LabChip. User s Manual. Copyright Caliper Life Sciences, Inc. All rights reserved. P/N Rev. 0

Transcription

1 LabChip 90 User s Manual Copyright Caliper Life Sciences, Inc. All rights reserved. P/N Rev. 0

2 Caliper Life Sciences, Inc. Product Warranty 2 Caliper Life Sciences, Inc. Product Warranty I. INSTRUMENTS Caliper Life Sciences, Inc. ( Caliper ) warrants your Caliper-manufactured instrument s hardware and firmware against defects in material and workmanship for a period of one (1) year from the date of shipment, subject to the exclusions set forth below and: (i) the warranty for TurboVap products shall be ninety (90) days from the date of shipment, (ii) the warranty for Limited-Life Parts (as defined below) shall be thirty (30) days from the date of installation, and (iii) the warranty for cosmetic surfaces shall be thirty (30) days from the date of installation (each, a Warranty Period ). Ongoing service support after the Warranty Period may be available at an additional expense. A. What is Included during the Warranty Period: unlimited emergency on-site repair services 1, parts and software updates that affect original functional design specifications, their associated labor and travel expenses. unlimited access to Caliper s Technical Support Center, which provides troubleshooting, repair instruction, service dispatching (other than for TurboVap and Twister I), replacement part information and shipment. one pre-scheduled, on-site Preventive Maintenance and Diagnostic Servicing (PM) performed by a Caliper Field Service Engineer 1. A PM visit includes the replacement of a pre-defined list of Limited-Life Parts exposed to fluidics, such as syringes, valves, seals and fittings. The Caliper Field Service Engineer will perform comprehensive analysis and testing to confirm that the equipment is left operating to factory specifications. Scheduling of the PM within the Warranty Period is flexible and determined by the customer after notification by the Caliper Field Service Engineer. The servicing is typically scheduled six months after the equipment installation, but equipment usage rate and availability may cause the date to vary. A completed Caliper Field Service Report provides thorough documentation of all maintenance and service work performed by the Caliper Field Service Engineer during an on-site visit. Documentation is not provided when Caliper provides service via telephone, fax or . Any failure of a product to conform to this Warranty shall be corrected by replacing or repairing the affected product or refunding the purchase price (as described below), in each case at Caliper s option. Parts replaced during the Warranty Period will be covered for the remaining term of the original Warranty Period, or for thirty (30) days from time of replacement, whichever is longer. Such replacement parts may, at Caliper s option, be new or remanufactured. All parts removed from warranted equipment become the property of Caliper. Caliper reserves the right to satisfy its warranty obligations in full by refunding the purchase price of any non-conforming product, minus any service, validation, or travel charges. B. Customer Responsibilities In order for a product to be covered under this Warranty, Customer must comply with the following terms: The equipment must be used under normal installation and application conditions as described in the product s User Manual. The equipment must be maintained as described in the User Manual. Only water or DMSO at a maximum concentration of 65% may be used as a system fluid in the Sciclone inl10. Any other system fluid must be approved by Caliper before use in the inl10. Customer is responsible for making the equipment available for a PM visit during the Warranty Period. Caliper will not be held liable for a missed PM visit due to restricted access to the equipment during the Warranty Period. When Caliper provides telephone, fax, or support, Customer is responsible for completing any necessary documentation of the service. If Customer maintains a change control/validation logbook as a permanent record, then Customer is responsible for entering all service documentation into such logbook. Customer must perform the appropriate level of revalidation required as a result of the maintenance or service provided. C. Exclusions Failure to comply with any of the Customer Responsibilities listed above will void this Warranty. Any alteration of hardware or software on products covered under this Warranty that are not performed by Caliper or an approved Caliper vendor will void this Warranty. A product that has been subject to misuse, accident, negligence or improper transportation, handling, installation, storage, use, or maintenance is not covered under this Warranty. Many Caliper products require the use of Caliper Automation Certified Disposables for proper operation. These may include, but are not limited to: pipet tips, seals, labels and filters. Use of a Caliper product with any disposables other than the specified Caliper Automation Certified Disposables will void this Warranty. This Warranty covers equipment manufactured by Caliper. Equipment purchased from other vendors is not covered by this Warranty. Damage to Limited-Life Parts caused by insufficient maintenance or cleaning practices or unauthorized applications are not covered under this Warranty. This Warranty applies only to the original buyer and delivery location. It is not transferable to other buyers or locations without Caliper s prior written approval. 1. TurboVap and Twister I are not eligible for on-site service or a PM visit, and must be returned to Caliper s Repair Depot for warranty service pursuant to the process set forth in Section E below. P/N Rev. 1 Caliper Life Sciences, Inc.

3 Caliper Life Sciences, Inc. Product Warranty 3 The Sciclone 384-channel low-volume head is warranted for one (1) year or 750,000 aspirate or dispense movements, whichever comes first. The Sciclone 100nL head is warranted for one (1) year or 600,000 aspirate or dispense movements, whichever comes first. The laser component of the LabChip 3000 is warranted for the earlier of one (1) year from the date of installation or 8,000 hours of use. The use in a Sciclone inl10 of DMSO above 65% concentration, or any other system fluid not sanctioned for use by Caliper, will invalidate this warranty as it relates to the pipetting head assembly. If Caliper performs service on equipment and determines that any of the exclusions set forth in this Warranty apply, then Caliper shall charge Customer its then-current current list prices for parts, labor and travel. D. Limited-Life Parts Limited-Life Parts are any parts that are exposed to solvents, reagents, or samples. Such parts include, but are not limited to syringes, valves, seals and fittings. A pre-defined list of Limited-Life Parts are routinely replaced by Caliper Field Service Engineers during a PM visit or during Caliper Repair Depot servicing. Otherwise, these parts are available from Caliper at current list prices and are designed for replacement by Customer. E. Equipment Return Policy In servicing situations requiring the return of equipment to Caliper, equipment must be returned to Hopkinton, MA, USA, or another facility designated by Caliper. Customer shall prepay shipping charges for equipment returned to Caliper, and Caliper will pay for return shipment to Customer. A Returned Material Authorization (RMA) must be obtained for any equipment being returned to Caliper. Contact the Caliper Technical Support Center by telephone at (508) , or via the Internet at techsupport@caliperls.com or by fax at (508) before returning any equipment to Caliper. Customer must complete a Caliper Chemical Questionnaire prior to the issuance of an RMA. All equipment returned to Caliper must first be decontaminated to meet Caliper and United States Department of Transportation procedures and standards for the safety of Caliper personnel. F. Hazardous Limitation Statement At no time will Caliper personnel perform service on unsafe equipment, perform service in unsafe environments or decontaminate equipment to make it safe. Prior to performing any service work, Caliper personnel will evaluate the condition of the equipment and the environment in which the equipment is located. If Caliper determines that the equipment and/or the environment could be hazardous to Caliper personnel, Caliper reserves the right to refuse to service the equipment. II. MICROFLUIDIC CHIPS Caliper warrants that microfluidic chips (each, a Chip ) purchased from Caliper by Customer will be free from defects in material and workmanship for a period of sixty (60) days from the date of shipment (the Warranty Period ). A defect for purposes of this Warranty is defined as any failure that occurs during analysis of the first one hundred (100) samples being run on a Chip. During the Warranty Period, if the Chip fails to comply with this Warranty, Caliper will repair or replace the Chip at its option and expense. If a Chip becomes damaged or its performance otherwise deteriorates due to solvents and or reagents other than those supplied or expressly recommended by Caliper, Caliper will replace the Chip at Customer s request and expense. No such replacement will extend the original Warranty Period. This Warranty does not extend to any Chip which has been (a) the subject of an accident, misuse, or neglect, (b) modified by a party other than Caliper, (c) used in a manner not in accordance with the instructions contained in the product User s Manual, or (d) used for an assay or application which has not been approved by Caliper. All claims under this Warranty must be made within thirty (30) days of the discovery of the defect. Caliper s obligations under this Warranty are limited to replacement as Caliper deems necessary to correct those failures of the Chip to comply with this Warranty of which Caliper is notified prior to expiration of the Warranty Period. III. GENERAL CALIPER SPECIFICALLY DISCLAIMS ANY AND ALL OTHER PROMISES, REPRESENTATIONS, AND WARRANTIES, EXPRESSED OR IMPLIED. THIS INCLUDES, BUT IS NOT LIMITED TO, THE PRODUCT S CONDITION, ITS CONFORMITY TO ANY DESCRIPTION OR REPRESENTATION, THE EXISTENCE OF ANY LATENT OR PATENT DEFECTS, AND IMPLIED WARRANTIES OF MERCHANTABILITY AND FITNESS FOR A PARTICULAR PURPOSE. IN NO EVENT WILL CALIPER BE LIABLE, WHETHER ARISING IN CONTRACT, TORT (INCLUDING NEGLIGENCE), STRICT LIABILITY OR OTHERWISE, FOR ANY LOSS OF PROFIT OR ANY OTHER COMMERCIAL DAMAGES, INCLUDING, BUT NOT LIMITED TO, SPECIAL, INCIDENTAL, CONSEQUENTIAL, OR OTHER AND IN NO EVENT SHALL CALIPER S LIABILITY EXCEED THE PURCHASE PRICE OF THE PRODUCT. THERE ARE NO ORAL STATEMENTS, PROMISES, REPRESENTATIONS OR OTHER WARRANTIES COLLATERAL TO OR AFFECTING THIS WARRANTY. THIS WARRANTY EXTENDS TO BUYER ONLY AND NOT TO PURCHASERS OR USERS OF BUYER S PRODUCTS. No agent, employee, or representative of Caliper has any authority to bind Caliper to any affirmation, representation, or warranty concerning a Caliper product that is not contained in Caliper s printed product literature or this Warranty. Any such affirmation, representation, or warranty made by any agent, employee, or representative of Caliper will not be binding on Caliper. P/N Rev. 1 Caliper Life Sciences, Inc.

4 Caliper Life Sciences, Inc. Software License Agreement 4 Caliper Life Sciences, Inc. Software License Agreement Thank you for purchasing our products. PLEASE READ THE TERMS AND CONDITIONS OF THIS LICENSE AGREEMENT CAREFULLY BEFORE OPENING THE PACKAGE CONTAINING THE SOFTWARE MEDIA, THE COMPUTER SOFTWARE CONTAINED THEREIN, AND THE ACCOMPANYING USER DOCUMENTATION (COLLECTIVELY REFERRED TO HEREIN AS THE "PROGRAM"). THE PROGRAM IS COPYRIGHTED AND LICENSED, NOT SOLD. BY OPENING THE PACKAGE CONTAINING THE PROGRAM, YOU ARE ACCEPTING AND AGREEING TO THE TERMS OF THIS LICENSE AGREEMENT. IF YOU ARE NOT WILLING TO BE BOUND BY THE TERMS OF THIS LICENSE AGREEMENT, YOU SHOULD PROMPTLY RETURN THE PROGRAM IN UNOPENED FORM, AND YOU WILL RECEIVE A REFUND. THIS LICENSE AGREEMENT REPRESENTS THE ENTIRE AGREEMENT CONCERNING THE PROGRAM BETWEEN YOU AND CALIPER LIFE SCIENCES, INC. (REFERRED TO AS "LICENSOR"), AND IT SUPERSEDES ANY PRIOR PROPOSAL, REPRESENTATION, OR UNDERSTANDING BETWEEN YOU AND LICENSOR. 1 License Grant. Licensor hereby grants to you, and you accept, a nonexclusive license to use the Program only as authorized in this License Agreement. The software included in the Program (the "Software") may be used only on a single computer owned, leased, or otherwise controlled by you; or, in the event of the inoperability of that computer, on a backup computer selected by you. If the Software is an applications package, you may use it on a single, local system controller. Neither concurrent use on two or more computers nor use in a local area network or other network is permitted without written authorization from licensor and the payment of additional license fees. You agree that you will not assign, sublicense, transfer, pledge, lease, rent, or share your rights under this License Agreement, except that you may permanently transfer all of your rights under this License Agreement, provided that you retain no copies, you transfer all of the Program, and the transferee agrees to the terms of this License Agreement. You agree that you may not reverse assemble, reverse compile, or otherwise translate the Software. Upon loading the Software into your computer, you may retain the Software media for backup purposes. In addition, you may make one copy of the Software on a second medium for the purpose of backup in the event the Software media is damaged or destroyed. Any such copies of the Software shall include Licensor's copyright and other proprietary notices. Except as authorized under this paragraph, no copies of the Program or any portions thereof may be made by you or any person under your authority or control. 2 Licensor s Rights. Licensor has invested significant time and money in developing the products purchased with the Program. The Program contains Licensor's trade secrets, which must be used in confidence. You acknowledge and agree that the program is proprietary to Licensor and protected under U.S. copyright law. You further acknowledge and agree that all right, title, and interest in and to the Program, including associated intellectual property rights, are and shall remain with Licensor. This License Agreement does not convey to you an interest in or to the Program, but only a limited right to use it, revocable in accordance with the terms of this License Agreement. In the event of a breach, the Licensor reserves the right to seek equitable relief. 3 License Fees. The license fees paid by you in connection with the purchase of the associated product(s) are paid in consideration of the licenses granted under this License Agreement. 4 Term. This License Agreement is effective upon your opening of this package and shall continue until terminated. You may terminate this License Agreement at any time by returning the Program, and all copies thereof and extracts therefrom to Licensor. Licensor may terminate this License Agreement upon the breach by you of any term hereof. Upon such termination by Licensor, you agree to return to Licensor the Program and all copies and portions thereof. 5 Limited Warranty. Licensor warrants, for your benefit alone, for a period of 90 days from the date of the commencement of this License Agreement (referred to as the "Warranty Period") that the Software Media in which the Software is contained are free from defects in material and workmanship. If, during the Warranty Period, a defect in the Software Media arises, you may return the Program to Licensor for either replacement or, if so elected by Licensor, refund of amounts paid by you under this License Agreement. You agree that the foregoing constitutes your sole and exclusive remedy for breach by Licensor of any warranties made under this License Agreement. EXCEPT FOR THE WARRANTIES SET FORTH ABOVE, THE PROGRAM IS LICENSED "AS IS," AND THE LICENSOR DISCLAIMS ANY AND ALL OTHER WARRANTIES, WHETHER EXPRESS OR IMPLIED, INCLUDING, WITHOUT LIMITATION, ANY IMPLIED WARRANTIES OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE. 6 Limitation of Liability. Licensor's cumulative liability to you or any other party for any loss or damages resulting from any claims, demands, or actions arising out of or relating to this License Agreement shall not exceed the license fee paid to Licensor for the use of the Program. In no event shall Licensor be liable for any indirect, incidental, consequential, special, or exemplary damages or lost profits, even if Licensor has been advised of the possibility of such damages. SOME STATES DO NOT ALLOW THE LIMITATION OR EXCLUSION OF LIABILITY FOR INCIDENTAL OR CONSEQUENTIAL DAMAGES, SO THE ABOVE LIMITATION OR EXCLUSION MAY NOT APPLY TO YOU. P/N Rev. 3 Caliper Life Sciences, Inc.

5 Caliper Life Sciences, Inc. Software License Agreement 5 7 Software Updates. Licensor periodically creates Software updates which will be made available to you at no charge. Thereafter, Licensor shall not be responsible for your use of, and shall not warrant or repair, superseded or outdated versions of Software. 8 Governing Law. This License Agreement shall be construed and governed in accordance with the laws of the Commonwealth of Massachusetts. Any dispute will be resolved in the state and federal courts of the Commonwealth of Massachusetts. 9 Severability. Should any term of this License Agreement be declared void or unenforceable by any court of competent jurisdiction, such declaration shall have no effect on the remaining terms hereof. 10 No Waiver. The failure of either party to enforce any rights granted hereunder or to take action against the other party in the event of any breach hereunder shall not be deemed a waiver by that party as to subsequent enforcement of rights or subsequent actions in the event of future breaches. P/N Rev. 3 Caliper Life Sciences, Inc.

6 Preface 6 Copyright Trademarks Content Preface Proper Equipment Operation This manual is published by Caliper Life Sciences, Inc., 68 Elm Street, Hopkinton, MA USA. Copyright , Caliper Life Sciences, Inc. All rights reserved. Reproduction by any means or in any form of this manual or the products it describes is prohibited. Caliper, LabChip, and the LabChip logo are registered trademarks of Caliper Life Sciences, Inc. The Caliper logo is a trademark of Caliper Life Sciences, Inc. Microsoft, Windows, and Windows NT are either registered trademarks or trademarks of Microsoft Corporation in the United States and/or other countries. All other trademarks and registered trademarks are the property of their respective holders. The information in this manual may contain typographical errors or technical inaccuracies and is subject to change without notice. Modifications may also be made to the product described in this manual at any time. WARNINGS Contact Us To reduce the risk of electric shock, do not remove the cover. No user serviceable parts are inside. Refer to qualified service personnel if help is required. Use this product only in the manner described in this manual. If the equipment is used in a manner not specified by the manufacturer, the protection provided by the equipment may be impaired. If you have a question about a product that is not answered in this manual or online Help, or if you need assistance regarding this product, please contact the Caliper Technical Support Center from 8:00 A.M. to 8:00 P.M., Eastern Time, Monday through Friday: Phone: (508) ; LabChip for LabChip products only Fax: (508) techsupport@caliperls.com Internet: For support in Europe contact Caliper Life Sciences LTD, Runcorn, UK or fax For more information contact your local Caliper representative. Before you call, you should have the following information available for the technical representative: Product serial number Software version (found by choosing About from the main Help menu) If applicable, the error code reference number shown on the product s LCD display or from the database event log.

7 Preface 7 Product Service and Customer Support Plans Training For Your Product CE Caliper offers a full range of services to ensure your success. From our original factory warranty through a comprehensive line of customer support plans, Caliper offers you Field Service Engineers and in-house Specialists who are dedicated to supporting your hardware, software and application development needs. Call: (508) Fax: (508) techsupport@caliperls.com Our programs can include such useful services as: Preventive maintenance Diagnostic servicing performed on-site by Caliper field service engineers or remotely via Technical Support Validation performed on-site by Caliper field service engineers Extended use of the Caliper Technical Support Center Software updates Parts, labor, and travel expense coverage Other customized services upon request Contact the Caliper Center for Training and Development for information about the availability of training courses for your product: Call: (508) Fax: (508) This device complies with all CE rules and requirements. NOTE Changes or modifications to this equipment not expressly approved by the party responsible for compliance could void the user s authority to operate the equipment.

8 Preface 8 Table of Symbols Table 1 contains symbols that identify particularly important information and alert you to the presence of hazards. These symbols may appear in this manual and/or on the product it describes. Table 1. Important Symbols Symbol Description DANGER: An imminently hazardous situation, which, if not avoided, will result in death or serious injury. WARNING: Caution, risk of danger. Refer to the User s documentation. NOTE: A cautionary statement; an operating tip or maintenance suggestion; may result in instrument damage if not followed. Hazardous voltage; risk of shock injury. Crush hazard. Risk of body parts, hair, jewelry, or clothing getting caught in a moving part. Risk of eye injury; wear safety glasses. Risk of fire. Risk of poison. Hazardous fumes. Laser light; avoid exposure. Risk of eye injury. Lifting hazard. May result in injury. Protective ground symbol. Ground symbol. Fuse. Alternating current. On (supply).

9 Preface 9 Table 1. Important Symbols (Continued) Symbol Description Off (supply). CE compliance mark. Signifies that the unit has passed safety tests for grounding, power line transience, and current leakage. Input. Output. Equipment labels are color coded: Yellow Caution, risk of danger Red Stop Blue Mandatory action Green Safe condition or information Helpful hints, additional information

10 Preface 10 Safety Chemical Hazard Warning WARNING CHEMICAL HAZARD. Some of the chemicals used with Caliper s instruments are potentially hazardous and can cause injury or illness. Read and understand the material safety data sheets (MSDSs) provided by the chemical manufacturer before you store, handle, or work with any chemicals or hazardous materials. Minimize contact with and inhalation of chemicals. Wear appropriate personal protective equipment when handling chemicals (e.g., safety glasses, gloves, or clothing). For additional safety guidelines consult the MSDS. Do not leave chemical containers open. Use only with adequate ventilation. Check regularly for chemical leaks or spills. If a leak or spill occurs, follow the manufacturer s cleanup procedures as recommended on the MSDS. Comply with all local, state/provincial, or national laws and regulations related to chemical storage, handling, and disposal. Chemical Waste Hazard Warning WARNING CHEMICAL WASTE HAZARD. Wastes produced by Caliper Life Sciences instruments are potentially hazardous and can cause injury or illness. Read and understand the material safety data sheets (MSDSs) provided by the manufacturers of the chemicals in the waste container before you store, handle, or dispose of chemical waste. Handle chemical wastes in a fume hood. Minimize contact with and inhalation of chemical waste. Wear appropriate personal protective equipment when handling chemicals (e.g., safety glasses, gloves, or clothing). After emptying the waste container, seal it with the cap provided. Dispose of the contents of the waste tray and waste bottle in accordance with good laboratory practices and local, state/provincial, or national environmental and health regulations.

11 Preface 11 About MSDSs Ordering MSDSs Laser Hazard Some of the chemicals used with this instrument may be listed as hazardous by their manufacturer. When hazards exist warnings are prominently displayed on the labels of all chemicals. Chemical manufacturers supply a current MSDS before or with shipments of hazardous chemicals to new customers and with the first shipment of a hazardous chemical after an MSDS update. MSDSs provide you with the safety information you need to store, handle, transport and dispose of the chemicals safely. We strongly recommend that you replace the MSDSs in your files each time you receive one packaged with hazardous chemicals. CHEMICAL HAZARD. Be sure to familiarize yourself with the MSDSs before using reagents or solvents. To order an MSDS for a chemical, call the chemical manufacturer. The AMS 90 SE/LabChip 90 instrument contains a diode laser with an output power nominally below 4.85 mw in the wavelength range nm. WARNING Use of controls or adjustments or performance of procedures other then those specified herein may result in hazardous radiation exposure. The following safety label is located on the AMS90SE/LabChip 90 Optics Module: Laser Safety Label Class 1 Laser Certification information is noted on the instrument identification label. This label can be found on the pedestal under the optics module.

12 Preface 12 Before Operating the Instrument Ensure that everyone involved with the operation of the instrument has: Received instruction in general safety practices for laboratories Received instruction in specific safety practices for the instrument Read and understood all related MSDSs

13 Table of Contents 13 Table of Contents Caliper Life Sciences, Inc. Product Warranty... 2 Caliper Life Sciences, Inc. Software License Agreement... 4 Preface... 6 Description of the System Hardware Overview Hardware Components Optics Module Chip Holder Plate Carrier Robot LabChip HT Software with PC Pressure Controller High-Voltage Controller Robot Controller Enclosure Safety Guidelines and Warnings General Warnings Specific Warnings Power System Main Power Switch Guidelines for Shutting Down and Powering Up Instrument Physical Specifications Description of the System Software Overview The LabChip HT Main Window Title Bar Menu Bar Tool Bar Plate and Ladder Stripwell Icons File and Assay Information Area Large Display Slab Gel View Results Table Small Display Error Message Area Status Bar Assay Properties Window Barcode Reader Window Edit Samples Window Export and Auto Export Windows File Properties Window Open Assay Window... 69

14 Table of Contents 14 Open Data File Window Options Window Data Files Tab Reader Tab Advanced Tab Print and Auto Print Windows Pressure Control Window Sample Information Tabs Sample Tab (for DNA Assays) Sample Tab (for Protein Assays) Sample Tab (for RNA Assays) Settings Tab Results Tab ErrorsTabs Start Window Saving Data and Assay Files Information About Your Computer Using Help Types of Help Available Preparing the System for Operation Overview Principles of Operation LabChip Kit and Instrument Guidelines General Reagents Chips Samples Preparing the System for Operation Aligning the Robot Preparing the Chip Preparing the Sample Plate Preparing Buffer and Ladder Strip Wells Installing the Chip, Sample Plate, and Strip Wells Installing the Chip Installing the Sample Plate Installing the Strip Wells Operating the System Overview Running the Assay Continuing the Read on a Previous Plate Preparing the System for Shutdown Storing the Chip Cleaning the Electrodes Data Analysis Overview How the Software Analyzes DNA Data

15 Table of Contents 15 How the Software Analyzes Protein Data How the Software Analyzes RNA Data Modifying Analysis Parameters Changing the Filtering Parameters Changing the Peak Find Parameters Changing the Time Window for Analysis Assigning Upper and Lower Marker Peaks for DNA Assays Aligning or Unaligning the Marker Peaks Assigning the Lower Marker Peak in a Protein or RNA Assay Using Expected Fragments/Expected Proteins Entering EFs and EPs Using the Edit Samples window Entering EFs and EPs Using the Sample Information Tab Viewing the EFs and EPs Changing the View of the Results Graph Overlay Changing to Gel View Forcing the Baseline to Zero Reanalyzing a Data File Using Sample Templates Data Handling and Printing Overview Organizing, Retrieving, and Backing-Up Data Files General Guidelines Printing a Report Printing Automatically at the End of a Run Printing Manually at Any Time Report Options Assay Summary Report Gel-Like Report Electropherogram Report The Combined Results Table Copying Information Choices for Copying Image Size Copying the Results Table Exporting Data To Export Files Automatically To Export Files Manually Maintenance and Service Overview Maintenance and Service Cleaning the Electrodes Cleaning the Electrode Block Cleaning the Priming Station Lid Aligning the Robot Removing a Capillary Blockage

16 Table of Contents 16 Troubleshooting Overview Troubleshooting the DNA Assay Additional Peaks or Spikes - DNA Broad Peaks - DNA Cross Contamination - DNA Fast Migrating Peaks - DNA Late Migrating Peaks - DNA Loss of Peak Resolution - DNA Low Peak Sensitivity - DNA Missing Peaks - DNA No Peaks and High Background Fluorescence - DNA No Peaks and Low Background Fluorescence - DNA Noisy Electropherogram - DNA Poor Chip Performance Detected - DNA Quantification Results Incorrect - DNA Sizing Results Incorrect - DNA Unstable Baseline - DNA Troubleshooting the Protein Assay Additional Ladder or Sample Peaks - Protein Baseline Humps - Protein Broad Peaks - Protein Cross Contamination - Protein Dips - Protein High Baseline Fluorescence - Protein Low Ladder Peaks - Protein Low Sample Peaks - Protein Missing Peaks - Protein Poor Chip Performance Detected - Protein Poor Reproducibility - Protein Quantitation Results Incorrect - Protein Spikes - Protein Wrong Alignment of Ladder - Protein Sizing Results Incorrect - Protein Troubleshooting the RNA Assay Missing Peaks - RNA Migration Time Shift - RNA Hardware Error Messages Poor Chip Performance Chip Not Detected Door Open Poor Focus Detected Current Leak Detected Software Error Messages Cannot Save File No Assays Listed on Assays Menu Fewer Than Two Valid Peaks

17 Table of Contents 17 General Error Instrument Calibration Error Instrument Connection Error Instrument Error During an Assay Run Marker Peaks Cannot Be Aligned Problem with the Ladder Data Recovering From Software Crashes Glossary Index

18 Description of the System Hardware 18 Description of the System Hardware Overview This chapter provides an overview of the LabChip 90 system features and components. Topic See Page Hardware Components 19 Safety Guidelines and Warnings 23 Power System 24 Instrument Physical Specifications 25

19 Description of the System Hardware 19 Hardware Components Optics Module Within the metal housing are the laser, lens, and detector. The laser is disabled when the chip holder is opened. Located at the bottom of the optics module is the electrode cartridge. This cartridge contains seven electrodes that fit into the wells of the chip. WARNING Use of controls or adjustments or performance of procedures other than those specified herein may result in hazardous radiation exposure. Enclosure Optics module Robot Plate carrier Pressure Controller High-voltage Controller Robot Controller Figure 1. LabChip 90 Front View

20 Description of the System Hardware 20 Chip Holder The chip holder fastens beneath the electrode cartridge under the optics module. When the latch is released, the holder is able to drop and swing out for chip loading and unloading. The holder contains a recessed space designed to fit the chip. The capillary fits through a hole at the base of the holder, enabling it to sip from the wells of the microplate. The holder is keyed so that the chip cannot be loaded incorrectly. Chip holder Holder latch Figure 2. Chip Holder Plate Carrier The plate carrier contains a platform that holds one 96- or 384-well sample plate and two strip wells that hold a wash buffer and control sample strip well. Buffer strip well Ladder strip well A1 Position Plate Platform Figure 3. Plate Carrier

21 Description of the System Hardware 21 Robot The plate carrier is attached to the robot. The robot uses three highspeed, high-precision translation stages to load and unload the sample plate. WARNING MOVING PARTS HAZARD. Physical injury can result from contacting moving parts of the System. To prevent injury, the enclosure door must be closed during operation. The robot is disabled when the door opens. LabChip HT Software with PC Pressure Controller High-Voltage Controller Robot Controller The LabChip HT software provides pressure, current, voltage, and robot control for the instrument. The pressure controller provides regulated pressure to the chip using a syringe pump. Lights on the front panel and corresponding indicator in the software show the status of power to the unit and its run condition. This unit supplies controlled electrical current or voltage to the electrodes that fit into wells of the chip. Lights on this unit and a corresponding indicator in the software show the power status as well as indicators for Ready and Run. The high-voltage light comes on when high voltage is being delivered to the electrodes. This unit provides three-axis robotic control through the PC. A power light shows the status of main power to the unit. The Enable button can be pressed to disable software control of the robot. Pressing this button stops a run in progress, allowing it to be used as an emergency stop button. While the power light and the enable button appear to be very similar, the power light is an indicator only and cannot be pressed. The Enable button can be pressed to enable or disable the robot.

22 Description of the System Hardware 22 Enclosure The enclosure covers the work area of the robot and controllers and contains the robotic arms. A sensor attached to the door of the enclosure determines whether it is open or closed. If you open the enclosure door during a run, all robotic movements stop, aborting the run in progress if one is running. The door must be closed to operate the system.

23 Description of the System Hardware 23 Safety Guidelines and Warnings General Warnings The instrument contains FAST MOVING PARTS, HIGH-VOLTAGE components, and potentially DANGEROUS LIGHT SOURCES. Use caution when operating the system and its subsytems. WARNING ELECTRICAL SHOCK HAZARD. Severe electrical shock, which could cause physical injury or death, can result from contacting parts of this System when the high-voltage power supply is operating. To avoid electrical shock, disconnect the power supply to the System, unplug the power cord, and wait at least 1 minute before moving the System. WARNING MOVING PARTS HAZARD. Physical injury can result from contacting moving parts of the System. To prevent injury, the enclosure door must be closed during operation. The robot is disabled when the door opens. WARNING Specific Warnings Do not mix reagents or prepare chips and plates in the instrument. The instrument contains a low voltage laser that is housed in the detection chamber. This laser is set to automatically turn off when the chip holder is disengaged. NOTE If any of the components have lost power or are malfunctioning, contact Caliper Technical Support immediately. Do not reset fuses or otherwise work on the system before contacting Caliper. See Contact Us on page 6.

24 Description of the System Hardware 24 Power System Main Power Switch The main power switch on the instrument is located on the power strip on the back on the system. Guidelines for Shutting Down and Powering Up General Recommendations Powering Down the System System Power Up If the system is used on a regular basis, it is recommended that you leave the main power switch on all the time. If the system needs to be moved, please contact Caliper Life Sciences. To power down the system: 1 Close the LabChip HT software. 2 From the desktop, click Start and then select Shut Down. Select Shutdown the computer when prompted. Wait until you see a message indicating that it is safe to turn off the computer, then power-down the computer. 3 Turn off the main power switch on the power strip located on the back of the instrument. To restore power to the system: 1 Turn on the main power switch located on the back of the instrument. 2 Turn on the computer. 3 Open the LabChip HT software.

25 Description of the System Hardware 25 Instrument Physical Specifications Specification Value / Measurement Plate format 96- and 384-well Dimensions 147 cm (58 in) H x 57 cm (22.5 in) W x 61 cm (24 in) D Weight 136 kg (300 lbs) Power requirements V AC, 47/63 Hz Power consumption 360 VA (360 W) Operating humidity 5 85% relative, noncondensing range Operating temperature C ( F) range For indoor use only. Use up to altitudes of 2000 M. Use in Pollution Degree 2 environments. Considered Installation Category II. NOTE Maximum relative humidity 80% for temperatures up to 31 C, decreasing linearly to 50% relative humidity at 40 C.

26 Description of the System Software 26 Description of the System Software Overview This chapter provides a detailed description of the features and functions of the LabChip HT software. Topic See Page The LabChip HT Main Window 27 Assay Properties Window 53 Barcode Reader Window 63 Edit Samples Window 64 Export and Auto Export Windows 66 File Properties Window 68 Open Assay Window 69 Open Data File Window 70 Options Window 71 Print and Auto Print Windows 74 Pressure Control Window 75 Sample Information Tabs 76 Start Window 80 Using Help 83

27 Description of the System Software 27 The LabChip HT Main Window Title bar Menu bar Tool bar Start button File Information Assay Information Plate and Ladder Display Large Display Small Display Error Display Status bar Figure 4. LabChip HT Main Window The main window of the LabChip HT software includes a title bar, menu bar, tool bar, plate and ladder display, file information, assay information, large display area, small display area, error area, and status bar. Title Bar The title bar displays the name of the application on the left and the name of the assay or data file on the right. Dragging the title bar repositions the window on the screen. The buttons at the right end of the title bar can be used to minimize the window, maximize the window, or to close the window.

28 Description of the System Software 28 Menu Bar The menu bar is directly below the title bar. Clicking a menu name displays a list of commands to access software functions. The contents of these menus are described below. File Menu The File menu contains the following commands: Item Open Save Save As... Export Print Page Setup XX... Properties 1 10 Recent Files Exit Description Opens a previously saved data file. Saves an unsaved or changed data file. Saves a data file under a new name. Opens the Export window to choose the type of data to export. Opens the Print window to choose the items to send to the printer. Opens the Page Setup window to change or review the settings for the current printer and to choose a different printer. Opens the File Properties window for the current data file. The data files (up to 10) that were opened most recently (the most recent file is labeled 1). Closes the software. If you have unsaved data, you are prompted to save it before closing.

29 Description of the System Software 29 Edit Menu The Edit menu contains the following commands: Item Copy Gel Copy Graph Copy Results Table Edit Samples Description Copies a large gel picture (such as would be seen if you viewed a large gel display) to the computer's clipboard with the lane labels as part of the graphic. You can paste this gel picture into another document, such as a word processing, graphics, or other program file. The size of the image that is placed on the clipboard when copying a graph depends on the display mode at the time you choose this command. If you choose Copy Graph from the Edit menu while viewing a single well display, a large-sized graphic (the same size as that shown in the large display) of the well graph is placed on the clipboard. If you are viewing a gel in the large display and choose this command, a graphic that is the size of the small well graph is placed on the clipboard. Copying the results table causes ASCII information to be placed on the clipboard. See Results Table Example on page 30 for an example of results table data that was copied from a sample DNA assay. Opens the Edit Samples window. See Edit Samples Window on page 64 for more information.

30 Description of the System Software 30 Results Table Example DNA_ _ Sample 1 Peak Mig.Time (secs) Height Area Size(BP) Conc. (ng/ml) Molarity 7(nmol/L) The first line shows the name of the saved data file followed by the name of the sample from which the results table data came. The second line provides the headers for the rest of the information which includes the peak number, the migration time in seconds, the peak area, the size of the peaks in base pairs, the concentration in nanograms per microliter, and the molarity in nanomoles per liter, followed by upper and lower marker information in the last column.

31 Description of the System Software 31 View Menu The View menu contains the following commands: Item Single Well All Wells View Gel View Slab Gel Previous Well Next Well Gel Color Standard Curve Focus Data Description Shows the single well view (of the selected well) in the large display. Shows all 12 wells in the large display. Shows the gel in the large display. Shows all of the plate data in a simulated gel view. In any display, decrements the view to the previous well or lane. If you are viewing Sample 1, clicking this button (or pressing the left arrow button on the keyboard) takes you to the ladder well/lane. If you are viewing the ladder well, clicking this button (or pressing the left arrow button on the keyboard) takes you to the last (sample 12) well/lane. In any display, increments the view to the next well or lane. If you are viewing the ladder, clicking this button (or pressing the right arrow button on the keyboard) takes you to Sample 1. If you are viewing sample 12, clicking this button (or pressing the right arrow button on the keyboard) takes you to the ladder well/lane. This pullout menu presents different color choices for viewing a gel in the large display. The colors are designed to approximate various actual gel staining and imaging techniques. Blue on White, for example, simulates a Coomassie gel often used with proteins. The Pseudo color choice provides more detail (1,280 colors) since it maps the signal into a larger color space than is available with the other monochrome options (256 levels of brightness). Opens the Standard Curve window, allowing you to view the ladder as a curve with a point-topoint fit. Opens the Focus Data window that displays the measured fluorescence vs. time for that well.

32 Description of the System Software 32 Graph Menu The Graph menu contains the following commands: Item Limit Scaling to Sample Scale to Selected Scale All Undo Zoom Undo Zoom Completely Peak Info Show Data Points Show Marker Label Show Found Fragments Show Found Proteins Show Gridlines Copy Graph Description When this is checked, wells are scaled to the largest sample peak in the well. Marker and/or system peaks are ignored. Sets current graph to full scale and scales all other graphs to the same as the selected well. Scales each well to itself allowing all of the peaks to be visible. Holding down the Shift key and choosing Scale All causes all of the wells to be scaled relative to each other. Undoes the last zoom in (returns to the previous zoom level). Useful if you have zoomed in quite far and wish to zoom out only a little. Doubleclicking in the display performs the same action. Returns to the standard (unzoomed) view of the single well. Specifies the information that is shown in the Results Table of the single well display. The default is Peak Number. (Not available for RNA assays.) Enables/disables the display of the data points used to generate the graph. Data points are visible only in the single-well display. Enables/disables the display of marker arrows that indicate the Lower Marker peak, Upper Marker peak and the selected sample peak (in the results box of Single Well view). Enables/disables the display of arrow markers and gel-lane markers that indicate found peaks (entered as expected fragments in the Edit Samples window). Enables/disables the display of arrow markers and gel-lane markers indicating the expected proteins that were found during the run. Displays grid lines on the graph in the large display. The grid lines mark finer increments as you zoom in on the graph. Copies the graph displayed onto the clipboard for pasting into another application.

33 Description of the System Software 33 Assay Menu The Assay menu includes the default assays that are shipped with the LabChip HT software, as well as any assays that have been created and saved in the Assays folder and subfolders. The Assay menu contains the following commands: Item dsdna Description HT-DNA K/5K/15K/SE30/SE55 The dsdna assays included with the LabChip HT software are designed for separation of DNA. The parameters for this assay can be edited and the assay saved as a new assay for future use. Protein HT Protein 100/200 The protein 100/200 assays included with the LabChip HT software are designed for the separation of proteins. The protein 100 assay should be used for sizing proteins 100 kda and smaller. The protein 200 assay should be used for sizing proteins smaller than 200 kda. The HT-Protein Express 100/200 High Sensitivity scripts provide 2X the sensitivity of the standard scripts, but the sizing is less robust to varying buffer conditions. RNA HT RNA Std Sens/High Sens/ Std Sens mrna/std Sens Prokaryote The RNA Std Sens assays are intended for use with 2uL of RNA sample with concentration range of 25 to 250ng/uL. The RNA High Sens assay gives higher sensitivity, but requires 6uL of RNA sample in the range of 5 to 50ng/uL. Demos Demo HT-DNA 5000 The demo assay is included as an example of data from a DNA assay. Demo HT Protein Express The demo assay is included as an example of the type of data from a protein assay. Demo HT RNA Eukaryote Total RNA This demo assay is included as an example of the type of data from an RNA assay.

34 Description of the System Software 34 Item xx... Properties Open Assay... Save Assay Save Assay As... Start/Stop Description This menu shows the name of the current assay. Choosing this command opens the Assay Properties window that displays the settings used to determine the peaks/fragments and other settings required for analysis. The settings on the first three tabs of this window are not changeable by the user. Settings on the last two tabs, Global Peak Find and Alignment are changeable. Opens the Open window, allowing you to open an assay that is not currently shown in the Assays menu (residing in another location). When you are working with an assay that you have created or altered previously (not one of the assays shipped with the software) and you made an additional change to the properties, choose this command to save the assay file with the updated properties. This command is not availableif the current assay has not been changed. Choosing this command while using one of the default software assays displays a warning message that the assay is read-only, and then opens the Save As window. Opens the Save As window to save the assay as it is currently configured under a different assay name. Opens the Start window to enter information about the run. Clicking Start in this window starts the run. The Start window also allows you to change the file prefix that is used to automatically name the data file. When an assay is in progress, this menu item changes to Stop.

35 Description of the System Software 35 Tools Menu The Tools menu contains the following commands: Item Turn Off/On Analysis View Log File Sipper Alignment Pressure... Load Plate Unload Plate Options... Description The default is analysis on which causes the marker peaks run with the samples to be aligned to marker peaks in the ladder. Choosing this command turns analysis off which removes the marker peak alignment. Two types of log files are maintained by the LabChip HT software: System Log Maintains a running record of all events that occurred with the instrument while it is online with the software (including the dates and times that the system went on and offline, the software version history, etc.). Run Log Includes information about the particular run including the date and time of the run, any problems that occurred, the assay that was used to generate the data, and the name for the saved data file. There are three options to choose from: Last Alignment Opens a message box displaying information about the last alignment files that were created. Alignment Wizard Opens the Sipper Alignment Wizard, which leads you through the procedure to create a new alignment file. Select Alignment File Opens the Select Alignment File window and allows you to browse and open a saved alignment file. Opens the Pressure window. Moves the plate carrier into the instrument. Moves the plate carrier into position to allow you to remove the plate from the instrument (enabled only after a plate is loaded). Opens the Options window that contains three tabs: Data Files, Reader and Advanced.

36 Description of the System Software 36 Help Menu The Help menu contains the following commands: Item Contents and Index About LabChip HT... Description Opens the Content/Index page for the LabChip HT Help file. Opens the About LabChip HT window, showing the software version number, hardware versions, etc.

37 Description of the System Software 37 Tool Bar Each button on the tool bar represents a menu command (and is a shortcut to activating that command). The tool bar contains the following buttons: Button Name Description Open Opens the Open Data File window (see page 70) to open a previously saved data file. Save Saves the currently open data file. Raw data cannot be changed; only the items that affect how that data is displayed are resaved. Print Opens the Print window to choose the elements to print. View Single Well Switches to a view of the selected well in the large display. View 12-Well Switches to a view of all 12 wells in the large display. View Gel Switches to a view of a simulated gel in the large display. Click the small arrow to the right of this button to open a drop-down menu allowing you to choose the coloring of the simulated gel image. View Slab Gel Opens a new window. The upper portion of this window displays a view of a simulated gel in a large display for the whole plate. If the data does not fit in the window, you can scroll horizontally. In the lower part of this window, you are able to change the alignment settings and gel width and able to print, export or copy the slab gel as it appears on-screen. Turn Off/On Analysis Scale to Selected Well Scale All Wells Turns analysis (alignment of markers to the ladder) off and on (toggle). The state of this setting is saved, even when the instrument is powered down, until you change it again. Sets the current graph to full scale and scales all other graphs to the same scale as the selected well. Causes all of the data to be visible within the individual well windows by scaling each well to itself. If you hold down the Shift key while clicking this button or choosing this command, each well is scaled to the well data having the highest peak.

38 Description of the System Software 38 Button Name Description Undo Zoom Undo Zoom Completely Show/Hide Data Points Show/Hide Marker Label Show/Hide Found Fragments/ Proteins Select Previous/ Select Next Well Select Previous/ Select Next Row Plate and Ladder Stripwell Icons If you have zoomed in for a closer view of a single well, this tool zooms out to the previous zoom level. This is useful when you have made multiple zooms. If you have zoomed in for a closer view of a single wells, this tool zooms out completely and returns to the standard view. Drop-down menu showing labeling options for the single-well view graph. Turns the display of data point on and off for singlewell view. Enables/disables the display of arrows that mark the Lower Marker peak, Upper Marker peak and currently selected peak (in the Single Well View). Enables/disables the display of arrows that mark the expected peaks found (listed in the Edit Samples window). Disabled for RNA assays. On the single-well display, allows you to move backward and forward through the wells. On the single-well display, allows you to move upward or downward through the wells. A microplate and ladder stripwell graphic is displayed on the left side of the window. The icon of the microplate is more than a picture: the well that is currently selected is shown with a circle around it; click a different well on the plate and the rest of the displays update to reflect the new well choice. The ladder stripwell to the left of the microplate shows all wells containing fluid. Regardless of which row is being read, the system always sips from the topmost well to obtain a ladder. The virtual sipping display shown indicates which of the rows is being read when the ladder sipping occurs. In a protein assay, additional data for the ladder well LI is shown. Before a plate is run, all wells selected to run in the microplate icon are blue. After reading, wells are shown in white. During a run, the well that is currently being assayed blinks.

39 Description of the System Software 39 File and Assay Information Area The file and assay information areas are located above the large display and show the following information: Left side - Data File information: Name of the data file (if saved, otherwise the area on the left is blank) Date and time the data file was created. Right Side - Assay File information: The name of the assay file that generated the data If an error occurs with data collection during an assay, a red circle with an X in it displays to the left of the data file information. A third line of data file information displays the type of error that occurred. Click the red error circle to view possible causes for the problem and suggested solutions. You can add or amend notes for the run by accessing the chip notes section of the Data File Properties window: double-click the filename shown above the large display. The Data File Properties window also contains a button allowing you to view the Run Log for that data file. Double-click the assay name shown on the right side above the large display to open the Assay Properties window. The tab labeled Global Peak Find in this window allows you to change the peak find settings.

40 Description of the System Software 40 Large Display Large 12-Well Graph View This area of the workspace shows a 12-well view (default) of the data assayed from one row of the microplate (the row containing the currently selected well, visible in the microplate icon below the Start button), a single-well display of the selected well, or a gel view. When the 12-well or single-well views are selected, the small display area shows the gel view and vice versa. This is the default view when you start the LabChip HT software. As data is acquired, the selected well (Sample 1, in the example above) increments to the well that is currently being run, and the data displays in real time. The figure above shows the large display after all data for the run has been collected. To see a single-well view, either select a well and click the singlewell display button in the tool bar, or double-click the desired well in the 12-well view. You can change the names of the sample wells to any names you wish. To change a name, highlight the existing name and type over it. When the file is saved, the new well names are saved with the file.

41 Description of the System Software 41 Large Single-Well Graph View Peak Number is the default peak label for the single-well display of DNA and Protein assays. The labels for RNA assays can only be selected when the Settings tab is selected in the lower right corner of the window. To change the information displayed, select the desired label in the Graph and Peak Info list on the tool bar. The available options are: No Peak Info Peak Number Peak Time-Center Peak Height Peak Area Peak Size (DNA and Protein) Peak Concentration (DNA) Peak Molarity (DNA) Peak Rel. Concentration (Protein) Holding the cursor over a peak in the Single-Well Graph View for displays the selected peak information. RNA Assays display the RNA Fragment label when any tab other than the Settings tab is selected.

42 Description of the System Software 42 Large Single-Well Graph View (Continued) The lower and upper markers are displayed in the single-well view with large green arrows labeled LM and UM respectively. The currently selected peak in the Results Table displayed a small blue arrow above the peak. Click the Show Marker Label button on the tool bar to display or hide these markers. When the large display shows a single well, you can zoom in to see the data in the graph more closely. Drag the mouse in a rectangle that bounds the area you want to view in more detail. This area enlarges to fill the large display area. You can continue to zoom in until the display reaches its limit of magnification. The dividing line between the graph and the Results Table can be moved vertically, giving more or less space to the Results Table. This allows you to view all of the results at once in wells having several peaks. You can also overlay the graphs from more than one well in a single-well display. To learn more about this feature, see Graph Overlay on page 122.

43 Description of the System Software 43 Large Gel View In this view, the gel that is typically shown in small format on the left side of the window is displayed in the large display and a small single well graph is shown on the left side of the window. One lane of the large gel view is surrounded by a box. This is the selected lane (one lane is always selected) that corresponds to a selected well in the plate icon; the small display shows the well graph corresponding to the selected lane. Clicking a different lane selects that lane and the small display updates to show the new well graph. When a new run is started, the gel display is blank and the first lane, which corresponds to the ladder well, is selected. As data is acquired in the first and subsequent lanes/wells containing samples, the selection box increments to show the well and lane that is currently acquiring data. If you select a lane/well that is earlier in sequence than the current well, the display no longer increments as new data is acquired but remains on the selected lane/well. The top of the large-gel view shows the name of the ladder used for the alignment of the lanes and the names of the samples.

44 Description of the System Software 44 Large Gel View (Continued) Holding the cursor over a gel in the large display displays detailed information next to a crosshair icon (see Figure 5). For a DNA assay, the numbers shown represent the base pair measurements for the area of the lane beneath the tip of the pointer. If the cursor is positioned over a recognized band, the concentration and molarity are also shown (see the example below). For a protein assay, the numbers shown represent the molecular size (kda) for the area beneath the center of the crosshair. If the crosshair is positioned over a recognized band, the molecular size, concentration (mg/ml) and % purity information are also displayed. For RNA assays, the fragment name, fragment area, and % of total RNA area are displayed. Figure 5. DNA, Protein and RNA Gel Views The display of the gel can be changed to a number of different color combinations. Select the desired color using the arrow to the right of the gel button in the tool bar or using the View menu.

45 Description of the System Software 45 Slab Gel View The top part of this window displays the slab-gel image. The options selected in the Assay Properties window are used when displaying the large slab-gel view. The horizontal scroll bar displays if the contents of the gel view are too large to display completely on the window. Alignment The alignment options available here are same ones available in the Alignment Tab of the Assay Properties window. In addition, you can turn off the alignment by selecting Alignment Off. Click Apply to apply your changes and to view them on the window.

46 Description of the System Software 46 Slab Gel View (Continued) NOTES The alignment feature is not active for protein assays. If all lanes are aligned to one ladder, the ladder is shown as the first lane and is always visible to allow comparison of any lane to the ladder. Changes made to the alignment performed in the Assay Properties window are saved in the assay portion of the dataassay file. Changes to the alignment performed in the Slab Gel window are saved in the data section of the data file. For the Slab Gel window, changes in the data section take precedence over changes in the assay section. View Gel Width can adjust the width of the gel lanes. To see a gap between gel lanes, checkmark Show Gap between Gel Lanes. Changes to these settings are visible immediately. Sample Information This area displays the data file name and assay type. Export Button Clicking this button opens the Export window, which is used to export the slab gel picture. Copy Button Clicking this button copies the currently displayed slab gel to the clipboard. It does not copy the data and assay information onto the clipboard. Print Button Clicking this button opens the Print window, which allows you to print the currently viewed slab gel. It also prints additional information such as the data file, assay, barcode, read/modified date, LabChip HT version and copyright.

47 Description of the System Software 47 Results Table The Results Table displays below the single well view in the large display area. The results table displays the data in the electropherogram in numeric format. The Results tables for DNA and Protein display similar data and are described below. The Results table for RNA assays display different data and are described in Results Table for RNA Assays on page 49. Results table for DNA and Protein Assays Figure 6. Results Table for DNA and Protein Assays The Results table for DNA and Protein assays displays the following information: Peak Number Order in which the peaks were detected. The letters EF and EP identify the expected fragments and expected proteins respectively. See Using Expected Fragments/Expected Proteins on page 118 for more information. Mig. Time (seconds) Amount of time from injection to the peak apex in seconds. Height Value at the apex of the peak minus the local baseline start value. Area Peak area is calculated as the sum of the parallelograms of a point-to-point fit down to absolute zero minus the parallelogram of the local baseline down to absolute zero. If the sample has been aligned, the area of the aligned peaks is reported.

48 Description of the System Software 48 Results table for DNA and Protein Assays (Continued) Size: DNA Assay (bp) The fragment size as calculated from a sizing curve generated by the known size of the ladder peaks (in base pairs). These peak sizes are located in the Ladder tab of the Assay Properties window for the ladder. Protein Assay (KDa) The protein size as calculated from a sizing curve generated by the known size of the ladder peaks (in kda). These peak sizes are located in the Ladder tab of the Assay Properties window for the ladder. Concentration (DNA Assay (ng/µl)) The concentration calculated relative to the ladder and marker peak concentrations. The ladder concentration is located in the Analysis tab of the Assay Properties window. Rel. Concentration (Protein Assay (µg/ml)) The concentration entered in the Analysis tab of the Assay Properties window (µg/ml), the same for all ladder peaks. % Purity (Protein Assay) The quantity of protein, expressed as a percentage of the total protein, found in a particular peak. Molarity (nmol/l)(dna assays only) where molarity is measured in nanomoles per liter (nmol/l). Observation The expected fragments/proteins found and their size in base pairs (BP) or kilodaltons (kda). See Edit Samples for more information on expected fragments. For protein assays, the system peak (fluorescence peak due to a buffer constituent) is automatically identified and displayed in the observation column.

49 Description of the System Software 49 Results Table for RNA Assays Figure 7. Results Table for RNA Assays The Results table for RNA assays displays the following information: Fragment Order in which the peaks were detected. Name The name of the fragment based on its size falling within a certain range set in the assay definition. (These definitions are visible only to assay developers). Start Time (seconds) Amount of time from injection to the leading edge of the fragment in seconds. End Time (seconds) Amount of time from injection to the falling edge of the fragment in seconds. Fragment Area Fragment area is calculated as the sum of the trapezoids between the fragment start and end times. The trapezoid base is a line drawn across the fragment base from the start point to the end point. % of Total Area The area of the RNA fragment divided by the total area. The total RNA area is computed using trapezoidal integration of the electropherogram from the end of the lower marker to the baseline end time. The baseline for the total area is a straight line drawn from the baseline start time to the baseline end time. The height of the baseline at these points is the average signal value over a 5 second region around these points.

50 Description of the System Software 50 Small Display This area of the workspace shows either a gel view of the data (default) or a single-well display, if the large display is showing a gel view. Small Gel View One lane of the small gel view is outlined with a box. This is the selected lane, which corresponds to a selected well in the plate icon (or in the 12-well display). Click on a different lane to select it and, if a single well is shown in the large display, the large display updates to show the currently selected well data. When a new run begins, the display is blank and the first lane/ladder well is selected. As data is acquired in lanes/wells, the selection rectangle around the lane increments to show the lane that is currently acquiring data. If you select a lane/well that is earlier in sequence than the current well, however, the display no longer increments as new data is acquired but remains on the selected lane/well. Small Graph View When you select a gel view in the large display, the small display shows a single-well view of the data in the selected well.

51 Description of the System Software 51 Error Message Area The area of the window just above the Status Bar is where most error messages are displayed. Clicking the error icon button opens a message relating to the error, allowing you to view additional information about the problem and how to correct it. Error messages can result from hardware or software problems. Most are the result of peaks not being located by the analysis algorithms of the software. This can be due to a sample or ladder peak not appearing as expected. The software settings (in the Global Peak Find tab or the individual well settings) can also cause peaks to go undetected, which can cause errors. Additionally, manually excluding a peak from analysis (from the Results Table) or changing the start or end times for a run can cause errors with the peak find algorithm.

52 Description of the System Software 52 Status Bar The Status Bar is located at the bottom of the LabChip HT main window and displays information about the current status of the instrument. The status bar is divided into the following sections: Action Area Progress Bar Sample Count Online Status Power Pressure Auto Export Status Auto Print Status Mode When the software is ready to run an assay, the action area of the Status Bar displays Ready. When an assay begins, the status bar displays each step as it starts, including the total amount of time a step requires and how much time remains for the step to complete. Note that the Ready state also displays in offline mode. The progress bar on the Status Bar provides a graphical representation of the same information. When an assay is not in progress, this area is blank. Sample count keeps a tally of samples since the chip was inserted. Ladders are not included in the sample count total. The sample count resets once the chip door is opened. The online status area shows whether or not an instrument is connected and communication is functioning properly. The last two sections of the Status Bar show whether Auto Export and/or Auto Print are enabled. If enabled, the text is black. If disabled, the text is gray (dimmed).

53 Description of the System Software 53 Assay Properties Window The Assay Properties window displays the settings used to determine the ladder peaks and other values required for analysis. These settings are read-only and cannot be changed. Only the settings on the Global Peak Find tab can be edited. To open the Assay Properties window, select <Assay Name> Properties from the Assay menu. The Assay Properties window contains the following tabs: Summary Tab (see page 54) Ladder Tab (see page 55) Analysis Tab (see page 56) Global Peak Find Tab (see page 58) Alignment Tab (see page 61) The Save As button on the tabs in the Assay Properties window allows you to save the values entered as the defaults for a new assay or to save the current values over an existing assay (except those assays that are shipped with the software and are read-only). The default assay folder is shown, but you can save the assay in any folder. NOTE Settings that can be changed and are saved with an assay file are the peak find settings, gel color default, and well names. Settings on the other tabs of the Assay Properties window are still read-only.

54 Description of the System Software 54 Summary Tab The Summary tab displays the name, title, and version number of the open assay. Also displays copyright and keyword information. This tab is same for DNA, protein, and RNA assays.

55 Description of the System Software 55 Ladder Tab Use the Ladder tab to view the ladder values for the open assay. The settings on this tab are view-only and cannot be edited. The size of the markers in base pairs (for DNA), kda (for protein), or nucleotides (nt) (for RNA) displays in the left column. When the Use Standard Area check box is selected, the standard area for each marker is shown in the right column. These values are the assay defaults that are used when a ladder is not run (see Global Peak Find Tab on page 58 for more information).

56 Description of the System Software 56 Analysis Tab Use the Analysis tab to view the settings used to analyze the data. These settings cannot be changed. The Analysis tab contains the following settings: Item Assay Class Baseline Well Total Ladder Conc Peak Sizing Quantitation Marker Description Shows the family of assays to which the open assay belongs. Displays the number of seconds into the reading that the software waits before establishing the start of a baseline. Total area of the assay is determined between the baseline start value and this value set for the well total. Concentration in ng/μl for the ladder. (Same as Marker Conc for Protein assays.) Specifies which part of the peak is used as the size calibration point. Leading Edge - If selected, uses the start of the peak. (May be better for data where sample peaks are broader than ladder peaks.) Center - If selected, uses the top of the peak. (May be better for data where leading edge is not well-defined.) Determines whether the upper or lower marker is used for quantitation.

57 Description of the System Software 57 Item Qualitative Assay Align to Upper Marker Align to Lower Marker Standard Curve System Peak Detection Marker Conc Dilution Ratio Description When enabled, determines that the assay is a qualitative, not quantitative, assay. When enabled, aligns the data from the reading to the upper marker (analysis on by default). This is enabled for DNA assays only. Protein and RNA assays use only the lower marker. When enabled, aligns the data from the reading to the lower marker (analysis on by default). This is enabled for DNA, protein, and RNA assays. Determines whether the fit for the standard curve is done on a point-to-point or log basis. Typically, DNA and RNA assays are set for point-to-point curve fits and protein assays are set for log. Ladder Ratio Time at which ladder peaks are detected as ladder peaks rather than system peaks. If the ladder ratio is set to 1.28, the software multiplies the lower marker migration time by 1.28 seconds and then begins ladder peak identification. For example, if the lower marker elutes at six seconds, the ladder peak detection starts at 6 x 1.28 or 7.68 seconds. Sample (kda) Protein size at which peaks are identified as sample peaks rather than system peaks. (Not available for DNA or RNA assays.) Concentration in ng/μl for the upper and lower markers for DNA assays and the lower marker for protein and RNA assays. (Not available for DNA or RNA assays.) Correction factors for ladder and sample preparation variances. Ladder Reflects a dilution of ladder in a total volume of ladder and sample buffer according to the LabChip Kit User Guide. Sample Reflects a dilution of sample in a total volume of sample, water, and sample buffer according to the LabChip Kit User Guide.

58 Description of the System Software 58 Global Peak Find Tab The settings on the Global Peak Find tab of the Assay Properties window determine how peaks are detected and are shown in the display. Any changes made on this tab apply to all wells. (To change settings for individual wells, see Settings Tab on page 78.) The Global Peak Find tab contains the following settings: Item Min Peak Height Min Peak Width Slope Threshold Start Time Description Specifies the height limit below which a peak is not detected. In the single-well display with the Settings tab selected, the minimum peak height is indicated by horizontal green lines on the graph. Specifies the width limit (in seconds) under which a peak is not detected. Specifies the amount of change in absorbance units over time required to indicate that a peak has occurred. Differentiates between an eluting peak and baseline noise. Changing this setting may cause peaks that were previously detected to be ignored or to interpret noise as peaks. Specifies the time in seconds after the start of the run when the first peak can appear (any peaks appearing before this time are ignored). The graph in the single-well display starts at this time.

59 Description of the System Software 59 Item End Time Filter Width Baseline Plateau Polynomial Order Exclude Ladder Exclude System Peaks Description Specifies the time when peak detection stops. The graph in the single-well display ends at this time. Defines the data window, given in seconds, used for polynomial averaging. The broader the filter width, the more raw data points are used for averaging. As a result, the noise level decreases, but peaks become lower and broader. When changing this setting, ensure that the value is less than twice the width of the peaks of interest or the peaks will be distorted. This is a baseline parameter for peak finding. The signal is at baseline whenever the slope of the data is less than the slope threshold setting (positive or negative) for longer than the time specified for Baseline Plateau. This setting rejects brief, low slope areas such as between non-baseline-resolved peaks. (Not available for RNA assays.) This value is read-only and cannot be changed. A filter algorithm is used to filter the data, increasing the signal-to-noise ratio. The data is convolved with a polynomial to produce filter data and a filter slope and decrease the background or baseline noise and/or spikes in the signal. The higher the number, the more the fit function follows the noisy raw data curve. As a result, the noise level of the filtered curve increases. DNA and RNA assays - If selected, excludes the ladder from any changes to the peak find settings (the default ladder settings from the assay are used instead). Protein assay - If selected, excludes the system peak (peak due to buffer constituent) from any changes you make to the peak find settings (the most recent custom ladder settings are used instead).

60 Description of the System Software 60 Item Baseline Algorithm Description Default is Zero Baseline. None - No correction. Zero Baseline - Offsets all graphs to zero baseline but does not affect analysis. Baseline Subtraction - A dynamic subtraction of the baseline that corrects for drifting baseline. This algorithm creates a smooth line fit to the baseline data points and subtracts this smooth fit from the data. The threshold for the baseline correction allows you to determine how much the baseline fit tries to follow changes in the data. Lowering the Threshold below the default value of 20 allows the baseline fit to ignore regions that are slow changes of real signal peaks and not baseline drift. The Reset button on the Global Peak Find tab changes the settings on the tab back to the last saved values.

61 Description of the System Software 61 Alignment Tab The Alignment tab of the Assay Properties window displays the settings for row alignment to a ladder. Any row of the plate can be aligned with a ladder from another row. The Alignment tab is not available for Protein assays. The Alignment tab contains the following settings: Item Align Rows to Corresponding Ladder Align Rows with Bracketing Ladders Align all the Rows to Ladder (A-P) Align Rows Based on the Rules in the Following Table Description If selected, each row is aligned to the ladder at the beginning of the row. This option is not available for DNA assays. This option should always be used for RNA assays. If selected, each row is aligned with the ladder at the beginning of the row and a ladder at the end of the row. If selected, all rows are aligned to the same selected Ladder row. If selected, each row is aligned with the ladder selected in the table. To turn off alignment, select Turn Off Analysis from the Tools menu or click the Turn Off/On Analysis button on the tool bar (the menu icon shows a red X indicating analysis is off).

62 Description of the System Software 62 Alignment Line Colors If a row is not aligned with a ladder of the same row, then the color of the alignment line on the ladder lane is displayed in a different color than the color of the alignment line for the samples. The color used for alignment is based on the gel color. The table below shows the colors that are used for the various gel colors. Gel Color Color of Alignment Lines when Rows are Aligned to their Corresponding Ladder Color of Alignment Lines when Rows are not Aligned to their Corresponding Ladder Black on Green Orange White White on Green Orange Black Blue on White Red Green Green on Red Blue Black Magenta on Green Blue White Red on Black Green Blue Pseudo Color Green Black

63 Description of the System Software 63 Barcode Reader Window Use the Barcode Reader window to enter the barcode of a plate before running an assay. You can scan the barcode if the system is equipped with a hand-held barcode reader, or you can type the barcode into the window. Click the Reader button in the Start window to open the Barcode Reader window. Item Reset button Unload Plate button OK button Cancel button Description Resets the barcode reader and clears the text box to try reading the barcode again. Moves the plate out to access the plate if the plate has already been loaded into the instrument. Closes the Barcode Reader window and displays the barcode in the Start window. Closes the Barcode Reader window without changing the barcode in the Start window.

64 Description of the System Software 64 Edit Samples Window Use the Edit Samples window to specify the names of the samples, enter comments about samples, enter the Expected Fragment /Expected Protein sizes, or import the values from a sample template into the Edit Samples window. If the Edit Samples After Start check box on the Start window is selected, the Edit Samples window opens automatically after starting the run. To open the Edit Samples window after the run is complete, select Edit Edit Samples on the menu bar. Figure 8. Edit Samples window for DNA and Protein Assays The Edit Samples window is used to: Change the default names of the samples to names of your choosing. Select the existing name (e.g., Sample 1) and type a new name (e.g., PhiX174/BsuR1). Sample names display on the printout. You can enter a comment about the sample, if desired. Type in your comment in the Sample Comment field next to the sample. Any associated comments display on the printout. You can enter the Expected Fragment /Expected Protein size for up to 10 expected fragments/proteins. Type in each fragment size separated by semi-colons (any expected fragments over 10 are ignored). (Not available for RNA assays.)

65 Description of the System Software 65 Edit Samples Window (Continued) To allow for small variation in expected fragment/protein size, a tolerance value can be entered (in the column +/- %). This value is specified as % of the expected size for that fragment/protein. During the run, when fragments/proteins are detected, the result is shown in the Results and Sample tab in the lower-right corner of the main window in Single view (i.e., Sample Information tabs). The Results Table (see page 47) also shows the expected fragments/proteins found in the Observation column and in the Peak column with the label EF or EP. The expected fragments display on the printout. See Using Expected Fragments/Expected Proteins on page 118 for more information. NOTE For DNA assays, you can also indicate that the expected fragments are from a restriction digest. At the bottom of the window you can enter notes about the chip or the run in general and they are included in the printed report. You can create a sample template to use for a run and import it into the Edit Samples window. The template is first created in Microsoft Excel and then saved as a *.csv (comma-separated values) text file: To open the template in LabChip HT, click Import, navigate to the desired template and click Open. Information entered in this window can also be exported to a *.csv file by clicking Export and specifying a filename. See Using Sample Templates on page 127 for more information. The current barcode saved with the sample displays in the Barcode field (e.g., the barcode information entered in the Start window). If you import a sample template already having a barcode, a warning message displays asking you if you want to use the barcode present in the current sample or the barcode of the imported sample. If you click Yes, then the barcode from the import is used. If you click No, the existing barcode is used.

66 Description of the System Software 66 Export and Auto Export Windows Use the Export window to export data at any time. To open the Export window, select File Export on the main menu bar. Use the Auto Export window to automatically export data at the end of each run. To open the Auto Export window, click the Settings button next to Auto Export in the Advanced tab on the Options window (see page 71). Item Results Tables Description If selected, the data in the results table is exported. You can select the following values to export: Exclude Markers When selected, the upper and lower markers are not exported. Include Ladder When selected, the ladder data is included in the export file. Include Peak Statistics When selected, the peak statistics data are included in the export file. To obtain peak statistics, the samples you want to compare must have the same name. For example, if you want to compare the peaks for wells A2, C2, and E2, they must all have the same name. To rename these samples, use the Edit Samples window. Because of space constraints, the peak statistics cannot be shown in the Results Table on-screen. When the data are exported, the peak statistics are displayed at the end of the file. An example is shown in Figure 9 on page 67.

67 Description of the System Software 67 Figure 9. Peak Statistics Data Item Format Raw Data Export Directories Description One Column Exports the results table for each well in a single column text format. Plate-Like Exports the results table for each well in the order the plate was run, across multiple columns in the text file. Table Exports the results table for each well in the order the plate was run, down multiple rows in the text file. If selected, exports the raw data in *.csv (comma-separated values) format that is suitable for opening with Microsoft Excel or pasting into another program. This option exports signal data as one file per well. See Data Handling and Printing on page 129 for more information. Default If selected, the exported file is saved in the indicated directory. Custom If selected, the exported file is saved in the specified directory. Create Daily Subdirectories If selected, a new directory is created each day and exported data is saved to the daily directory.

68 Description of the System Software 68 File Properties Window The File Properties window presents statistics and information about the open data file. NOTE The Run Log button is only visible if a run log file exists.

69 Description of the System Software 69 Open Assay Window When you select Open Assay from the Assay menu, the Open Assay window opens to select the desired assay. Assay files have an.asl file extension. You can open any previously saved assay file, including files that have not been saved into the directories that are automatically shown in the Assay menu. A checkbox at the bottom of the window allows you to open the assay as read-only, prohibiting you from making changes and saving the changed file to the same name.

70 Description of the System Software 70 Open Data File Window When you choose Open from the File menu, the Open Data File window opens. Data files have a.cla file extension. Choose a data file name from the list box and click the Open button or double-click a file name to open the data file. The Open as Read Only check box opens the file as read-only and Open read-only is displayed after the filename in the title bar. A read-only file can be edited but cannot be saved with the same filename. If you attempt to save an edited, read-only file, an error message displays explaining that the file is a read-only file. Clicking OK in the error box opens the Save As window. The benefit of opening a file as read-only is to prevent changes to the file. Since the LabChip HT software allows you to open data files, reanalyze them using different parameters, and save the new parameters with the file, you may want to ensure that the original parameters that were used to create the file are not altered.

71 Description of the System Software 71 Options Window Data Files Tab To open the Options window, select Options from the Tools menu. The Options window contains the following tabs: Data Files Tab Reader Tab Advanced Tab The Data Files tab specifies how data files are automatically named. You can include a prefix of your choice, the date and/or the time of the run, the barcode of the plate, and/or the chip ID. For example: including the prefix LabChip HT, the date and time of the run, and the barcode, would create file names such as LabChip_HT_ _ _Barcode. If you choose not to use the time of the run as part of the data file name, the LabChip HT software automatically appends a 1, 2 etc., for each subsequent run made that day. This tab also allows you to choose the directory in which data files are stored. The default stores them in a directory with the software files but you can create and use a custom directory, if desired. You can also choose to have daily subdirectories created for file storage each day. Saved files can be changed and resaved, or saved with a new name, if desired.

72 Description of the System Software 72 Reader Tab Item Serial Port Instrument Identification Description Specifies the serial (COM) port to which the HV controller is connected (the default setting is COM1). A setting of None appears if you're running the software alone without a connection to an instrument. This setting allows you to give the instrument a label for internal use, such as the Capital Equipment tag number or the instrument s lab location. This name is saved with the instrument.

73 Description of the System Software 73 Advanced Tab Item Display Raw Data Gel Mobility Correction Keep Packet files for the last X Runs Auto Print Auto Export Description If selected, the single-well view displays raw (unfiltered) data. Plots each point against mobility, instead of time, where mobility equals distance/time. By plotting against the reciprocal of time (1/time), the separation of the peaks is proportional to the mobility and is more comparable to a photo of a real gel. Applying mobility correction expands the fast peaks and compresses the slower ones. Specifies the number of runs to keep the packet files. The packet files contain raw data suitable for troubleshooting (typically requested by Caliper Technical Support). If selected, a report is automatically printed at the end of each run. Click the Settings button to choose the desired print settings in the Auto Print window. For more information, see Printing a Report on page 131. If selected, data is automatically exported at the end of each run. Click the Settings button to choose the desired export settings in the Auto Export window. For more information, see Exporting Data on page 139.

74 Description of the System Software 74 Print and Auto Print Windows Use the Print window to print assay information at any time. To open the Print window, select Tools Print on the main menu bar. Use the Auto Print window to automatically print the selected assay information at the end of each run. To open the Auto Print window, click the Settings button next to Auto Print on the Advanced tab in the Options window (see page 71). The Print and Auto Print windows provide the following options for printing: Assay Summary Gel Like Electropherogram Combined Result Table You can choose from one to all four options; choosing Electropherogram or Combined Result Table enables choices in the lower half of the window (range of rows, etc.). Clicking OK opens a Print window with printer-dependent settings to send the print request. See Printing a Report on page 131 for more information.

75 Description of the System Software 75 Pressure Control Window The Pressure Control window opens when you choose Pressure from the Tools menu. It provides connection and status information about the Pressure Controller. The three buttons at the top of the window correspond to selections on the Pump menu in the menu bar: Pressure Control (default), Piston Position Control, and Communication. Item Pump Initialize Release Pressure Set Pressure Set Point Pressure Active Pressure Description Allows you to control the pump pressure. Clicking this button releases the pressure, if any, and then initializes the pump module. Releases the currently applied pressure. Sets the pressure (in PSI) shown in the text box to the right of the button. To set the pressure, enter the desired value in the text box and then click the Set Pressure button. If a positive number between 1 and 5 is entered, the pump module creates a positive PSI pressure; if the value is between 1 to 5, the pump creates a vacuum. Shows the pressure setting entered previously (the current set point). Shows the actual pump pressure.

76 Description of the System Software 76 Sample Information Tabs The Sample Information tabs display next to the Results Table at the bottom of the single well view. The tabs that are displayed and the information they contain depend on the type of assay in use and whether or not run data for the particular well being viewed is available. Sample Tab (for DNA Assays) You can change the sample name, add a comment about the sample, indicate that the assay is a restriction digest and/or enter the number of base pairs expected in the sample fragments up to ten total fragments. Each fragment should be separated by a semicolon. Indicating that a sample is a restriction digest allows the software to flag peaks that may have co-migrated. Since it is assumed that the molarity of all the fragments in a restriction digest should be the same, any peaks or clusters having a molarity that is significantly larger than the rest are flagged as potentially co-migrating peaks, allowing you to examine them in more detail. Possible Co-Migration of peaks is displayed in the Observations column of the Results table. NOTE Any information entered on the Edit Samples window when the run began is shown here and can be updated or changed.

77 Description of the System Software 77 Sample Tab (for Protein Assays) You can change the sample name, add a comment about the sample and enter the size of up to 10 total expected proteins in the sample. Each protein should be separated by a semi-colon. Sample Tab (for RNA Assays) You can change the sample name or add a comment about the sample.

78 Description of the System Software 78 Settings Tab The Settings tab specifies the peak find settings for the selected well. Change the settings for the selected well by clicking Apply or for all wells in the row by clicking Apply to Row. Change the settings for all wells by clicking the Global button to open the Assay Properties window. Item Min Peak Height Min Peak Width Slope Threshold Baseline Start Time Baseline End Time Global button Reset button Apply Apply to Row Description Specifies the height limit below which a peak is not detected. For each peak, the difference between the peak start time and the peak apex must be greater than the Min Peak Height value. Specifies the width limit (in seconds) under which a peak is not detected. Specifies the amount of change in response over time required to differentiate between an eluting peak and baseline noise. (RNA assays only) Specifies the start time for the baseline. The baseline start is shown in the large graph as a vertical green dashed line when the Results tab is selected. (RNA assays only) Specifies the end time for the baseline. The baseline end is shown in the large graph as a vertical green dashed line when the Results tab is selected. Opens the Global Peak Find tab (see page 58) in the Assay Properties window. Changes made on this tab affect all wells. Resets the values back to the default global properties. Applies changes to the selected well. Applies changes to the entire row.

79 Description of the System Software 79 Item Apply to all Ladders Description Displayed when a ladder well is selected. Applies the changes to the entire ladder. Results Tab This tab displays the peaks found in that well. ErrorsTabs This tab displays any errors associated with the data from that well, if any.

80 Description of the System Software 80 Start Window Figure 10. Start Window for DNA and Protein Assays The Start window opens when you click the Start button above the plate icon. To begin the run, click the Start button. Item File Prefix Assay Operator Chip ID Barcode Plate Samples Ladders Description Specifies the file prefix for the names of the data files. You can enter a file prefix other than the one shown in the window. Displays the name of the open assay. The name cannot be changed. Specifies the system operator s name. Specifies the ID of the chip. Specifies the plate barcode. Type the barcode of the plate manually, or if using a barcode reader, click on the Reader icon and scan the barcode. Specifies the type of plate (96- or 384-well). For protein assays, only 96-well plates can be selected. Using the Row and Column buttons, choose the wells that you want to run. For DNA and RNA assays, choose a ladder option: One Ladder Per Plate, or One Ladder Per Row. For protein assays, there is only one option, One Ladder Per Row.

81 Description of the System Software 81 Item Run Options Description Skip Priming If checked, skips the priming step when the run is started. Skip Warming If checked, skips the warming step when the run is started. Skip Focus If checked, skips the focus step when the run is started. Continue Previous Plate Select this option to finish reading a plate that was partially read in a previous experiment. See Continuing the Read on a Previous Plate on page 98 for more information. Repeat Plate Select this option to read the plate more than once. When selected, the Repeat Plate Options window opens: Edit Samples After Start Skip Priming If selected, the priming step is skipped in repeat reads. Skip Warming If selected, the warming step is skipped in repeat reads. Skip Focus If selected, the focus step is skipped in repeat reads. If selected, the Edit Samples window opens after clicking Start and before the assay begins.

82 Description of the System Software 82 Saving Data and Assay Files File data is saved automatically at the end of a run. Files are named according to the specifed prefix, serial number, date, and time of the run (see the Options Window on page 71 for more information). You can also save files manually by choosing File > Save or File > Save As. NOTE After data has been acquired and you make changes to the file display, the raw data acquired from the instrument is NOT changed only the display of the data is changed and saved. If you change the data shown after it has been saved and try to exit the software or acquire new data, a window opens asking whether you want to save the changed file. Information About Your Computer A System Info feature is installed with the LabChip HT software. This feature can be used to examine the computer and show information about the operating system, fonts, printing, display, and more. This information may be useful to the Technical Support representative if you call for assistance. To view information using System Info: 1 On the Help menu, click About LabChip HT. 2 Click the System Info button. 3 Click a category for the type of information you want. You can save or print information from the System Info window.

83 Description of the System Software 83 Using Help Contents and Index Context-Sensitive Help The Help system enables you to retrieve the information you need quickly and then return to your work. Help appears in a separate window. For quick access, you can keep the Help window displayed on top of or behind the application. You can print specific topics from the online Help system. Context-sensitive help is also available. When you click Contents and Index from the Help menu, the Help window opens, allowing you to do one of the following things: Click the Contents tab to display conceptual and how-to information. Click Index to search by a name or concept. The context-sensitive Help displays information that is relevant to the current window displayed in the software. To access contextsensitive help, click the Help button or press the F1 key. NOTE Positioning the cursor over a tool on the tool bar or over other items in the workspace and leaving it there momentarily displays a Tool Tip, listing the name of the tool. Often this is enough to describe the item's function. Printing Help You can print specific help topics or print entire sections of online help. To... Print an entire section Print a single help topic Do this... From the Contents page, click the Print button at the bottom-right side of the window. Go to the topic and click the Print button at the top of the window.

84 Description of the System Software 84 Types of Help Available General Help Context-Sensitive Help Hotspots and Links By clicking the Help menu and choosing Contents and Index, you can view the Help topics that are grouped by subject matter into books or search for a keyword and jump to a topic. Pressing the F1 key provides help that is specific to the active window. Certain items in the help files are links to other types of help. You will see single- or double-underlined words. Clicking the doubleunderlined word(s) takes you to another topic providing more information about the underlined item(s). Clicking the singleunderlined word(s) opens a small window, providing a short description of the underlined item. Click anywhere in the small window to close it.

85 Preparing the System for Operation 85 Preparing the System for Operation Overview This chapter contains the information and procedures for preparing the LabChip 90 system for operation. Topic See Page Principles of Operation 86 LabChip Kit and Instrument Guidelines 89 Preparing the System for Operation 91 Installing the Chip, Sample Plate, and Strip 93 Wells

86 Preparing the System for Operation 86 Principles of Operation The assay is based on traditional gel electrophoresis principles that have been transferred to a chip format. The chip format dramatically reduces separation time and provides automated sizing and quantitation information in a digital format. The chip contains an interconnected set of microchannels that join the separation channel and buffer wells. One of the microchannels is connected to a short capillary that extends from the chip at a 90- degree angle. The capillary is designed to sip from the wells of a microplate during the assay. Some of the channels in the chip are larger than others. The larger channels are meant to contain buffer. During the chip preparation, the smaller channels and some of the wells are filled with sieving gel and buffer. DNA Chip Protein Chip Figure 11. Chip Schematics Once the channels are filled, the chip functions as an integrated electrical circuit. The circuit is driven by the seven electrodes in the electrode cartridge that contact solutions in the chip s wells when the chip holder is fastened beneath the optics unit. Each electrode is connected to an independent power supply that provides maximum control and flexibility. The polymer filling the smaller channels in the chip is designed to sieve DNA/RNA fragments or proteins by size as they are driven through it by means of electrophoresis, similar to using agarose or polyacrylamide gels. The sample and sieving buffers also contain a fluorescent dye that gets brighter upon binding to double-stranded DNA, RNA, or protein/sds complex.

87 Preparing the System for Operation 87 Principles of Operation (Continued) In the chip, each sample is sipped by negative pressure until a sufficient quantity is loaded in the chip. The sample is then moved electrophoretically into the central channel. As the fragments move down the central channel, they separate by size, finally passing the laser that excites the fluorescent dye bound to the molecule. The software plots the fluorescence intensity versus time and produces electropherograms for each sample (see below). Figure Well Graph View The data can also be plotted in a gel-like format to achieve the appearance of a slab gel (several different color variations are available). Figure 13. Large Gel View

88 Preparing the System for Operation 88 Principles of Operation (Continued) Quantitating the concentration and accurately sizing each fragment are achieved by comparing against a sizing ladder and running internal standards or markers with each sample. Internal standards of known concentration are mixed with the sample to aid in quantitation. The amount of sample injected into the chip depends on ph, salt concentration, and buffer additives. The internal standards normalize these factors so that the software can use the ratio of the area of the curve of the standard to the unknown peak to determine concentration directly. The internal standards lie slightly outside the assay range so they do not interfere with analysis.

89 Preparing the System for Operation 89 LabChip Kit and Instrument Guidelines This section lists best practices for handling tools, chips, reagents, and samples. To ensure proper assay performance, please follow the important handling practices described below. Failure to observe these guidelines may void the LabChip Kit product warranty. It is important to keep particulates out of the chip wells, channels and capillary. Many of the following guidelines are designed to keep the chips particulate-free General Reagents Allow the chip, sample plate and all reagents to equilibrate to room temperature before use (approximately 20 min.). Clean the chip priming station weekly and the electrodes daily. Refer to the Maintenance and Service section for procedures. Avoid use of powdered gloves. Use only non-powdered gloves when handling chips, reagents, sample plates and when cleaning the instrument electrodes and electrode block. Calibrate laboratory pipettes regularly to ensure proper reagent dispensing. Avoid use of laboratory wipes. With the exception of cleaning the chip window, do not use laboratory wipes on chip wells, the priming station or electrodes. Follow the cleaning technique described in Maintenance and Service on page 143. Do not use transfer pipettes or other liquid handling tools that can contaminate liquids with fibers or other debris. Water used for reagent and chip preparation procedures must be micron filtered, deionized and molecular biology grade (or better). Store reagents at 4 C when not in use Protect the dye, gel/dye mixture and marker from light. Store in dark and at 4 C when not in use. The gel/dye mixture expires three weeks after preparation. Caliper recommends reverse pipetting to dispense gel, gel/dye and destaining solutions for accurate delivery into chip wells. If the level in the chip wells is too low, the liquid does not make contact with the electrodes. This can cause electrical issues, resulting in poor assay performance. Make sure the marker is pipetted accurately. If there is not enough marker in well 4, the marker will run out prior to the completion of a plate. Data collected without marker peaks cannot be analyzed by the software.

90 Preparing the System for Operation 90 Chips Samples Use vacuum to remove fluids from the chip wells. Aspirating with a pipette can leave used reagents in the chip wells. For this reason, it is recommended that you vacuum the wells instead. Rinse and aspirate the chip wells prior to priming and storage. Each active well (1,2, 3, 4, 7, 8, 9 and 10) should be rinsed and aspirated three times using filtered deionized water. During each aspiration step, carefully vacuum all liquid by reaching all the way to the bottom of the well. Perform this procedure even on new chips. After priming, be sure to remove any liquid on the tops of the chip wells using a vacuum. Keep the chip detection window free of fingerprints, debris and smudges. Prior to inserting a chip into the instrument, inspect the chip window. If needed, clean both sides of the chip window a laboratory tissue dampened with water or isopropanol. Avoid contacting the chip wells with the tissue. Insert chips into the instrument as soon as possible after preparation to reduce the time that they are exposed to laboratory particulates. Do not submerge the chip in any solution. The entire chip surface must be thoroughly dry before use. The sipper must be kept wet at all times and should not be exposed to an open environment for longer than one minute. Use care in chip handling to prevent sipper damage. Damage to the sipper can result in inconsistent sampling. Avoid exposing the chips to dust. Prepared sample plates should be free of gas bubbles and particulate debris, both of which may inhibit sipper flow. Sample plates containing gas bubbles and/or particulate debris should be spun down at 3,000 rpm prior to analysis. Insert plates into the instrument as soon as possible after preparation to reduce the time they are exposed to laboratory particulates.

91 Preparing the System for Operation 91 Preparing the System for Operation Aligning the Robot Preparing the Chip These procedures provide a general outline for aligning the robot, preparing the chip, sample plate, and strip wells. Specific details will vary depending on the assay you are performing and the chip you are using. Please refer to the application protocol included in the LabChip Kit User Guide for specific details on reagent and chip preparation. The robot should be aligned during the initial system installation and on a monthly basis thereafter. Alignment should also be performed any time a new type or brand of microplate is used on the system. The software stores both of the previous alignments for the last 96- and 384-well plates. To align the robot: 1 Have the sipper alignment tool and an empty microplate ready. 2 In the LabChip HT software main window, select Sipper Alignment from the Tools menu. 3 Follow the on-screen wizard instructions. Every chip has eight wells but not all of them are connected by channels. The specific chip determines which wells are connected to the main channel. On all chips, the well in the number one position is connected to the vacuum/pressure-waste source. A DNA chip and channel schematic are shown below. DNA Capillary Chip 1 10 Vacuum well Polymer/dye wells Marker well Capillary

92 Preparing the System for Operation 92 Preparing the Chip (Continued) Each chip is packaged in a box designed to prevent the capillary and channels from drying out. You can prepare the chip and leave it in the original box until you are ready to install it on the system. To prepare and prime the chip: 1 Follow the application protocol located in the reagent kit. Prepare all reagents according to the LabChip Kit User Guide located in the reagent kit. 2 Open the chip package, exposing the top of the chip. Allow the chip and reagents to reach ambient temperature. 3 Aspirate out the fluid from all wells using vacuum. To avoid introducing bubbles, aspirate one well at a time before refilling. 4 Fill the active wells with your assay reagents. NOTE The location of the reagents and or dye is dependent on the assay you are performing. Refer to the LabChip Kit User Guide in the reagent kit for proper location and volumes of reagents. 5 Replace the cover on the chip package. Preparing the Sample Plate We recommend using only 96- and 384-well plates with conical wells on the system. Please consult the application protocol for details regarding your specific assay for sample plate preparation. Preparing Buffer and Ladder Strip Wells The purpose of the strip wells is to provide a source of buffer and calibration standard or ladder. The instrument uses two strip wells; one for wash buffer and one for ladder.

93 Preparing the System for Operation 93 Installing the Chip, Sample Plate, and Strip Wells Installing the Chip The chip is held in place by a movable holder located on the underside of the optics module. It is secured by a latch. Accessing the holder requires that the plate platform be in the unload position. To install the chip: 1 Press down on the latch. The cartridge lowers as shown in Figure 14. Figure 14. Cartridge Latch 2 When the cartridge is released, rotate the cartridge toward the front of the instrument as shown in Figure 15. Figure 15. Cartridge Open 3 Use a lint-free laboratory tissue to remove any condensed moisture on the glass surface of the chip. Grasp the chip by the edges and place the chip in the cartridge as shown in Figure 16 so the capillary slides through the hole in the cartridge.

94 Preparing the System for Operation 94 Installing the Chip (Continued) Figure 16. Chip in Cartridge 4 Being careful not to touch the capillary, rotate the cartridge back into place and snap it into place. Installing the Sample Plate Place the sample plate on the plate carrier so that the A1 position on the plate lines up with the A1 marker on the plate carrier. A1 position Installing the Strip Wells 1 Install the buffer strip well in the plate carrier trough labelled B. 2 Install the ladder strip well in the plate carrier trough labelled L. Align the ladder strip so that the tab that extends vertically next to well H fits in the notch to the left of the ladder strip well.

95 Operating the System 95 Operating the System Overview This chapter provides all the procedures and information required to run an assay on the LabChip 90 system. Running the Assay Topic See Page Running the Assay 95 Continuing the Read on a Previous 98 Plate Preparing the System for Shutdown 100 To start an assay to read a plate: 1 Insert a chip into the chip holder. Install the buffer and ladder troughs. 2 Place the prepared plate on the plate carrier, then select Load Plate from the Tools menu to load the plate into the instrument. 3 Select the assay that you want to run from the Assay menu. 4 Click the Start button (above the microplate icon) to open the Start window. The name of the open assay displays in the Start window. The assay name cannot be changed. Figure 17. Start Window

96 Operating the System 96 Running the Assay (Continued) 5 Type the Operator and Chip ID information. 6 To change the names of the samples and add comments and notes about the run, select the Edit Samples After Start box. 7 Type or scan in the plate barcode, if desired. If using a barcode reader, click on the Reader button and scan the barcode. If the barcode does not scan properly, click on the Reset button to clear the code and scan again. If the plate is in the load position and you need access to scan the barcode, click on Unload Plate. The plate automatically moves to the load position prior to sampling. 8 Select the plate type, either 96- or 384-well. (384-well is not available for the Protein assays.) 9 Under Samples, select the rows and columns of the wells to sample. The wells are sampled row-by-row. 10 Leave Skip Priming, Skip Warming, and Skip Focus options UNCHECKED in the Start window. These functions should be performed on new chips or chips that have just been prepared. 11 Click the Start button. 12 The run begins. If the Edit Samples After Start box was selected, the Edit Samples window opens and displays the current settings for the open assay. Figure 18. Edit Samples Window-DNA Assay 13 If desired, change the names of the samples, add comments or notes, and specify the size of expected fragments or proteins for DNA or Protein assays.

97 Operating the System 97 Running the Assay (Continued) 14 When done, click OK. 15 The warming, priming, and focusing steps are performed first. The warming step allows the heater plate located in the chip holder to regulate chip temperature to 30 C. The priming step fills the channels of the chip with reagent. The focus step focuses the laser across the channels of the chip. Data collection begins after the warm up, prime, and focus steps are completed. Data is saved to a file with the name shown above the graph window. 16 After the run begins, the Start button on the LabChip HT window changes to Stop. To end the run for any reason, click the Stop button. A message box confirms that you want to end the run in progress. Click Yes to stop the run. The file information at the top of the window displays a red error circle and a third line of file information displays Status: Aborted by the user. 17 To view the results for individual wells as data is acquired or after the run is finished, click a well in the plate icon, a single well displayed on the large 12-well display, or a lane in the small gel. Data from the selected well displays in the Results Table and on sample information tabs at the bottom of the main window. For more information see The LabChip HT Main Window on page When the assay is complete, the Unload Plate window opens.

98 Operating the System 98 Running the Assay (Continued) 19 Click the OK button. 20 Remove the plate and remove the chip. Continuing the Read on a Previous Plate If a run is stopped before it is complete and the data file was saved, you can restart the run. When you continue the run, the reading starts from the beginning of the last row that was not completed. NOTE This procedure assumes that you have a partially complete run for which you saved the data file. To continue the read: 1 Click Start... in the upper-left corner of the main window to open the Start window. This procedure uses a DNA assay as the example. Figure 19. Start Window 2 Select Continue Previous Plate in the Run Options pane. The Continue Previous Plate window opens as shown in Figure 20 on page 99.

99 Operating the System 99 Continuing the Read on a Previous Plate (Continued) Figure 20. Continue Previous Plate Window 3 In the Continue Previous Plate window, click the Browse button and navigate to the data file that you want to continue. 4 Select the desired data file and click Open. In the Continue Previous Plate message window, click OK. 5 In the Start window that reopens, Continue Previous Plate is checked and the row and column where the run will begin is selected under Samples. If an Operator, Chip ID and Barcode were entered for the original run, those entries display. Click Start to begin. The run starts at the beginning of the last row that was not completed in the previous run.

100 Operating the System 100 Preparing the System for Shutdown Storing the Chip When you have finished using the system, the following tasks are required to properly shut down the system: Storing the chip Cleaning the electrodes To store the chip: 1 Remove the chip from the instrument. 2 Using a pipette, remove the reagents from all wells of the chip. 3 Add water or storage buffer to all active wells. Refer to the LabChip Kit User Guide for a procedure. 4 Check the fluid level in the sipper reservoir of the chip package and refill with deionized water as needed. 5 Place the chip into the storage box and close the lid. NOTE Cleaning the Electrodes Chip damage can occur if the reagent wells or the sipper become dry during storage. It is recommended that the electrodes on the instrument be cleaned at the end of each day. Please refer to Maintenance and Service on page 143 for instructions.

101 Data Analysis 101 Data Analysis Overview This chapter provides instruction for analyzing the data obtained from the LabChip 90 system. Topic See Page How the Software Analyzes DNA Data 102 How the Software Analyzes Protein Data 104 How the Software Analyzes RNA Data 107 Modifying Analysis Parameters 111 Using Expected Fragments/Expected Proteins 118 Changing the View of the Results 122 Reanalyzing a Data File 125 Using Sample Templates 127

102 Data Analysis 102 How the Software Analyzes DNA Data The purpose of the LabChip HT DNA assay is to calculate the size and concentration of nucleic acid fragments. Results for a particular well are calculated after all data for that well has been read. The data analysis process for DNA assays consists of the following steps: 1 Raw data is read and stored by the system for all of the individual wells. 2 The data is filtered and the resulting electropherograms of all wells are plotted. You can change the settings of the filtering algorithm after the run and reanalyze your data. 3 Peaks are identified for all wells and are tabulated by migration time. You can change the settings of the peak find algorithm and reanalyze the data after the run has finished. Peak find settings can be changed for all or only certain wells. 4 A sizing ladder (see Figure 21), which is a mixture of DNA fragments of different known sizes, is run first from the ladder well in the trough. The concentrations and sizes of the individual base pairs are preset in the assay and cannot be changed. Figure 21. DNA Ladder 5 A standard curve of migration time versus DNA size is plotted from the DNA sizing ladder by interpolation between individual DNA fragment size/migration points. The standard curve derived from the data of the ladder well should resemble Figure 22 on page 103.

103 Data Analysis 103 How the Software Analyzes DNA Data (Continued) Figure 22. Standard Curve - DNA 6 Two DNA fragments are run with each of the samples, bracketing the DNA sizing range. The lower marker and upper marker are internal standards used to align the ladder data with data from the sample wells. Figure 23 shows an example of assigned marker peaks in a sample well. The software performs alignment by default. Turning the alignment off suspends data evaluation until you turn it on again. Figure 23. Marker Peaks - DNA 7 The standard curve and the markers are used to calculate DNA fragment sizes for each well from the migration times measured. 8 To calculate the concentration of the individual DNA fragments in all sample wells, the upper marker, in conjunction with a calibration curve plotting assay-specific concentration against base-pair size, is applied to the individual sample peaks in all sample wells.

104 Data Analysis 104 How the Software Analyzes DNA Data (Continued) The software allows you to define upper and lower markers. Changing the markers causes quantitative changes in the calibration procedure, however, and therefore in the entire data evaluation. How the Software Analyzes Protein Data The data analysis process for protein assays consists of the following steps: 1 Raw data is read and stored by the system for all of the individual wells. 2 The data is filtered and the resulting electropherograms of all wells are plotted. You can change the settings of the filtering algorithm after the run and reanalyze your data. 3 Peaks are identified for all wells and are tabulated by migration time. You can change the settings of the peak-find algorithm and reanalyze the data after the run has finished. Peak-find settings can be changed for all or only certain wells. 4 A sizing ladder (see Figure 24), which is a mixture of protein fragments of different known sizes, is run first from the ladder well in the trough. The concentrations and sizes of the individual base pairs are preset in the assay and cannot be changed. Figure 24. Protein Ladder

105 Data Analysis 105 How the Software Analyzes Protein Data (Continued) 5 A standard curve of migration time versus mobility is plotted from the ladder by interpolation between the individual protein size/migration points. The standard curve derived from the data of the ladder well should resemble Figure 25 on page 105. Figure 25. Standard Curve - Protein 6 A Lower Marker is the internal standard used to align the ladder data with data from the sample wells. Figure 23 shows an example of the assigned Lower Marker peak in a sample well. NOTE The software performs alignment by default. Turning alignment off suspends data evaluation until you turn it on again. Figure 26. Lower Marker - Protein

106 Data Analysis 106 How the Software Analyzes Protein Data (Continued) 7 Before calculating either the protein size or concentration, the sample data is processed relative to the two ladders that bracket every 12 samples. First, all of the data is aligned to the lower marker and then stretched, relative to the highest molecular weight protein in the ladder. 8 The concentration of the sample proteins is determined relative to the bracketed ladders. There are seven proteins in the ladder (not including the lower marker). The ladder concentration is specified on the Analysis tab of the Assay Properties window and cannot be changed. NOTES The seven proteins are assumed to represent the average staining behavior of proteins and use them to determine an average peak area per μg/ml of protein. This factor is used to convert sample peak areas into relative concentration. The total relative concentration of all proteins is calculated by addition of the relative concentration of the individual proteins and is displayed in the Results tab. Since this conversion factor is determined for each ladder, the factor can be linearly interpolated for each sample and thus account for any assay drift. The software allows you to define the lower marker. Changing the selection of the markers will lead to quantitative changes in the calibration procedure, however, and therefore in the entire data evaluation.

107 Data Analysis 107 How the Software Analyzes RNA Data The purpose of the LabChip HT RNA assay is to determine the quality of the RNA sample by measuring the relative amounts of know RNA fragments relative to the total RNA present in the sample. Results for each well are calculated after all data for the well has been read. The data analysis process for RNA assays consists of the following steps: 1 Raw data is read and stored by the system for all of the individual wells. 2 The data is filtered and the resulting electropherograms of all wells are plotted. A curve spline fit to the data is performed to generate a baseline above which RNA fragment peaks are detected. This baseline is displayed as a blue trace on the electropherogram when the Settings tab is selected. You can change the settings of the filtering algorithm after the run and reanalyze your data. 3 Peaks extending above the baseline are identified for all wells and are tabulated by migration time. You can change the settings of the peak find algorithm and reanalyze the data after the run has finished. Peak find settings can be changed for all or only certain wells. 4 A sizing ladder (see Figure 27 on page 108), which is a mixture of RNA fragments of different known sizes, is run first from the ladder well in the trough. The concentrations and sizes of the individual nucleotides present in the ladder are predetermined by the assay and cannot be changed.

108 Data Analysis 108 How the Software Analyzes RNA Data (Continued) Figure 27. RNA Ladder 5 A standard curve of migration time versus RNA size is plotted from the RNA sizing ladder by interpolation between individual RNA fragment size/migration points. The standard curve derived from the data of the ladder well should resemble Figure 28. Figure 28. Standard Curve - RNA 6 A dye matching the lowest peak in the ladder is run with each of the samples. This lower marker, labeled LM in the RNA sample (see Figure 29 on page 109) is used to align the ladder data with data from the sample wells. The software performs alignment by default. Turning the alignment off suspends data evaluation until you turn it on again. If the lower marker is set incorrectly, it can be assigned manually by right clicking on the appropriate peak in the peak table.

109 Data Analysis 109 How the Software Analyzes RNA Data (Continued) Figure 29. Lower Marker - RNA 7 The standard curve and the markers are used to calculate RNA fragment sizes for each well from the migration times measured. 8 The total RNA present is computed by finding the area under the electropherogram trace. The baseline for this integration is a straight line starting at the end of the lower marker and ending at the baseline end time. The height of the baseline endpoints is computed from an average of a five second window around the baseline start and end times (shown on the Settings tab). This baseline can be viewed and adjusted by selecting the Result tab and using the mouse to drag the left and right vertical dashed lines to areas that more properly reflect the signal baseline. Figure 30. Baseline Start and End Lines - RNA

110 Data Analysis 110 How the Software Analyzes RNA Data (Continued) 9 The total RNA concentration in the sample is computed from the ratio of the RNA area in the sample to that found in the ladder multiplied by the ladder concentration specified in the analysis settings. 10 Assay defined RNA fragments are identified from the peaks in the peak table. Fragments are located by finding the largest peak within a size range associated with the fragment. For Eukaryote RNA assays, 5S, 18S and 28S fragments are located. For Prokaryote assays, 5S, 16S and 23S fragments are identified. 11 Total RNA Assay: The Fast area is computed by integrating the signal between the 5S and 18S (or 16S) fragments. RNA quality metrics: rrna Area and Height Ratios (28S/18S or 23S/16S) and Fast/Total RNA area ratio are computed. Figure 31. Total RNA Assay Results Table 12 Messenger RNA Assay: The RNA contamination ratio is computed. This is the ratio of the area of all the fragments to total RNA area. Figure 32. Messenger RNA Assay Results Table

111 Data Analysis 111 Modifying Analysis Parameters Different sets of parameters can be changed in the software to modify the data evaluation for sample analysis: Filtering parameters (see below) Peak find parameters for all wells/peak height for individual wells (see page 112) Time window for analysis (see page 114) Assigning upper and lower marker peaks for DNA assays (see page 115) Aligning or unaligning marker peaks (see page 116) Assigning the lower marker peak in a protein assay (see page 117) These settings can be made before a new run is started or when reanalyzing a previously saved data file. Changing the Filtering Parameters The first step in analyzing the raw data is to apply data filtering. The following filtering parameters can be changed: Filter Width To change the Filter Width setting for the open assay: 1 Select Assay Assay Name Properties to open the Assay Properties window (see Figure 33), then click the Global Peak Find tab. Figure 33. Global Peak Find Tab - DNA Assay

112 Data Analysis Change the Filter Width parameter as necessary. (See Global Peak Find Tab on page 58 for more information.) 3 Click the OK button to save the setting. Changing the Peak Find Parameters After data filtering, the peak find algorithm locates the peaks and calculates the local peak baselines. The algorithm begins by finding all the peaks above the noise threshold to determine the baseline, after which any peaks below the noise threshold are rejected. A local baseline is calculated for each peak to allow for baseline drift. The following peak find parameters can be changed: Min Peak Height Min Peak Width Slope Threshold Baseline Plateau See Global Peak Find Tab on page 58 for more information about the parameters available on this tab. To change the peak find parameters for all wells in the open assay: 1 Select Assay Assay Name Properties to open the Assay Properties window (see Figure 33), then click the Global Peak Find tab. Figure 34. Global Peak Find Tab - DNA Assay 2 Change the parameters as necessary. 3 Click the OK button to save the setting.

113 Data Analysis Changing any of these settings while custom settings have been applied (on the Settings tab for a single well) opens a window asking whether to override custom well settings: 5 Choosing Yes causes any changes made to the peak find settings for individual wells to be discarded and applies the settings globally to all wells. Choosing No causes individual wells to retain changed peak find settings. The changes are not applied to any wells. To change peak find settings, except Baseline Plateau, for individual wells: 1 Choose View > Single Well and select one of the sample wells (or click a well in the plate icon, a single well displayed on the large 12-well display, or a lane in the small gel). The lower right pane of the window displays four data tabs. The Settings tab shows the peak find settings currently in effect for that well. 2 Changing the settings on the Settings tab only affects the selected well.

114 Data Analysis 114 Changing the Time Window for Analysis The Start and End time parameters in the Global Peak Find tab define the time window within which fragments are found. To change the Start Time and End Time parameters for all wells in the open assay: 1 Select Assay Assay Name Properties to open the Assay Properties window (see Figure 33), then click the Global Peak Find tab. Figure 35. Global Peak Find Tab - DNA Assay 2 Change the parameters as necessary. 3 Click the OK button to save the setting.

115 Data Analysis 115 Assigning Upper and Lower Marker Peaks for DNA Assays For each DNA sample, the upper and lower marker peaks are assigned first and then the data is aligned so that the well markers match the ladder markers in time, allowing the size and concentration of the sample peaks to be determined. See Assigning the Lower Marker Peak in a Protein or RNA Assay on page 117 for Protein or RNA assays. For DNA assays, the first peak is assigned to be the lower marker and is then offset to match the lower marker in the ladder. The upper marker is then assigned to the last peak in the sample well or to the peak nearest the ladder s upper marker. See Aligning or Unaligning the Marker Peaks on page 116 for an example of assigned marker peaks. If you get unexpected peaks in the ladder analysis or the markers have been set incorrectly, you can manually exclude peaks from the ladder or set a peak to be used as a marker. NOTES Excluding a peak or manually setting a peak to be an upper or lower marker for a DNA assay can cause errors with analysis. You can move the boundary between the Results Table and the well graph up or down to increase or reduce the amount of space allotted to the Results Table, making it possible to see all of the results at once. Right-clicking in the Results Table of a ladder well when the Settings tab is selected opens a pop-up menu with the following commands: Exclude Peak from Ladder Copy Results Table Right-clicking in the Results Table of a sample well from a DNA assay when the Settings tab is selected opens a pop-up menu with the following commands: Manually Set Lower Marker Manually Set Upper Marker Exclude Peak Copy Results Table Right-clicking on a peak in the electropherogram displays the same menu.

116 Data Analysis 116 Aligning or Unaligning the Marker Peaks The upper and lower markers are then aligned to the ladder markers by resampling the well data in a linear stretch or compression using a point-to-point fit. Data before Alignment Markers aligned to Ladder If the sample marker peaks are either more than twice as far apart or less than half as far apart as the ladder markers, they are assumed to be the wrong peaks, and analysis of the well stops, producing the error Marker peaks not detected. With DNA assays, the height of marker peaks is assay dependent. Ladder peaks are analyzed to calculate a marker peak threshold that is used to locate the marker peaks in the sample wells. If the marker peaks found using this calculated method fail to align with those of a sample, the LabChip HT software will use the minimum peak height threshold setting instead (if this value is lower than the value for the marker peak). For example, the calculated threshold might be too high to find the sample's markers if they happen to be very small for some reason. Either no markers will be found or the wrong peaks will be assumed to be markers and these may not align with the ladder markers. Consequently, the software attempts to use the minimum peak height threshold that, if it is set low enough, will catch the real markers, allowing the sample to align.

117 Data Analysis 117 Assigning the Lower Marker Peak in a Protein or RNA Assay For each protein or RNA sample, the lower marker peak is assigned first and then the data is aligned so that the well markers match the ladder markers in time, allowing the size and concentration of the sample peaks to be determined. The first peak is assigned to be the lower marker and is then offset to match the lower marker in the ladder. If you get unexpected peaks in the ladder analysis or the marker has been set incorrectly, you can manually exclude peaks from the ladder or set a peak to be used as a marker. NOTES Excluding a peak or manually setting a peak to be the lower marker for a protein assay can cause errors with analysis. You can move the boundary between the Results Table and the well graph up or down to increase or reduce the amount of space allotted to the Results Table, making it possible to see all of the results at once. Right-clicking in the Results Table of a ladder well when the Settings tab is selected opens a pop-up menu with the following commands: Exclude Peak from Ladder Copy Results Table Right-clicking in the Results Table of a sample well of a protein or RNA assay when the Settings tab is selected opens a pop-up menu with the following commands: Manually Set Lower Marker Exclude Peak Copy Results Table Right-clicking on a peak in the electropherogram displays the same menu.

118 Data Analysis 118 Using Expected Fragments/Expected Proteins You can track up to 10 expected DNA fragments (EFs) or proteins (EPs) for any sample in a DNA or Protein assay, respectively. There are two ways of entering the EFs and EPs: In the Edit Samples window In the Sample Information tab Entering EFs and EPs Using the Edit Samples window To enter an EF or EP via Edit Samples: 1 Select Edit Samples from the Edit menu. 2 In the column labeled Expected Fragments or Expected Proteins, enter the size of the expected fragment/protein in bp or kda respectively for the appropriate sample. NOTE You can enter up to 10 EFs or EPs per sample. Fragments/proteins greater than 10 are ignored. 3 In the +/- % column, if desired, enter a tolerance value to allow for small variations in the expected fragment/protein size. This value is specified as % of the expected size for that fragment/protein. The default is 10%. Figure 36. Edit Samples Window, DNA and Protein Assays NOTE If there are multiple peaks within the tolerance range, the largest peak is labeled as the EF or EP, even if it is not the exact size specified.

119 Data Analysis 119 Entering EFs and EPs Using the Sample Information Tab To enter an EF or EP using the Sample Information tab: 1 Select Single Well View from the View menu. 2 Click on the Sample tab to the right of the Results Table at the bottom of the single well view window. 3 In the Exp. Fragment (bp) (for DNA assays) or Exp. Proteins (kda) (for protein assays) text box, enter the sizes of the expected fragment or proteins. Separate each by a semicolon. You can enter up to 10 EFs or EPs per sample. Fragments/proteins greater than 10 are ignored. 4 If the assay has already run, click Apply on the bottom of the Sample tab to re-analyze the sample to tag the fragments. NOTE To enter a tolerance value for size variation, you must use the Edit Samples window. Figure 37. Sample Information Tab, DNA and Protein Assays

120 Data Analysis 120 Viewing the EFs and EPs To display the EFs or EPs in the electropherogram, mini gel and slab gel, select Show Found Fragments/Found Proteins from the Graph menu or click the Show Found Fragments/Show Found Proteins button on the tool bar. If there are multiple peaks within the tolerance range, the largest peak is labeled as the EF or EP, even if it is not the exact size specified. EFs/EPs on the Electropherogram Expected Fragments and Expected Proteins are identified in the electropherogram by open triangles over the peaks. EFs/EPs on the Mini Gel and Slab Gel Expected Fragments and Expected Proteins are indicated in the mini gel and slab gel by colored bands; red for DNA assays and green for protein assays. Figure 38. Expected Fragments and Expected Proteins on a Mini-Gel

121 Data Analysis 121 Figure 39. Expected Fragments and Expected Proteins on a Slab Gel EFs/EPs on the Results Table Expected Fragments and Expected Proteins are identified in the Results Table with the label EF or EP in the Peaks column. Figure 40. Results Table, DNA Assay Figure 41. Results Table, Protein Assay

122 Data Analysis 122 Changing the View of the Results Graph Overlay A number of different options are available for viewing the data after it has been acquired by the instrument. These options do not change the raw data but rather provide different means of displaying the data. Data from multiple wells can be overlaid in the large single well graph for visual comparison. Hold down the CTRL key and then leftclick on other wells in the plate icon or small gel view. If overlaying wells in the same row, bounding boxes display around the gel lanes signifying which wells are shown overlaid. Each peak graph is shown in a different color and line style with a legend at the top of the window. You can remove wells from the overlay by CRTL+ clicking the corresponding wells (the bounding box disappears).

123 Data Analysis 123 Changing to Gel View To compare the electropherograms generated with the instrument with results from a slab gel device, switch to the gel view. From the menu bar, click View > View Gel. The large display shows the results in a format similar to that generated by a slab gel device. Figure 42. Large Gel View Different gel display colors are available by choosing View > Gel Color and then choosing one of the color schemes from the dropdown menu. The colors are designed to approximate various actual gel staining and imaging techniques. Blue on White, for example, simulates a Coomassie gel often used with proteins. The Pseudo color choice provides more detail (1,280 colors) since it maps the signal into a larger color space than is available with the other monochrome options (256 levels of brightness).

124 Data Analysis 124 Forcing the Baseline to Zero All electropherograms produced with the instrument show some amount of background fluorescence. By default, the LabChip HT software enables the zero baseline function. To remove the zeroing, select Tools > Options to open the Options window, click the Advanced tab, and uncheck the Zero Baseline box. Zero Baseline Non-Zero Baseline

125 Data Analysis 125 Reanalyzing a Data File Occasionally you may need to open and view or reanalyze a data file that was run and saved previously. The raw data values are saved in the data file, along with the analysis settings that were chosen for the run, so that the data can be reanalyzed with different settings. NOTE You can always reanalyze data generated by older versions of the software with newer, updated versions of the software. Once you have saved the assay/data with the newer software you cannot read them with the older software. See File Properties Window on page 68 for how to get information about the software versions used to generate/analyze a data file. The following analysis parameters can be changed: Global peak find settings Individual sample peak find settings chosen in the sample information pane to the right of the Results Table in the single well view window Gel color Sample names and comments Exclude peaks from analysis Reassign upper/lower markers Alignment or no alignment with ladder peaks Assay you can save the changed settings under a new assay name, if desired To reanalyze a data file: 1 Select Open from the File menu. 2 Select the filename from the list of data files. 3 Click OK. NOTE If you have no unsaved data currently open, the selected file opens allowing you to view/edit the results. If you have unsaved data open, a window prompts you to save the current data first.

126 Data Analysis 126 Reanalyzing a Data File (Continued) 4 Make appropriate parameter changes. 5 Save the file. NOTE Settings such as gel color, well names, and peak find settings are saved. To preserve the original file, choose Save As and rename the file or save it in another location.

127 Data Analysis 127 Using Sample Templates Sample templates (provided with the instrument software) can be used to import sample information (sample name, comments, expected peaks, size tolerance, and plate barcode) into the Edit Samples window. The following templates are provided: 384 well sample template DNA.csv 96 well sample template DNA.csv 96 well sample template Protein.csv 96 well sample template RNA.csv The template files are located in the main Caliper LabChip HT directory. To enter sample information, open the template in Microsoft Excel. DNA Templates Sample information for DNA assays should be entered as follows (the instructions are also included in the templates): Column Description A Enter sample name. This should be in text only. B Enter sample comments. This should be in text only C Enter the expected fragment base pair size. These values should be numeric only (250, 720 etc.). Up to 10 fragments can be entered. Separate fragment values by a semicolon, no spaces. Example: 312;541;772;1050. D Determines whether expected fragments from column C are highlighted with a green hollow arrow in the graph. Enter 1 for yes or 0 for no. Default is 0. E Enter the size percent variation to allow (numeric entry only). Default is 10%. Enter the percentage as whole numbers, for example: enter 5 for 5%, 20 for 20%.

128 Data Analysis 128 Column Description Cell A98 Cell A99 and up Protein Templates Enter barcode directly after BARCODE=. Do not enter spaces after the equal sign. You can add additional notes (text only) in cells A99 and up on 96-well templates or cells A387 and up on 384- well templates. Sample information for protein assays should be entered as follows (the instructions are also included in the template). Column Description A Enter sample name. This should be in text only. B Enter sample comments. This should be in text only C Enter the expected protein size in kda. These values should be numeric only. Up to 10 proteins can be entered. Separate each protein value by a semicolon, no spaces (e.g., 20;40;60). D Not used. E Enter the protein percent variation you want to allow (numeric entry only). Default is 10%. Enter the percentage as whole numbers. For example, enter 5 for 5%, 20 for 20%. Cell A98 Enter barcode directly after BARCODE=. Do not enter spaces after the equal sign. Cell A99 and up You can add additional notes (text only) in cells A99 and up on 96-well templates.

129 Data Handling and Printing 129 Data Handling and Printing Overview This chapter provides the instructions on handling and printing LabChip 90 system data files. Topic See Page Organizing, Retrieving, and Backing-Up Data 130 Files Printing a Report 131 Report Options 133 Copying Information 137 Exporting Data 139

130 Data Handling and Printing 130 Organizing, Retrieving, and Backing-Up Data Files General Guidelines As you begin to work with the LabChip HT software, it's a good practice to organize your files. Create a directory in which to save the data files. Have each person save files to their own directory. This helps to locate a particular file to re-examine the data. Review your files periodically, even if only one person uses the LabChip HT software, and archive files you are no longer using but wish to save, and discard unneeded files. Have each user in the laboratory specify a particular prefix to easily differentiate data files from each other. Additionally, you can specify a new directory be created each day for storage of that day's runs. To do this, choose Tools > Options and then place a checkmark in the box at the bottom of the Data Files tab of the Options window next to Create daily subdirectories. Figure 43. Data Files Tab on Options Window Archive files to a backup disk for safekeeping and/or to remove files from your hard disk periodically. Depending on the amount of hard disk space available to the LabChip HT software, you may need to clear space on the hard drive to ensure there is enough room to save upcoming assay data.

131 Data Handling and Printing 131 Printing a Report Printing Automatically at the End of a Run To automatically print a report at the end of each run: 1 Select Tools > Options to open the Options window, and then click the Advanced tab. Figure 44. Advanced Tab on Options Window 2 Select the Auto Print check box. 3 Click the Settings button next to the check box to open the Auto Print window. Figure 45. Auto Print Window 4 Select the check boxes next to the items to be printed. Choosing to print Electropherograms or the Combined Results Table enables the items under Options. 5 Click the OK buttons on each window to save the settings.

132 Data Handling and Printing 132 Printing Manually at Any Time Choosing File > Print opens a window which offers the same choices as the Auto Print window: it allows you to print the same four types of information. The selections you make for manual printing are separate from the Auto Print selections, they do not affect each other. The settings are saved and are used when the software is restarted. You can print just one or any combination of the items shown in the Print section by enabling the checkboxes next to the desired items.