Prepare a new chip. Lower vortexing speed or mix samples manually.

Least probable causes Solution Laser broken. Perform Laser, optics, and autofocus tests as described in Hardware Diagnostics on

page 29. If tests fail, contact Agilent Technologies at www.agilent.com/genomics/contact.

Autofocus failure or high voltage power supply defective.

Check autofocus and high voltage power supply by means of the Hardware Diagnostics on page 29. If diagnostic fails, contact Agilent Technologies at www.agilent.com/genomics/contact.

Maintenance and Troubleshooting 81

6 Troubleshooting the RNA Application Symptoms (RNA)

Missing RNA Fragment

Back to Symptoms (RNA) on page 69.

Most probable causes Solution

Sample salt concentration is too high. Refer to the maximum sample buffer salt limits as specified in the Kit Guide. Dilute samples with deionized RNase free water, if necessary,

Probable causes Solution

RNase contamination of electrodes or reagents.

Clean electrodes with RNaseZAP. Follow cleaning procedure, see How to Clean the Pin Set of the Electrode Cartridge on page 128. Prepare a new chip with fresh reagents. Wear powder-free gloves when preparing the chip.

82 Maintenance and Troubleshooting

Troubleshooting the RNA Application 6 Symptoms (RNA)

Wavy Baseline

Back to Symptoms (RNA) on page 69

Most probable causes Solution Contamination with genomic DNA.

Check RNA isolation protocol. To remove genomic DNA, perform DNase treatment.

Leak currents due to contaminated electrodes.

Clean the electrode cartridge as described in How to Clean the Pin Set of the Electrode Cartridge on page 128. Prepare a new chip.

Leak currents due liquid spillage on top of the chip (detergents in sample buffer lower surface tension in the wells of the chip).

Prepare a new chip. Lower vortexing speed or mix samples manually

Chip not properly primed. Clogged priming station or wrong priming station settings.

Check chip priming station as described in Checking the Chip Priming Station for Proper Performance - Seal Test on page 141. Clean/replace syringe, gasket and plastic adapter, if necessary. Check if clip and base plate of priming station are in the correct position (see Kit Guide).

Probable causes Solution Leak currents due to wet cartridge.

Use only 350 L of water in the cleaning chip. Ensure the humidity in the room is below 70% at 15-27C (59-81F).

Least probable causes Solution Changes in ambient temperature of more than 5C during the run.

Place the 2100 Bioanalyzer instrument in a thermally stable environment.

Autofocus failure or high voltage power supply defective.

Check autofocus and high voltage power using the Overview on page 30. If a diagnostic test fails, contact Agilent Technologies at www.agilent.com/genomics/contact.

Maintenance and Troubleshooting 83

6 Troubleshooting the RNA Application Symptoms (RNA)

Late Migration

Back to Symptoms (RNA) on page 69

Most probable causes Solution Chip not properly primed. Clogged chip priming station or wrong priming station settings.

Check chip priming station as described in Checking the Chip Priming Station for Proper Performance - Seal Test on page 141. Clean/replace syringe, gasket and plastic adapter, if necessary. Check if clip and base plate of priming station are in the correct position (see Kit Guide).

Vortex speed too high. Vortex chip for 1 minute. Only use the IKA vortexer. Ensure speed is adjusted to the set point.

Leak currents due to contaminated electrodes.

Clean electrodes as described in How to Clean the Pin Set of the Electrode Cartridge on page 128.

Leak currents due to liquid spillage on top of the chip (detergents in sample buffer lower surface tension in the wells of the chip).

Prepare a new chip. Lower vortexing speed or mix samples manually.

Probable causes Solution Vortex adapter not connected tightly. Press vortex adapter tightly on mount (vortex adapter must not rock).

Replace vortex adapter as described in Changing the Adapter on page 152.

Loss of gel separation properties. Gel or gel-dye mix expired or stored incorrectly. Check Kit Guide for proper storage of gel and gel-dye mix. Use gel-dye mix within indicated time. Do not use expired reagents.

84 Maintenance and Troubleshooting

Troubleshooting the RNA Application 6 Symptoms (RNA)

Error Message: No data received since 5 seconds

Most probable causes Solution

Disrupted communication between instrument and computer.

Please refer to Troubleshooting the Instrument Communication on page 15 for troubleshooting instrument communication issue. Ensure the Agilent USB-Serial Adapter cable, black cable (5188-8031) for 2100 Expert Software version B.02.08 and greater is used to connect the 2100 Bioanalyzer instrument to the computer through a USB port. See USB to Serial Adapter on page 21.

Maintenance and Troubleshooting 85

6 Troubleshooting the RNA Application Symptoms (RNA)

RNA Integrity Number (RIN): N/A

Most probable causes Solution Incorrect Assay selected. Check the sample type. Prepare a new Chip and re-run the samples

selecting the correct Assay. Incorrect assignment of the Lower Marker. Please refer to Chapter Troubleshooting the RNA Application on

page 67 to solve the problem. Prepare a new Chip and re-run the samples. Incorrect assignment of the ribosomal bands. Please refer to Chapter Troubleshooting the RNA Application on

page 67 to solve the problem and re-run the samples. Samples not heat denatured. Heat the samples 2 min at 70 C as described in the Kit guide. Prepare a

new Chip and re-run the samples. Un-expected signal anomalies detected in the samples (Error message Unexpected signal).

Modify predefined thresholds for anomaly detection to clear the error message and get RIN displayed.

86 Maintenance and Troubleshooting

Troubleshooting the RNA Application 6 Symptoms (RNA)

NOTE In the Errors tab, an error Unexpected signal in can appear. The error message will indicate the anomaly that caused the problem. Adjust the corresponding anomaly threshold in Advanced mode within the RIN Setpoints group.

NOTE RIN N/A is a warning that the RIN may not be reliable for a particular sample (such as due to unusual noise/signals, ribosomal ratio and other factors). Clearing the critical error message may yield a RIN, but Agilent does not guarantee the accuracy of this value. It is recommended to also perform a visual inspection of the data.

Maintenance and Troubleshooting 87

6 Troubleshooting the RNA Application Symptoms (RNA)

88 Maintenance and Troubleshooting

Maintenance and Troubleshooting

7 Troubleshooting the Protein Application Overview 90 Symptoms (Protein) 91

Residual Gel in Spin Filter after Centrifugation 92 Too High Quantitation Results 92 Too Low Quantitation Results 93 Wrong Sizing Result 94 Chip Not Detected 95 Run Aborted 96 Unexpected Run Time 97 Additional Sample or Ladder Peaks 101 Missing Upper Marker 103 Broad Variability of the Lower Marker 104 Missing Peaks 105 Spikes 106 Poor Reproducibility 107 Low Signal Intensity 108 Low Ladder Peaks 109 Broad Peaks 110 Baseline Dips 111 Late Migration 112 Error Message: No data received since 5 seconds 113

Symptoms (High Sensitivity Protein) 114 Saturation of Lower Marker or Sample Peaks Optical Signal too High 115 Low Signal Intensity 116

89Agilent Technologies

7 Troubleshooting the Protein Application Overview

Overview

Error messages appearing on the screen describe a problem that has occurred with either the hardware or the software.

Additional information regarding the nature of a problem may be found in the Run Log for the data file. Select the Log Book tab in the Data and Assay context. The Run Log lists all the actions and errors that occurred during the run.

In rare cases, results generated by the 2100 Bioanalyzer system might not be as expected. To help find the reason for the discrepancy, see Symptoms (Protein) on page 91.

For most observations, there will be at least one corresponding example, depicting a typical electropherogram, gel-like image or result table. Once the observation that resembles the outcome of the experiment has been identified, a set of assigned causes will be listed by priority.

The causes are grouped into three levels:

Most probable cause

Probable cause

Least probable cause

A list of solutions that help fix the problem are assigned to the causes. For successful troubleshooting, go through all the solution hints listed by probability.

90 Maintenance and Troubleshooting

Troubleshooting the Protein Application 7 Symptoms (Protein)

Symptoms (Protein)

Click to go straight to the troubleshooting hints.

Residual Gel in Spin Filter after Centrifugation on page 92

Too High Quantitation Results on page 92

Too Low Quantitation Results on page 93

Wrong Sizing Result on page 94

Chip Not Detected on page 95

Run Aborted on page 96

Unexpected Run Time on page 97

Additional Sample or Ladder Peaks on page 101

Missing Upper Marker on page 103

Broad Variability of the Lower Marker on page 104

Missing Peaks on page 105

Spikes on page 106

Poor Reproducibility on page 107

Low Signal Intensity on page 108

Low Ladder Peaks on page 109

Broad Peaks on page 110

Baseline Dips on page 111

Late Migration on page 112

Error Message: No data received since 5 seconds on page 113

Maintenance and Troubleshooting 91

7 Troubleshooting the Protein Application Symptoms (Protein)

Residual Gel in Spin Filter after Centrifugation

Too High Quantitation Results

Back to Symptoms (Protein) on page 91

Most probable causes Solution

Gel was filtered at insufficient g-value. Refer to the Kit Guide for proper centrifuge settings.

Cooled centrifuge was used for gel filtration.

Repeat centrifugation step at room temperature.

Gel was too cool or viscous. Reagents must be equilibrated at room temperature for 30 minutes prior to use.

Most probable causes Solution

Alignment of upper marker not set properly.

Manually set upper marker. Follow instructions for Manual Marker Assignment on page 98 .

Pipetting error during preparation of reagent mixes or chip.

Refer to the Kit Guide for proper preparation of reagents. Check dilution procedure and calibration of pipette.

Chip preparation error. Prepare new chip. Always insert the pipette tip to the bottom of the well when dispensing the liquid. Use appropriate pipette and tips.

Probable causes Solution

Upper marker is degraded by proteases.

Treat sample with protease inhibitors prior to sample preparation.

Sample salt concentration is too high. Check maximum sample buffer salt limits in the compatible buffer list in the Kit Guide. Dilute the sample prior to the sample preparation or use a different buffer, if possible.

Improper denaturation of sample. Use fresh sample aliquot. Heat sample or denaturing solution for 5 minutes at 100C. Use 0.5 mL tubes for denaturation.

Least probable causes Solution

Loaded chip kept too long before run. Prepared chips must be used within 5 minutes of preparation.

92 Maintenance and Troubleshooting

Troubleshooting the Protein Application 7 Symptoms (Protein)

Too Low Quantitation Results

Back to Symptoms (Protein) on page 91

Most probable causes Solution

Alignment of upper marker not set properly.

Manually set upper marker. Follow instructions for Manual Marker Assignment on page 98.

Pipetting error during preparation of reagent mixes or chip.

Refer to the Kit Guide for proper preparation of reagents. Check dilution procedure and calibration of pipette.

Chip preparation error. Prepare new chip. Always insert the pipette tip to the bottom of the well when dispensing the liquid. Use appropriate pipette and tips.

Probable causes Solution

Sample concentration too high. Use sample concentration according to the specifications in the Kit Guides. Do not forget to dilute samples with deionized water after heat denaturation.

Diluted samples are degraded. Use diluted samples within one day. Store samples at 4C when not in use for longer than 1 hour.

Least probable causes Solution

Loaded chip kept too long before run. Prepared chips must be used within 5 minutes of preparation.

Maintenance and Troubleshooting 93

7 Troubleshooting the Protein Application Symptoms (Protein)

Wrong Sizing Result

Back to Symptoms (Protein) on page 91

Most probable causes Solution

Incorrect assignment of ladder peaks. Check assignment of ladder peaks. For details, please refer to the Online Help or Users Guide.

Incorrect assignment of upper and/or lower marker.

Store sample buffer and denaturing solution according to the instructions given in the Kit Guide. Check assignment of markers. Follow instructions for Manual Marker Assignment on page 98.

Ladder degraded. Use diluted ladder within one day. Store ladder at 4C when not in use for longer than 1 hour.

Probable causes Solution

Improper denaturation of ladder. Use fresh ladder aliquot. Heat ladder for 5 minutes at 100C. Use 0.5 mL tubes for denaturing.

Least probable causes Solution

Incomplete reduction of samples. Due to disulfide bonds, some proteins will not migrate according to their molecular weight if they are not reduced properly. Proteins will migrate higher than the expected molecular weights. Check preparation of denaturing solution described in the Kit Guide.

Protein characteristics Glycosylation and other post-translational modifications may disturb micelle formation around the protein. The proteins will migrate higher than the expected molecular weights. This effect is reproducible.

94 Maintenance and Troubleshooting

Troubleshooting the Protein Application 7 Symptoms (Protein)

Chip Not Detected

Back to Symptoms (Protein) on page 91

Most probable causes Solution

Insufficient volume in well(s) or chip is empty.

Check Kit Guide on amount of liquid to be pipetted. Ensure all wells contain sample, ladder or buffer.

Air bubbles at the bottom of the well. Always insert the pipette tip to the bottom of the well when dispensing the liquid. Remove large air bubbles with a pipette tip (small bubbles on top of well do not affect the assay).

Probable causes Solution

No communication between the instrument and PC.

Test the PC-instrument communication as described in Verify the Instrument Communication on page 16.

Chip not properly primed. Air bubble in chip.

Prepare a new chip. Check chip priming station as described in Checking the Chip Priming Station for Proper Performance - Seal Test on page 141. Clean/replace syringe, gasket, and plastic adapter, if necessary. Check if clip and base plate of priming station are in the correct position (see Kit Guide).

Least probable causes Solution

High voltage power supply defective. Check high voltage power supply using the Overview on page 30. If the power supply is defective, contact Agilent Technologies at www.agilent.com/genomics/contact.

Maintenance and Troubleshooting 95

7 Troubleshooting the Protein Application Symptoms (Protein)

Run Aborted

NOTE In the logbook, an error will appear: Run aborted on port x. Instrument error occurred on port x, Unusual high or low voltage or current was detected during the start phase of the on-chip analysis. The marked wells will indicate the wells on the chip that caused the problem.

Most probable causes Solution Insufficient volume in well(s). Check Kit Guide for the amount of liquid to be pipetted. Ensure all wells contain

sufficient ladder, samples or buffer. Air bubble at the bottom of the well, obstructing access to microchannels.

Always insert the pipette tip to the bottom of the well when dispensing the liquid. Remove large air bubbles with a pipette tip (small bubbles on top of the well will not affect the assay).

Dirty electrodes. Clean electrodes according to instructions in How to Clean the Pin Set of the Electrode Cartridge on page 128.

Chip not properly primed. Clogged chip priming station or wrong priming station settings.

Prepare a new chip. Check chip priming station as described in Checking the Chip Priming Station for Proper Performance - Seal Test on page 141. Clean/replace syringe, gasket, and plastic adapter, if necessary. Check if clip and base plate of priming station are in the correct position (see Kit Guide).

Probable causes Solution Chip prepared with cold reagents.

Prepare a new chip. Allow all reagents and reagent mixes to warm up to room temperature for 30 minutes prior to use. Store chips at room temperature.

Least probable causes Solution High voltage power supply defective.

Check high voltage power supply using the Overview on page 30. If the power supply is defective, contact Agilent Technologies at www.agilent.com/genomics/contact.

96 Maintenance and Troubleshooting

Troubleshooting the Protein Application 7 Symptoms (Protein)

Unexpected Run Time

Unexpected Run Time

Back to Symptoms (Protein) on page 91

Most probable causes Solution

Lower and/or upper markers are called incorrectly.

Turn off alignment and check which bands are the correct lower and upper markers. For more details see Manual Marker Assignment on page 98 .

Least probable causes Solution

Bent electrode pin. Check if electrode pins are bent or damaged. Replace electrophoresis cartridge.

Maintenance and Troubleshooting 97

7 Troubleshooting the Protein Application Symptoms (Protein)

Manual Marker Assignment

1 Turn alignment off . Check the gel-like image to identify which bands are the correct lower and upper markers.

2 Turn the alignment back on . Check the electropherogram of each sample. Go to the Peak Table tab to adjust the markers.

98 Maintenance and Troubleshooting

Troubleshooting the Protein Application 7 Symptoms (Protein)

3 To adjust the lower marker, right click on the correct peak, and choose Manually Set Lower Marker.

4 To adjust the upper marker, right click on the correct peak, and choose Manually Set Upper Marker.

The data is properly aligned after the markers are called correctly.

Maintenance and Troubleshooting 99

7 Troubleshooting the Protein Application Symptoms (Protein)

100 Maintenance and Troubleshooting

Troubleshooting the Protein Application 7 Symptoms (Protein)

Additional Sample or Ladder Peaks

Most probable causes Solution

Sample or ladder not denatured properly.

Prepare fresh sample aliquot. Heat sample or denaturing solution and ladder for 5 minutes at 100C

Improper tubes used for denaturing samples.

Use 0.5 mL tubes for denaturing sample or denaturing solution.

Chip or gel-dye mix contaminated with particles.

Prepare new chip with new gel-dye mix. Wear powder-free gloves only. Do not touch the underside of the chip. Do not touch the wells of the chip. Clean the electrodes, see How to Clean the Pin Set of the Electrode Cartridge on page 128. Load the chip immediately after taking it out of its sealed bag.

Probable causes Solution

Sample degraded or contaminated. Always wear gloves when handling chips and samples.

Ladder degraded. Refer to the Kit Guide for proper ladder storage. Optional: Prepare ladder aliquots and use a new aliquot.

Chip prepared with cold reagents. Prepare a new chip. Allow all reagents and reagent mixes to warm up to room temperature for 30 minutes before use. Store chips at room temperature.

Maintenance and Troubleshooting 101

7 Troubleshooting the Protein Application Symptoms (Protein)

Back to Symptoms (Protein) on page 91

Dye agglomerates present in the gel-dye mix.

Use dye concentration according to the Kit Guide. Let the dye warm up to room temperature for 30 minutes before preparing the gel-dye mix. Protect the dye from light during this time. After centrifugation, the gel-dye mix should be taken up from the top of the tube.

Vibration of 2100 Bioanalyzer instrument.

Do not touch the 2100 Bioanalyzer instrument during a run. Remove vibration devices, such as vortexers and vacuum pumps, from bench.

102 Maintenance and Troubleshooting

Troubleshooting the Protein Application 7 Symptoms (Protein)

Missing Upper Marker

Back to Symptoms (Protein) on page 91

Most probable causes Solution

Upper marker was called incorrectly. Check upper marker assignment. Follow instructions for Manual Marker Assignment on page 98.

Improper preparation of sample buffer or denaturing solution.

Refer to the Kit Guide for instructions on storage and preparation of the sample buffer or denaturing solution.

Incompatible sample component. Some components of the buffer, e.g. CHAPS, TFA, etc. may interfere with the upper marker and decrease sensitivity.

See Kit Guide for a list of compatible buffers and buffer compounds. For an updated list, refer to the web site www.agilent.com/genomics/bioanalyzer. If necessary, dilute, dialyze or desalt the sample. It is recommended to perform a serial dilution with water or compatible buffer to determine the optimal dilution.

Diluted samples are too old. Use diluted samples within one day. Store samples at 4C when not in use for more than 1 hour.

Probable causes Solution

Digestion of upper marker by proteases.

Add protease inhibitor cocktails to cell lysate samples.

Improper denaturation of samples. Use fresh sample aliquot. Heat samples with denaturing solution for 5 minutes at 100C Use 0.5 mL tubes for denaturing samples.

Maintenance and Troubleshooting 103

7 Troubleshooting the Protein Application Symptoms (Protein)

Broad Variability of the Lower Marker

Back to Symptoms (Protein) on page 91

NOTE If the lower marker is detected, the assay performance is not affected by lower marker or system peak variability.

Most probable causes Solution

Buffer components of the sample , e.g. salts, detergents, other additives etc. may interfere with the lower marker.

Ionic strength of the sample buffer may affect the lower marker intensity. See Kit Guide for a list of compatible buffers and buffer compounds. For an updated list, refer to the web site www.agilent.com/genomics/bioanalyzer. If necessary, dilute, dialyze or desalt the sample. It is recommended to perform a serial dilution with water or compatible buffer to determine the optimal dilution.

104 Maintenance and Troubleshooting

Troubleshooting the Protein Application 7 Symptoms (Protein)

Missing Peaks

Back to Symptoms (Protein) on page 91

Most probable causes Solution

Gel-dye mix was loaded in the destain well.

Prepare a new chip.

Probable causes Solution

Fingerprint on focusing lens or on the backside of the chip.

Clean lens as described in Cleaning the Lens on page 145. Do not touch the underside of the chip.

Least probable causes Solution

Defective laser. Check the laser stability using the Overview on page 30. If the laser test fails, contact Agilent Technologies at www.agilent.com/genomics/contact.

Autofocus failure. Check autofocus using the Overview on page 30. If autofocus test fails, contact Agilent Technologies at www.agilent.com/genomics/contact.

High voltage power supply defective. Check high voltage stability using the Overview on page 30. If the high voltage stability test fails, contact Agilent Technologies at www.agilent.com/genomics/contact.

Maintenance and Troubleshooting 105

7 Troubleshooting the Protein Application Symptoms (Protein)

Spikes

Back to Symptoms (Protein) on page 91

Most probable causes Solution

Chip, gel-dye mix, destaining solution, or electrodes contaminated.

Prepare new chip with new gel-dye mix and new destaining solution. Wear powder-free gloves only. Do not touch the underside of the chip. Do not touch the wells of the chip. Clean the electrodes as described in Maintenance of the Electrode Cartridge on page 117. Load the chip immediately after taking it out of its sealed bag.

Gel-dye mix or destaining solution not properly prepared.

Refer to the Kit Guide for proper preparation of the gel-dye mix and destaining solution. Let the dye warm up to room temperature for 30 minutes before preparing the gel-dye mix. Protect the dye from light during this time.

Probable Causes Solution

Vibration of 2100 Bioanalyzer instrument.

Do not touch the 2100 Bioanalyzer instrument during a run. Remove vibration devices, such as vortexers and vacuum pumps, from the bench.

106 Maintenance and Troubleshooting

Troubleshooting the Protein Application 7 Symptoms (Protein)

Poor Reproducibility

Back to Symptoms (Protein) on page 91

Most probable causes Solution

Wrong peak alignment. Check lower and upper marker assignment. Follow instructions for Manual Marker Assignment on page 98.

Improper denaturation of sample(s). Use fresh sample aliquot. Heat samples with denaturing solution for 5 minutes at 100C Use 0.5 mL tubes for denaturing samples.

Samples not prepared similarly, i.e. reducing agent (BME or DTT) was not added to all samples.

Refer to the Kit Guide for proper sample reduction.

Dirty electrodes. Thoroughly clean the electrodes as described in Maintenance of the Electrode Cartridge on page 117.

Probable causes Solution

Diluted samples are too old. Use diluted samples within one day.

Incompatible buffer component. See Protein Kit Guide for a list of compatible buffers and buffer compounds. For an updated list, refer to the web site www.agilent.com/genomics/bioanalyzer. If necessary, dilute, dialyze or desalt the sample.

Maintenance and Troubleshooting 107

7 Troubleshooting the Protein Application Symptoms (Protein)

Low Signal Intensity

Back to Symptoms (Protein) on page 91

Most Probable Causes Solution Protein concentration is too low. Follow specifications given in the Kit Guide. Sample salt concentration is too high.

Salt concentration strongly affects the sensitivity of the assay. If necessary dilute, dialyze or desalt the sample. It is recommended to perform a serial dilution with water or compatible buffer to determine the optimal dilution.

Insufficient dissolution of SDS in the dye.

Allow the dye to equilibrate to room temperature for 30 minutes before use. Protect dye from light during this time. Check for undissolved SDS crystals in the tube. Vortex dye well before use. If necessary, heat the sample buffer to 37C for 2 minutes.

Samples were not diluted prior to chip loading

Dilute samples according to protocol given in the Kit Guide.

Probable Causes Solution Improper denaturation of samples. Prepare fresh sample aliquot. Heat sample and denaturating solution for 5 minutes at

100C. Use 0.5 mL tubes for denaturation.

Pipetting error during preparation of reagent mixes.

Check dilution procedure and check calibration of pipette(s).

Least Probable Causes Solution Samples dissolved in acidic buffer. Neutralize samples with appropriate buffer or dilute samples in deionized H2O.

Alternatively, dialyze samples against buffer with medium pH.

108 Maintenance and Troubleshooting

Troubleshooting the Protein Application 7 Symptoms (Protein)

Low Ladder Peaks

Back to Symptoms (Protein) on page 91

Most probable causes Solution

Ladder degraded. Refer to the Kit Guide for proper ladder storage. Optional: Prepare ladder aliquots and use a new aliquot.

Ladder not diluted after denaturing. Refer to Kit Guide for proper chip preparation.

Probable causes Solution

Improper denaturation of ladder. Use fresh ladder aliquot. Heat ladder for 5 minutes at 100C. Use 0.5 mL tubes for denaturation.

Diluted ladder is too old. Use diluted ladder within one day.

Pipetting error during preparation of reagent mixes.

Check dilution procedure and calibration of pipette.

Maintenance and Troubleshooting 109

7 Troubleshooting the Protein Application Symptoms (Protein)

Broad Peaks

Back to Symptoms (Protein) on page 91

Most probable causes Solution Lower and/or upper markers are called incorrectly.

Turn off alignment and check which bands are the correct lower and upper markers. For more details see Manual Marker Assignment on page 98.

Air bubbles at the bottom of the well. Always insert the pipette tip to the bottom of the well when dispensing the liquid. Remove large air bubbles with a pipette tip (small bubbles on top of the well will not affect the assay).

Chip not properly primed. Clogged chip priming station or wrong priming station settings.

Prepare a new chip. Check chip priming station as described in Checking the Chip Priming Station for Proper Performance - Seal Test on page 141. Clean/replace syringe, gasket, and plastic adapter, if necessary. Check if clip and base plate of priming station are in the correct position (see Kit Guide)

Leak currents due to contaminated electrodes.

Clean electrodes with analysis-grade water and a toothbrush, see How to Clean the Pin Set of the Electrode Cartridge on page 128. Do not leave chip in instrument after run. Clean electrodes with the electrode cleaner chip for 10 seconds after each run.

Probable causes Solution Sample was not denatured properly. Use fresh sample aliquot. Heat sample and denaturing solution for 5 minutes at

100C. Samples not prepared similarly, i.e. reducing agent (BME or DTT) was not added to all samples.

Refer to the Kit Guide for proper sample reduction.

110 Maintenance and Troubleshooting

Troubleshooting the Protein Application 7 Symptoms (Protein)

Baseline Dips

Back to Symptoms (Protein) on page 91

NOTE If the lower marker is detected, the assay performance is not affected by dips.

Most probable causes Solution

Sample contains additional detergents and dyes.

See Kit Guide for a list of compatible buffers and buffer compounds. For an updated list, refer to the web site www.agilent.com/genomics/bioanalyzer. If necessary, dilute, dialyze or desalt the sample.

Gel-dye mix or destaining solution not properly prepared.

Refer to the Kit Guide for proper preparation of the gel-dye mix and destaining solution. Let the dye warm up to room temperature for 30 minutes before preparing the gel-dye mix. Protect the dye from light during this time.

Maintenance and Troubleshooting 111

7 Troubleshooting the Protein Application Symptoms (Protein)

Late Migration

Back to Symptoms (Protein) on page 91

Most probable causes Solution

Chip not properly primed. Clogged chip priming station or wrong priming station settings.

Prepare a new chip. Check chip priming station as described in Checking the Chip Priming Station for Proper Performance - Seal Test on page 141. Clean/replace gasket, syringe and plastic adapter, if necessary. Check if clip and base plate of priming station are in the correct position (see Kit Guide)

Protein chips expired. Check expiration date on chip box.

Protein concentration in samples too high.

Use protein concentration accorting to specifications given in the Kit Guide.

Least probable causes Solution

Defective heater plate. Run the temperature test by using the Overview on page 30. If the heater plate is defective, contact Agilent Technologies at www.agilent.com/genomics/contact.

112 Maintenance and Troubleshooting

Troubleshooting the Protein Application 7 Symptoms (Protein)

Error Message: No data received since 5 seconds

Most probable causes Solution

Disrupted communication between instrument and computer.

Please refer to Troubleshooting the Instrument Communication on page 15 for troubleshooting instrument communication issue. Ensure the Agilent USB-Serial Adapter cable, black cable (5188-8031) for 2100 Expert Software version B.02.08 and greater is used to connect the 2100 Bioanalyzer instrument to the computer through a USB port. See USB to Serial Adapter on page 21.

Maintenance and Troubleshooting 113

7 Troubleshooting the Protein Application Symptoms (High Sensitivity Protein)

Symptoms (High Sensitivity Protein)

Click to go straight to the troubleshooting hints.

Saturation of Lower Marker or Sample Peaks Optical Signal too High on page 115

Low Signal Intensity on page 116

114 Maintenance and Troubleshooting

Troubleshooting the Protein Application 7 Symptoms (High Sensitivity Protein)

Saturation of Lower Marker or Sample Peaks Optical Signal too High

Back to Symptoms (High Sensitivity Protein) on page 114

Most probable causes Solution Insufficient dilution of ladder or samples.

Follow instructions in the Kit Guide. Dilution of the labeling reaction by 1:200 is recommended.

Probable causes Solution Chip prepared with cold reagents. Prepare a new chip.

Allow all reagents and reagent mixes to warm up to room temperature for 30 minutes prior to use. Store chips at room temperature.

Maintenance and Troubleshooting 115

7 Troubleshooting the Protein Application Symptoms (High Sensitivity Protein)

Low Signal Intensity

Back to Symptoms (High Sensitivity Protein) on page 114

Most probable causes Solution

Insufficient labeling of ladder or samples.

Follow instructions in the Kit Guide. Labeling occurs between pH 8-9. Proteins must contain lysines for labeling reaction. Check buffer compatibility in the Compatibility List for the Labeling Reaction in the Kit Guide.

Insufficient sample present. Follow instructions for protein concentration in the Kit Guide. Low abundant proteins may require pico labeling (Technical Note 5990-3703EN).

116 Maintenance and Troubleshooting

Maintenance and Troubleshooting

8 Maintenance of the Electrode Cartridge Overview 118 DNA and Protein Assays 119

Cleaning the Electrodes after each DNA and Protein Assays 120 RNA Nano Assay 121

Cleaning the Electrodes before each RNA Nano Assay 122 Cleaning the Electrodes after each RNA Nano Assay 123 RNase Decontamination of the Pin Set 124

RNA Pico or Small RNA Assay 125 Cleaning the Electrodes before each RNA Pico or Small RNA Assay 126 Cleaning the Electrodes after each RNA Pico or Small RNA Assay 127 RNase Decontamination of the Pin Set 127

How to Clean the Pin Set of the Electrode Cartridge 128

117Agilent Technologies

8 Maintenance of the Electrode Cartridge Overview

Overview

The cleaning procedure of the electrode cartridge depends on the assay that is run on the 2100 Bioanalyzer system. For details see How to Clean the Pin Set of the Electrode Cartridge on page 128. Table 4 on page 118 gives an overview on the different cleaning procedures.

Table 4 Maintenance of the Electrode Cartridge

Assay Before each run After each run Monthly or after liquid spill: pin set cleaning

DNA and Protein

Optional: Electrode cleaner: deionized H2O for 10 seconds.

Electrode cleaner: deionized H2O for 10 seconds.

With brush: deionized H2O or isopropanol.

RNA Nano Electrode cleaner: RNaseZAP for 60 seconds. RNase free H2O for

10 seconds.

Electrode cleaner: RNase free H2O for

10 seconds.

RNase decontamination with brush: RNaseZAP. RNase free H2O.

RNA Pico and Small RNA

Electrode cleaner: RNase free H2O for

5 minutes.

Electrode cleaner: RNase free H2O for

30 seconds.

RNase decontamination with brush: RNaseZAP RNase free H2O

NOTE Electrode cleaner should be filled with 350 L of solution (H20 or RNaseZAP).

NOTE Electrode Pinset must be completely dry before adding a chip and initiating a new run.

118 Maintenance and Troubleshooting

Maintenance of the Electrode Cartridge 8 DNA and Protein Assays

DNA and Protein Assays

Assay Before each run After each run Monthly or after liquid spill: Pin set cleaning

DNA and Protein

Optional: Electrode cleaner: deionized H2O for 10 seconds.

Electrode cleaner: deionized H2O for 10 seconds.

With brush: deionized H2O or isopropanol

Maintenance and Troubleshooting 119

8 Maintenance of the Electrode Cartridge DNA and Protein Assays

Cleaning the Electrodes after each DNA and Protein Assays When the assay run is complete, immediately remove the used chip out of the 2100 Bioanalyzer instrument and dispose of it according to good laboratory practice. Then perform the following procedure to ensure that the electrodes are clean and no residues are left over from the previous assay.

1 Slowly fill one of the wells of the electrode cleaner with 350 L deionized analysis-grade water.

2 Open the lid and place electrode cleaner in the 2100 Bioanalyzer instrument.

3 Close the lid and leave it closed for about 10 seconds.

4 Open the lid and remove the electrode cleaner.

5 Wait another 10 seconds for the water on the electrodes to evaporate.

6 Empty the electrode cleaner after every cleaning procedure and refill the electrode cleaner.

7 After 25 Chip runs, replace the used electrode cleaner with a new one.

When After each DNA and Protein run.

Parts required # p/n Description

1 NA Electrode cleaner (required amount included in the kits) 1 NA Deionized analysis-grade water

CAUTION Leak currents between electrodes Liquid spill may cause leak currents between the electrodes.

Never fill more than 350 L of water in the electrode cleaner.

NOTE When switching between different assays, a more thorough cleaning may be required. For details, refer to How to Clean the Pin Set of the Electrode Cartridge on page 128.

120 Maintenance and Troubleshooting

Maintenance of the Electrode Cartridge 8 RNA Nano Assay

RNA Nano Assay

Assay Before each run After each run Monthly or after liquid spill: Pin set cleaning

RNA Nano Electrode cleaner: RNase ZAP for 60 seconds. RNase free H2O for

10 seconds.

Electrode cleaner: RNase free H2O for

10 seconds.

RNase decontamination with brush: RNase ZAP. RNase free H2O.

Maintenance and Troubleshooting 121

8 Maintenance of the Electrode Cartridge RNA Nano Assay

Cleaning the Electrodes before each RNA Nano Assay To avoid decomposition of the RNA sample, follow this electrode decontamination procedure on a daily basis before running any RNA Nano assay.

1 Slowly fill one of the wells of an electrode cleaner with 350 L RNaseZAP. 2 Open the lid and place electrode cleaner in the 2100 Bioanalyzer

instrument. 3 Close the lid and leave it closed for about 1 minute. 4 Open the lid and remove the electrode cleaner - label the electrode cleaner

and keep for future use. You can reuse the electrode cleaner for all chips in one kit.

5 Slowly fill one of the wells of another electrode cleaner with 350 L RNase-free water.

6 Place electrode cleaner chip in the 2100 Bioanalyzer instrument. 7 Close the lid and leave it closed for about 10 seconds. 8 Open the lid and remove the electrode cleaner. Label it and keep it for

further use. 9 Wait another 10 seconds for the water on the electrodes to evaporate before

closing the lid.

When Before each RNA Nano run.

Parts required # p/n Description

2 NA Electrode cleaner (required amount included in the kits) 1 NA RNase-free water 1 NA RNaseZAP (Ambion, Inc cat. no. 9780)

NOTE Perform the following RNase decontamination procedure on a daily basis before running any RNA Nano assays.

CAUTION Leak currents between electrodes Liquid spill may cause leak currents between the electrodes. Never fill more than 350 L of water in the electrode cleaner.

NOTE Remove the RNaseZAP and the RNase-free water from the electrode cleaner at the end of the day.

122 Maintenance and Troubleshooting

Maintenance of the Electrode Cartridge 8 RNA Nano Assay

Cleaning the Electrodes after each RNA Nano Assay When the assay is complete, immediately remove the used chip from the 2100 Bioanalyzer instrument and dispose of it according to good laboratory practice. Then perform the following procedure to ensure that the electrodes are clean and no residues are left over from the previous assay.

1 Slowly fill one of the wells of the electrode cleaner with 350 L RNase free water.

2 Open the lid and place electrode cleaner in the 2100 Bioanalyzer instrument.

3 Close the lid and leave it closed for about 10 seconds.

4 Open the lid and remove the electrode cleaner.

5 Wait another 10 seconds for the water on the electrodes to evaporate.

When After each RNA Nano run.

Parts required # p/n Description

1 NA Electrode cleaner (required amount included in the kits) 1 NA RNase-free water

NOTE Use a new electrode cleaner with each new kit.

CAUTION Leak currents between electrodes Liquid spill may cause leak currents between the electrodes.

Never fill more than 350 L of water in the electrode cleaner.

NOTE Remove the RNase-free water from the electrode cleaner at the end of the day.

Maintenance and Troubleshooting 123

8 Maintenance of the Electrode Cartridge RNA Nano Assay

RNase Decontamination of the Pin Set When the pin set of the electrode cartridge is suspected to be contaminated with RNases follow the instructions described in How to Clean the Pin Set of the Electrode Cartridge on page 128.

124 Maintenance and Troubleshooting

Maintenance of the Electrode Cartridge 8 RNA Pico or Small RNA Assay

RNA Pico or Small RNA Assay

Assay Before each run After each run Monthly or after liquid spill: pin set cleaning

RNA Pico or Small RNA

Electrode cleaner: RNase free H2O for

5 minutes.

Electrode cleaner: RNase free H2O for

30 seconds.

RNase decontamination with brush: RNaseZAP RNase free H2O

Maintenance and Troubleshooting 125

8 Maintenance of the Electrode Cartridge RNA Pico or Small RNA Assay

Cleaning the Electrodes before each RNA Pico or Small RNA Assay To avoid decomposition of the RNA sample, follow this electrode decontamination procedure on a daily basis before running any RNA Pico or Small RNA assay.

1 Slowly fill one of the wells of an electrode cleaner with 350 L RNase-free water.

2 Open the lid and place electrode cleaner in the 2100 Bioanalyzer instrument.

3 Close the lid and leave it closed for 5 minutes.

4 Open the lid and remove the electrode cleaner. Label the electrode cleaner and keep for future use.

5 Wait another 30 seconds for the water on the electrodes to evaporate before closing the lid.

When Before each RNA Pico or Small RNA run.

Parts required # p/n Description

1 NA Electrode cleaner (required amount included in the kits) 1 NA RNase-free water

NOTE To prevent contamination problems, it is strongly recommended to use a dedicated electrode cartridge for RNA Pico and Small RNA assays.

CAUTION Leak currents between electrodes Liquid spill may cause leak currents between the electrodes.

Never fill more than 350 L of water in the electrode cleaner.

126 Maintenance and Troubleshooting

Maintenance of the Electrode Cartridge 8 RNA Pico or Small RNA Assay

Cleaning the Electrodes after each RNA Pico or Small RNA Assay When the assay is complete, immediately remove the used chip out of the 2100 Bioanalyzer instrument and dispose of it according to good laboratory practice. Then perform the following procedure to ensure that the electrodes are clean and no residues are leftover from the previous assay.

1 Slowly fill one of the wells of an electrode cleaner with 350 L RNase-free water.

2 Open the lid and place electrode cleaner in the 2100 Bioanalyzer instrument.

3 Close the lid and leave it closed for 30 seconds.

4 Open the lid and remove the electrode cleaner.

5 Wait another 30 seconds for the water on the electrodes to evaporate before closing the lid.

RNase Decontamination of the Pin Set When the pin set of the electrode cartridge is suspected to be contaminated with RNases follow the instructions described in How to Clean the Pin Set of the Electrode Cartridge on page 128.

When After each RNA Pico or Small RNA run.

Parts required # p/n Description

1 NA Electrode cleaner (required amount included in the kits) 1 NA RNase-free water

CAUTION Leak currents between electrodes Liquid spill may cause leak currents between the electrodes.

Never fill more than 350 L of water in the electrode cleaner.

NOTE Replace the water in the electrode cleaner after each use. Use a new electrode cleaner after 12-13 electrode cleaning procedures and with each new kit.

Maintenance and Troubleshooting 127

8 Maintenance of the Electrode Cartridge How to Clean the Pin Set of the Electrode Cartridge

How to Clean the Pin Set of the Electrode Cartridge

The electrode cartridge, which includes the pin set, can be removed for cleaning.

1 Turn off line power to the 2100 Bioanalyzer instrument. The line switch is located at the rear of the instrument.

When On a monthly basis. Whenever the pin set is contaminated with liquid spill or salt deposition. When the pin set is contaminated with RNases.

Tools required p/n Description

NA Compressed oil-free air OR NA Desiccator

NA Beaker NA Soft brush

Parts required # p/n Description

1 NA Deionized analysis-grade water 1 NA RNase-free water 1 NA Unused chip to run the short circuit diagnostic test.

CAUTION Damage of electrodes and high voltage power supply.

Do not touch the electrodes while the cartridge is in the 2100 Bioanalyzer instrument, this could damage the electrodes and high voltage power supply.

128 Maintenance and Troubleshooting

Maintenance of the Electrode Cartridge 8 How to Clean the Pin Set of the Electrode Cartridge

2 Open the lid and pull the metal lever on the inside left of the lid to the vertical position as shown in Figure 1 on page 129. When the lever is in the vertical position, the cartridge is released from the lid by about 10 mm.

Figure 1 Remove/replace the electrode cartridge

3 Gently pull the cartridge out of the lid as shown in Figure 1 on page 129.

Maintenance and Troubleshooting 129

8 Maintenance of the Electrode Cartridge How to Clean the Pin Set of the Electrode Cartridge

4 Open the bayonet socket of the pin set by turning the plastic lever to the left, see Figure 2 on page 130.

Figure 2 Bayonet socket of the electrode pin set

130 Maintenance and Troubleshooting

Maintenance of the Electrode Cartridge 8 How to Clean the Pin Set of the Electrode Cartridge

5 Remove the cover of the bayonet socket by gently pulling the plastic lever. The pin set may stick to the electrode base. Remove it by carefully pulling it off, see Figure 3 on page 131.

Figure 3 Bayonet cover and pin set

6 Gently brush the pin set with a soft brush in deionized analysis-grade water or isopropanol. In case of RNase contamination, use RNaseZap (Ambion, Inc. cat. no. 9780).

7 In case of highly contaminated or dirty pins, the pin set may be autoclaved or sonicated. For autoclaving or sonicating the pin set, follow standard procedures for plastic material.

8 Rinse pin set thoroughly with deionized analysis-grade water when running DNA or Protein assays, or RNase-free water when running RNA assays.

CAUTION Damage of pin set Bending or misaligning the pins will lead to poor quality results or prematurely terminated assay runs.

Be careful not to bend or misalign the pins.

CAUTION Damp pin set

Make sure that the pin set is fully dry before placing it back into the electrode base. Even small amounts of liquid on the pin set can damage the high voltage power supply.

Maintenance and Troubleshooting 131

8 Maintenance of the Electrode Cartridge How to Clean the Pin Set of the Electrode Cartridge

9 Let the pin set completely dry in a desiccator overnight or use oil-free compressed air.

10 Place the pin set on the cartridge base and the bayonet cover over the pin set, see Figure 3 on page 131.

11 Lock the pin set to the electrode base by turning the plastic lever of the bayonet cover to the right, see Figure 2 on page 130.

12 Slide the electrode cartridge with the pin set into the 2100 Bioanalyzer instrument lid as shown in Figure 1 on page 129 and move the metal lever to the flat (closed) position.

13 Push the metal front of the electrode cartridge to ensure a tight connection to the 2100 Bioanalyzer instrument, see Figure 1 on page 129.

14 To verify that the electrodes are completely dry, perform the Short circuit diagnostic test from the Diagnostics tab in the Instrument context. This test takes approximately three minutes.

15 If the short circuit test fails, the electrode assembly may still be wet. Take the pin set out of the instrument, dry it with oil-free compressed air, then repeat the test.

CAUTION Damage of electrode cartridge Heat can permanently damage the electrode cartridge.

Do not dry the electrode cartridge in an oven.

132 Maintenance and Troubleshooting

Maintenance and Troubleshooting

9 Maintenance of the Chip Priming Station Overview 134 Replacing the Syringe 135 Cleaning the Syringe Adapter 136 Replacing the Syringe Adapter 138 Replacing the Gasket 139 Checking the Chip Priming Station for Proper Performance - Seal Test 141

133Agilent Technologies

9 Maintenance of the Chip Priming Station Overview

Overview

Regular cleaning procedures are necessary to maintain the performance of the chip priming station. The table below gives an overview on the different maintenance procedures.

Procedure Time interval Or if...

Replacing the syringe With each new kit Latest every 3 months

...syringe is broken, see Replacing the Syringe on page 135.

Cleaning the syringe adapter Every 3 months

Replacing the syringe adapter ...adapter is clogged with dried gel or damaged, see Replacing the Syringe Adapter on page 138.

Replacing the gasket Every 3 months ...gasket is damaged, torn or contaminated with dried gel, see Replacing the Gasket on page 139.

Checking the chip priming station for proper seal

Every 4 weeks ...gasket, syringe adapter or syringe was replaced, see Checking the Chip Priming Station for Proper Performance - Seal Test on page 141.

134 Maintenance and Troubleshooting

Maintenance of the Chip Priming Station 9 Replacing the Syringe

Replacing the Syringe

1 Unscrew the old syringe from the top of the chip priming station.

2 Remove clip from the old syringe. Dispose syringe according to good laboratory practices.

3 Slide new syringe into the clip. Ensure syringe and clip are flushed together.

4 Screw the syringe tight into the luer lock adapter.

5 Check the priming station as described in Checking the Chip Priming Station for Proper Performance - Seal Test on page 141.

When Quarterly or whenever it is clogged.

Parts required # p/n Description

1 NA Syringe kit that comes with each DNA, RNA and Protein kit 1 NA Deionized water

Maintenance and Troubleshooting 135

9 Maintenance of the Chip Priming Station Cleaning the Syringe Adapter

Cleaning the Syringe Adapter

1 Open the priming station.

2 Move the mounting ring holding the adapter in place to the left as shown in Figure 4 on page 136. The ring will come off.

Figure 4 Mounting ring of the syringe adapter

When Quarterly or whenever it is clogged.

Parts required # p/n Description

1 NA Syringe kit that comes with each DNA, RNA and Protein kit 1 NA Deionized water

136 Maintenance and Troubleshooting

Maintenance of the Chip Priming Station 9 Cleaning the Syringe Adapter

3 Press the syringe adapter out of its mount as shown in Figure 5 on page 137.

Figure 5 Removing/replacing the syringe adapter

4 Remove dried gel at the opening of the adapter with a needle. 5 Screw on syringe and flush water through the adapter several times. 6 Flush syringe with isopropanol. 7 Allow adapter to dry fully. 8 Insert the syringe adapter, see Figure 5 on page 137. 9 Follow the steps as described in Cleaning the Syringe Adapter on

page 136 to reassemble the priming station. 10 Close the chip priming station. 11 Screw a dry syringe tight into the luer lock adapter. 12 Check the priming station as described in Checking the Chip Priming

Station for Proper Performance - Seal Test on page 141.

Maintenance and Troubleshooting 137

9 Maintenance of the Chip Priming Station Replacing the Syringe Adapter

Replacing the Syringe Adapter

1 Follow the steps described in Cleaning the Syringe Adapter on page 136 to remove the syringe adapter.

2 Dispose the old syringe adapter.

3 Insert the syringe adapter, see Figure 5 on page 137.

4 Follow the steps as described in Cleaning the Syringe Adapter on page 136 to reassemble the priming station.

5 Check the priming station as described in Checking the Chip Priming Station for Proper Performance - Seal Test on page 141.

When If significantly clogged and unable to clean thoroughly.

Parts required # p/n Description

1 G2938-68716 Gasket kit

138 Maintenance and Troubleshooting

Maintenance of the Chip Priming Station 9 Replacing the Gasket

Replacing the Gasket

The silicone gasket, see Figure 6 on page 139, ensures a tight connetion between the chip and syringe adapter.

Figure 6 Syringe adapter with gasket

1 Remove the syringe adapter out of the chip priming station as described in Replacing the Syringe Adapter on page 138.

When Quarterly or when it is torn.

Parts required # p/n Description

1 G2938-68716 Gasket kit

Maintenance and Troubleshooting 139

9 Maintenance of the Chip Priming Station Replacing the Gasket

2 Pull out the old silicone gasket with your fingers or tweezers. See Figure 7 on page 140 for a disassembled adapter.

Figure 7 Syringe adapter with disassembled gasket

3 Insert a new silicone gasket and gently push into place.

4 Insert the syringe adapter into the chip priming station as described in Replacing the Syringe Adapter on page 138 and reassemble the priming station.

5 Check the priming station as described in Checking the Chip Priming Station for Proper Performance - Seal Test on page 141.

140 Maintenance and Troubleshooting

Maintenance of the Chip Priming Station 9 Checking the Chip Priming Station for Proper Performance - Seal Test

Checking the Chip Priming Station for Proper Performance - Seal Test

1 Make sure the syringe is tightly connected to the chip priming station.

2 Pull the plunger of the syringe to the 1.0 mL position (plunger pulled back).

3 Place an unused chip in the chip priming station.

4 Close the chip priming station. The lock of the latch will audibly click when it closes.

5 Press the plunger down until it is locked by the clip. This is shown in Figure 8 on page 141.

Figure 8 Locking the plunger of the syringe with the clip

When Every month or whenever a component of the priming station (syringe, adapter or gasket) was replaced.

Parts required Description

Unused DNA or RNA Chip

Maintenance and Troubleshooting 141

9 Maintenance of the Chip Priming Station Checking the Chip Priming Station for Proper Performance - Seal Test

6 Wait for 5 seconds and lower latch of the clip to release the plunger as shown in Figure 9 on page 142.

Figure 9 Releasing the plunger from the syringe

7 To indicate an appropriate sealing, the plunger should move back up at least to the 0.3 mL mark within less than 1 second.

NOTE If the plunger does not move up to the 0.3 mL mark within a second, the syringe-chip connection is probably not tight enough. Retighten the syringe or replace the syringe adapter, syringe or gasket to fix the problem.

142 Maintenance and Troubleshooting

Maintenance and Troubleshooting

10 Maintenance of the Agilent 2100 Bioanalyzer instrument Overview 144 Cleaning the Lens 145 Changing the Fuses 146

143Agilent Technologies

10 Maintenance of the Agilent 2100 Bioanalyzer instrument Overview

Overview

The 2100 Bioanalyzer instrument should be kept clean. Cleaning should be done with a damp lint-free cloth. Do not use an excessively damp cloth allowing liquid to drip into the 2100 Bioanalyzer instrument. The following table gives an overview on the different 2100 Bioanalyzer instrument maintenance procedures:

WARNING Pathogenic, toxic, or radioactive samples Handling and use of pathogenic, toxic, or radioactive samples and of genetically modified organisms holds risks for health and environment.

Ensure that all necessary safety regulations, guidelines, precautions and practices are adhered to accordingly.

Consult the laboratory safety officer for advise on the level of containment required for the application, and proper decontamination or sterilization procedures to follow if fluids escape from containers.

Procedure Time Interval Or if...

Cleaning the Lens on page 145 latest every 3 months ...lens is contaminated with liquid spill or noticeably dirty

Changing the Fuses on page 146 n/a ...status indicator is off and the cooling fan is not running

144 Maintenance and Troubleshooting

Maintenance of the Agilent 2100 Bioanalyzer instrument 10 Cleaning the Lens

Cleaning the Lens

Liquid spill may reduce the light throughput of the focusing lens underneath the chip. To avoid low intensity signals due to absorbent coatings on the lens, follow the procedure below.

1 Switch off the instrument. The line switch is located at the rear of the 2100 Bioanalyzer instrument.

2 Open the lid of the instrument.

3 Dampen a lens tissue with isopropanol and gently swab the surface of the lens. Repeat several times with clean tissues and alcohol each time.

4 Wait for alcohol to evaporate before use.

When Quarterly or after liquid has been spilled on the lens.

Parts required # p/n Description

1 NA Reagent-grade isopropanol 1 NA Lens tissue

CAUTION Damaging the instrument Liquid dripping into the instrument could cause a shock or damage the instrument.

Do not allow liquid to drip into the 2100 Bioanalyzer instrument.

Maintenance and Troubleshooting 145

10 Maintenance of the Agilent 2100 Bioanalyzer instrument Changing the Fuses

Changing the Fuses

1 Switch off the instrument. The line switch is located at the rear of the 2100 Bioanalyzer instrument.

2 Disconnect the power cable from the power input socket.

When If the status indicator is off and the cooling fan is not running.

Tools required p/n Description

NA Screw driver

Parts required # p/n Description

2 2110-0007 fuses 1A, 250 V

CAUTION Disconnect the 2100 Bioanalyzer instrument from line power before changing a fuse.

Use Agilent recommended fuses only.

146 Maintenance and Troubleshooting

Maintenance of the Agilent 2100 Bioanalyzer instrument 10 Changing the Fuses

3 To access the fuse drawer, gently lift the outer plastic housing of the power inlet socket using a screw driver, see Figure 10 on page 147.

Figure 10 Remove power inlet housing 4 Pull out the fuse drawer as shown in Figure 11 on page 147.

Figure 11 Remove fuse drawer

Maintenance and Troubleshooting 147

10 Maintenance of the Agilent 2100 Bioanalyzer instrument Changing the Fuses

5 Replace the two fuses.

6 Slide in the fuse drawer and push till it fits tightly as shown in Figure 12 on page 148.

Figure 12 Insert fuse drawer

NOTE Perform this procedure with care.

148 Maintenance and Troubleshooting

Maintenance of the Agilent 2100 Bioanalyzer instrument 10 Changing the Fuses

7 Close the fuse drawer housing (see Figure 13 on page 149), reconnect the instrument to the power line and switch it on.

Figure 13 Close fuse drawer housing

Maintenance and Troubleshooting 149

10 Maintenance of the Agilent 2100 Bioanalyzer instrument Changing the Fuses

150 Maintenance and Troubleshooting

Maintenance and Troubleshooting

11 Maintenance of the Vortexer Changing the Adapter 152

151Agilent Technologies

11 Maintenance of the Vortexer Changing the Adapter

Changing the Adapter

1 Release the 4 screws on top of the adapter.

2 Hold the base of the vortex mixer and pull up the head. Discard the old head according to good laboratory practices.

3 Place the new head adapter on the vortex mixer.

4 Insert and fix the 4 screws with the screw driver.

When Whenever the vortex adapter is damaged.

Tools required p/n Description

N/A Screw driver

Parts required # p/n Description

1 IKA 3428300 IKA vortex mixer adapter (for MS 3 vortexers) NOTE: This part must be purchased directly from IKA.

152 Maintenance and Troubleshooting

Maintenance and Troubleshooting

12 Spare Parts and Accessories Overview 154

153Agilent Technologies

12 Spare Parts and Accessories Overview

Overview

Overview on page 154 provides a list of spare parts and accessories that are available for the 2100 Bioanalyzer system.

To buy parts, please refer to the Agilent Online Store: www.agilent.com/genomics/bioanalyzer

Reorder number Part Description

5185-5990 Filters for gel matrix Extra filters for gel matrix. Contains 25 spin filters for the electrophoresis assays.

5188-8031 USB-serial adapter cable Connects RS232 cables to USB PC ports (for PCs without serial ports).

RS232-6101 RS232 cable Communication cable between PC and instrument.

2110-0007 Fuse Fuse for power supply.

5065-9951 Electrode cleaner kit Contains 7 electrode cleaners for the maintenance of the electrode cartridge.

5065-4401 Chip priming station Includes gasket kit and adjustable clip.

G2938-68716 Gasket kit Contains spare parts for chip priming station: 1 adapter, 1 mounting ring and 10 gaskets.

5042-1398 Adjustable clip For use with luer lock syringe.

5065-4413 Electrode cartridge Removable cartridge with detachable 16-pin electrode assembly for easy cleaning. For use with electrophoresis assays. NOTE: electrode pin set is not sold separately.

G2938-68300 Test chip kit for electrophoresis

Comprises 1 autofocus and 1 electrode/diode chips.

IKA 3428300 Vortex mixer adapter For IKA MS3 vortexer (must be ordered through IKA).

154 Maintenance and Troubleshooting

Index

Index

2 2100 expert software 16

A additional peaks

DNA 43 protein 101 RNA 74

artefact peaks high sensitivity DNA 63

B baseline

DNA 52, 53, 50, 51 protein 111 RNA 83, 79

bioanalyzer icons 16 bioanalyzer 14, 30, 144 broad peaks

DNA 49 RNA 80

C chip not detected

DNA 42 protein 95 RNA 72

chip priming station 10, 134, 141 chips 13 COM port 18, 20 contamination 9

D degraded RNA 76 demo port 20 diagnostic test 32 DNA

symptoms 62, 37 troubleshooting 36

E electrode cartridge 118 electrode cleaner 9 electrode cleaning

DNA 119 electrode cleaning

protein 119 RNA 125, 121

error message DNA 61 Protein 113 RNA 85

essential measurement practices 7

F fuse drawer 147 fuse 146

G gasket 10, 138, 139 gel-dye 11 gel 11

H handling

chips 13 gel-dye 11 gel 11 reagents 11 samples 12

hardware diagnostics 30 head adapter 152 high sensitivity DNA 62 high sensitivity protein 114

I installation qualification 26 instrument context 16

L ladder

protein 109 lens 145 license 18 log book 36 lower marker

protein 104 luer lock 135, 137

M marker assignment

DNA 58 protein 98

migration DNA 54 protein 112 RNA 84

missing peaks DNA 47

Maintenance and Troubleshooting 155

Index

RNA 81 mounting ring 136

N next generation sequencing 62

O optical signal

protein 115

P peak tailing

DNA 56 peaks broad

protein 110 peaks missing

protein 105 peaks

high sensitivity DNA 63 pin set cleaning 128 pipette tips 9 plunger 141 poor chip performance 74, 97 power inlet socket 147 power switch 16 proteases 92 protein

symptoms 91, 114 troubleshooting 90

Q quantitation

DNA 39, 38 protein 93, 92 RNA 71, 70

R reagents 11

reproducibility protein 107

RNA fragment 82 RNA pico 75 RNA

symptoms 69 troubleshooting 68

RNAse contamination 124, 127 RS232 cable 18, 20 run aborted 41, 73, 96 run log 36 run time

DNA 57 protein 97

S salt concentration 12 samples 12 saturation

protein 115 RNA 75

seal test 141 sensitivity

DNA 45 high sensitivity protein 116 protein 108 RNA 78

short circuit diagnostic test 132 signal intensity

DNA 45 high sensitivity protein 116 protein 108 RNA 78

sizing DNA 40 protein 94

small RNA 75 spikes

DNA 44

protein 106 RNA 77

split peaks high sensitivity DNA 64

status indicator 16 syringe adapter 136, 138, 139 syringe clip 10 syringe 9, 135

T test chips 30 tools and handling 9 troubleshooting

DNA 36 protein 90 RNA 68

U upper marker

DNA 48 protein 103

V validation 26 verification 26 vortexer adapter 152

156 Maintenance and Troubleshooting

Index

Maintenance and Troubleshooting 157

www.agilent.com

In this Book

This manual provides maintenance and troubleshooting information for the Agilent 2100 Bioanalyzer system. It includes essential measurement practices, troubleshooting hints for hardware, software and applications, maintenance procedures and a list of spare parts and accessories.

This manual is based on the 2100 Exp

Manualsnet FAQs

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