AG480F / E

1 L Assays

USER GUIDE

 2009 Beckman Coulter Biomedical GmbH. All Rights Reserved. Beckman Coulter AG480 User Guide. This User Guide as well as the system described in it may be used or copied only in accordance with the terms of copyright. The content of this User Guide is intended for instructional use only and is subject to change without notice. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical or otherwise, without the prior written permission of Beckman Coulter Biomedical GmbH. Please remember that existing artwork or images from this guide that you may want to include in your project may be protected under copyright law. The unauthorised incorporation of such material into your new work could be a violation of the rights of the copyright owner. Please be sure to obtain any permission required from the copyright owner. Any references to any company names or peoples names in sample templates and screens are for demonstration purposes only and are not intended to refer to any actual organisation or names of persons.

General support contact: Beckman Coulter Biomedical GmbH Sauerbruchstrae 50 D-81377 Munich Phone: +49-(0)89-579589-3540 Fax: +49-(0)89-579589-3503 E-Mail: singlecell@beckman.com

For all other regions go to www.advalytix.com for your nearest distributor. For research use only. Not for use in diagnostic procedures.

PowerPlex System and RNasin are trademarks of Promega GmbH, Germany. Polymerase Chain Reaction (PCR) process is covered by patents which are owned by Hoffmann-La-Roche Inc. and F. Hoffmann-La Roche Ltd. QIAGEN, HotStarTaq, Omniscript and Sensiscript are trademarks of QIAGEN GmbH, Germany. AmpliTaq, AmpliTaq Gold, GeneAmp, AmpF/STR SEFiler are Trademarks of Applied Biosystems, USA. FluoCycle and BlueTaq are trademarks of EuroClone SpA, Italy. Multipette is a trademark of Eppendorf GmbH, Germany. AutoRep is a trademark of Rainin Instrument, LLC., USA.

manufacturer

25C 15C

storage temperature

expiry date

refer to manual

REF

ordering information

LOT

Lot-number

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Table of Contents
01 Product Information Kit Components Storage Information Recommended Equipment Materials required but not provided Precautions before Use 02 AmpliGrid AG480F: Experimental Procedure 03 AmpliGrid AG480E: Experimental Procedure 04 Special Applications 05 Order Information Appendix A: Tested kits and components Appendix B: Troubleshooting 5 7 7 7 8 8 9 11 15 17 19 21

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User Guide - AmpliGrid AG480F

Intended Use
The AmpliGrid slides have been designed by Beckman Coulter to carry out a wide range of biological and biochemical reactions in a 1 L volume. This includes exponential amplification, isothermal amplification, whole genome amplification, RT approaches, hybridisations etc., using various sample materials like single cells, DNA/RNA dilutions or even material from forensic stains.

The AmpliGrid system is for research use only. Not for use in diagnostic procedures.

Please discard any previous versions of this User Guide !

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User Guide

01 Product Information
Kit Components Storage Information Recommended Equipment Materials required but not provided Precautions before Use
01 Product Information Kit Components Storage Information Recommended equipment Materials required but not provided Precautions before Use 02 AmpliGrid AG480F: Experimental Procedure 03 AmpliGrid AG480E: Experimental Procedure 04 Special Applications 05 Order Information Appendix A: Tested kits and components Appendix B: Troubleshooting

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01 Product Information

01 Product Information
The AmpliGrid slides (fig. 1) comprise 48 lithographically defined reaction sites, each holding 1 L of the reaction solution. Evaporation is prevented by covering the aqueous phase with a special sealing solution. All liquid components are safely contained and separated by structured chemical surfaces which form reaction sites surrounded by hydrophilic and hydrophobic circles (fig. 2). Components for biological or chemical reactions can be pre-deposited either as a master mix or sequentially with intermediate air-drying. All AmpliGrid products are manufactured and packed in state-ofthe-art class 100 clean rooms to ensure superior product quality. The AmpliGrid line substrate uses ultra-flat and extra-low fluorescence glass slides in the standard size of 75.6 mm x 25 mm x 1.0 mm containing reaction sites of 1.6 mm diameter with a centerto-center spacing of 4.5 mm. This common 384-well MTP pitch and the design of reaction sites on the slides means that it is possible to adapt processes to an automated liquid handling robot or fluorescence activated cell sorters. The unique optical quality allows for superior control of e.g. cell deposition as well as image acquisition using fluorescent scanners.

Figure 1: Dimensions [mm] of the AmpliGrid slide

Beckman Coulter offers two different formats of the AmpliGrid system:

Figure 2: Cross section of AG480F DNA-free microliter reaction site

AmpliGrid AG480F Features: 100% DNA-free with an inactivated surface to ensure lowest binding efficiency for biomolecules like polymerases or nucleic acids. Applications: 1 L amplification research assays (e.g. PCR / RT-PCR), single cell deposition using flow cytometry instruments, long term storage of DNA and cells (no covalent immobilisation), target for microdissection.

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01 Product Information

AmpliGrid AG480E Features: Highly reactive epoxy coated surface (fig. 3) for covalent immobilisation of both amino-modified and unmodified long oligos, cDNAs, proteins, cells and tissues. Research Applications: 1 L amplification and/or hybridisation assays (microarray based), solid phase PCR, immobilising biomolecules for downstream analysis, target for microdissection.
Figure 3: Cross section of AG480E epoxy coated microliter reaction site

Kit Components
Table 1: Kit components Order Number OAX 04503/04509 OAX 04505/04511 OAX 04504/04510 OAX 04506/04512 AmpliGrid slides 1x 5 1x 25 1x 5 1x 25 Sealing Solution 5x 500 L 25x 500 L 1x 4 mL 4x 4 mL Product Manual 1 1 1 1

Storage Information
To ensure constant quality, the AmpliGrid slides and the sealing solution are packed under inert gas in light-protective aluminum foil. Both components of the AmpliGrid system should be stored in the original package at room temperature (15 25 C). After removing the aluminum foil, both components should be used immediately or can be stored at room temperature in a dark, dry and dust-free place. Do not use any of the components after expiry date.

Recommended Equipment
Thermal Cycler All AmpliGrid experiments have been optimised with AmpliSpeed ASC200D slide cycler (fig. 4; Beckman Coulter, Order No. : OAX04102). Electronic Multi-step pipettes (e.g. Eppendorf Multipette Stream or Rainin AutoRep E). Manual multi-step pipettes might cause satellite drops when dispensing sealing solution. Pipette tips for multi-step pipette covering the range of 1- 5 L. Analysis system (e.g. capillary electrophoresis, polyacrylamide or agarose gel electrophoresis, UV-spectrometer).

Figure 4: AmpliSpeed ASC200D slide cycler

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01 Product Information

Materials required but not provided

Powder free gloves. Template (DNA / single cells / tissues). Amplification reagents (master mix, single components, polymerase). Note: Some commercially available DNA polymerases have not been designed for use in low volumes. Please refer to the Beckman Coulter support (see page 2) or Appendix A for a list of tested enzymes and kits.

Precautions before Use

Slide surface must not be touched. Please work in a clean environment as dust particles may interfere with the reaction. Always place the AmpliGrid slide with the correct side up, so that you can read the engraved markings. Amplification of human DNA (e.g. forensic strains) is subject to contaminations by very small amounts of non-template human DNA. Extreme care and good laboratory practice is necessary to avoid cross-contamination throughout all steps of the experiment. Each reaction drop should be covered with sealing solution as soon as possible. The loaded reaction volume (1 L per reaction site) may dry depending on room temperature, humidity etc.

Inadequate volumes will compromise the interpretation of results. A divided workflow is advisable. Wear powder free gloves and use aerosol resistant pipette tips. Do not reuse the AmpliGrid slide. Do not expose AmpliGrid AG480E slides to humidity or high temperatures before use.

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User Guide

02 AmpliGrid AG480F: Experimental Procedure

01 Product Information Kit Components Storage Information Recommended equipment Materials required but not provided Precautions before Use 02 AmpliGrid AG480F: Experimental Procedure 03 AmpliGrid AG480E: Experimental Procedure 04 Special Applications 05 Order Information Appendix A: Tested kits and components Appendix B: Troubleshooting

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02 AmpliGrid AG480F: Experimental Procedure

02 AmpliGrid AG480F: Experimental Procedure

Figure 5:Workflow AmpliGrid AG480F

1. Put the AmpliGrid slide on a dark surface to ensure good visualisation of the engraved circles marking the hydrophilic regions of the slide. 2. Optional: Up to 1 L of template solution e.g. primers, DNA dilutions or other sample material such as single cells, can be transferred onto each reaction site (fig. 5) and subsequently airdried at room temperature, then the reaction mix can be added as described in step 3. Sorter 3. Transfer 1 L of reaction mix onto each reaction site of the AmpliGrid AG480F. Due to the hydrophilic structure of the reaction sites, aqueous liquids will automatically move to the center on contact with the surface. 4. Cover each droplet with 5 L of sealing solution. NOTE: Ensure that there is no evaporation of the reaction mix before covering with the sealing solution. A divided workflow is advisable. 3 5. Incubate the AmpliGrid on the AmpliSpeed slide cycler according to your required thermal cycling profile specifications. It might be necessary to adapt established thermal profiles due to the superior performance of the AmpliSpeed instrument. 6. Retrieving sample from the AmpliGrid: Pipette the aqueous phase into a new repository 4 Add buffers for downstream processing e.g. gel loading dye, formamide etc. Purify by loading the whole sample, including sealing solution, onto a suitable column (e.g. Sephadex). 7. Sample is ready for analysis (capillary electrophoresis, PAGE, etc.).

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03 AmpliGrid AG480E: Experimental Procedure

01 Product Information Kit Components Storage Information Recommended equipment Materials required but not provided Precautions before Use 02 AmpliGrid AG480F: Experimental Procedure 03 AmpliGrid AG480E: Experimental Procedure 04 Special Applications 05 Order Information Appendix A: Tested kits and components Appendix B: Troubleshooting

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03 AmpliGrid AG480E: Experimental Procedure

03 AmpliGrid AG480E: Experimental Procedure

Figure 6:Workflow AmpliGrid AG480E

Deposit your selected capture molecules on the reactive surface of the reaction sites by pipette or automated instruments e.g. standard microarrayers. For detailed information on spotting parameters, please contact Beckman Coulter support (see page 2). Epoxy surfaces also show high adherence for tissues and might be used for immobilisation. Protocol for the immobilisation of DNA / oligonucleotides We recommend using AdvaSpot AT100 buffer (Beckman Coulter Biomedical GmbH, Ref: OAX05205) for depositing DNA molecules on the AmpliGrid surface. This ensures high quality spot morphology and binding efficiency. 3 Possible applications: DNA-Microarrays with integrated amplification, SNP-typing, antibiotic resistance screening of microorganisms. Inactivation of the slide surface and immobilisation of capture molecules. Incubate AmpliGrid AG480E slides over night at 42C in a humidity chamber with a saturated water atmosphere. 4 Wash slides for 2 min in 1 mM HCl. Wash slides for 10 s in ddH2O. Wash slides for 10 min in 100 mM KCl. Wash slides 2 times in ddH2O to prevent salt precipitation. 5 Air-dry or spin-dry the slides using the AdvaTube AT400 slide holder (Beckman Coulter, Order No.: OAX05216) in a 50 mL Falcon size swing-out bucket (3 min @ 500xg). 1. Put the AmpliGrid slide on a dark surface to ensure good visualisation of the engraved circles marking the hydrophilic regions of the slide. 2. Optional: Up to 1 L of template solution like e.g. DNA-dilutions, primer or other sample material such as single cells can be transferred onto each reaction site and subsequently air-dried at room temperature. Then the reaction mix can be added as described above (fig. 6). 3.Transfer 1 L of reaction mix onto each reaction site of the AmpliGrid AG480E. Due to the hydrophilic structure of the reaction sites, aqueous liquids will automatically move to the center on contact with the surface.

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03 AmpliGrid AG480E: Experimental Procedure

4. Cover each droplet with 5 L of sealing solution. NOTE: Ensure that there is no evaporation of the reaction mix before covering with the sealing solution. A divided workflow is advisable. 5. Incubate the AmpliGrid on the AmpliSpeed slide cycler according to your required thermal profile specifications. It might be necessary to adapt established thermal profiles due to the superior performance of the AmpliSpeed instrument. 6a. Retrieving sample from AmpliGrid: Pipette the aqueous phase into a new repository. Add buffers for downstream processing e.g. gel loading dye, formamide, etc. Purify by loading the whole sample including sealing solution onto a suitable column (e.g. Sephadex). Proceed with the protocol for direct image analysis (below).
Figure 7: Magnification of pipetted AmpliGrid slide

6b. Direct Image Analysis Pre-soak slides thoroughly in a 1 % SDS / 2 x SSC solution to remove most of the sealing solution. Subsequent washing steps should be performed according to standard microarray washing protocols. NOTE: Do not use SDS-based washing solutions after the first step as this might result in a higher background. Use of AdvaWash AW400 automated slide washing station (fig. 8; Beckman Coulter, Order No.: OAX05107) including the AdvaTube slide holder is recommended for reliable and reproducible washing conditions (tab. 2). Please refer to the corresponding manual for further information.

Figure 8: AdvaWash AW400 automated slide washing station

Table 2:Washing conditions for AG480E in AdvaWash Reagent 2 x SSC / 0.1 % Triton X-100 1 x SSC 1 x SSC 0.2 x SSC Soak time 5 min (Pulsing:Y) 40 s 4 min 20 s 5 min

After washing, transfer the AdvaTube into a centrifuge with 50 mL Falcon size swing-out buckets. Settings: 3 min ~ 500xg NOTE: Ensure that wet slides are processed without delay to avoid salt precipitation and a higher background.

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03 AmpliGrid AG480E: Experimental Procedure

Remove the slides from the AdvaTube and blow-off excess sealing solution with compressed nitrogen to reduce fluorescence background. Detection of fluorescent-labelled amplification fragments can be done in a standard microarray scanner. Protocol for the immobilisation of proteins Possible applications: Antibody testing Dilute the protein (e.g. antibody) to the appropriate concentration in 1 x PBS pH 7.4. Tested range is between 20 g/mL and 1 mg/mL. Pipet 1 L of protein solution onto the AmpliGrid reaction site. Incubate AmpliGrid over night in a humidity chamber. Ensure that droplets do not evaporate. Remove AmpliGrid slides from humidity chamber and allow the droplets to dry at room temperature. NOTE: Slides can be stored for several weeks at 4 C until use. Rehydrate slides in 0.1% Tween 20 / 1 x PBS for 3 x 2 min. Dry the slide by centrifugation at 500xg for 2 min. AmpliGrid slides can now be used for downstream experiments using 1 L volume per reaction site.

Washing and drying of AmpliGrid slides after protein experiments: Any kind of washing buffer can be used for washing the protein slides. For high reproducibility we recommend using the AdvaWash AW400 instrument (Beckman Coulter, Ref: OAX05107). For fast and efficient drying of the slides we recommend using the AdvaTube slide holder (Beckman Coulter , Ref: OAX05216) in a centrifuge with standard 50 mL Falcon size swing-out buckets at 500xg for 2-3 min.

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04 Special Applications

01 Product Information Kit Components Storage Information Recommended equipment Materials required but not provided Precautions before Use 02 AmpliGrid AG480F: Experimental Procedure 03 AmpliGrid AG480E: Experimental Procedure 04 Special Applications 05 Order Information Appendix A: Tested kits and components Appendix B: Troubleshooting

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04 Special Applications

Reagents and template material can be dispensed onto the AmpliGrid by standard liquid handling platforms (fig. 9) or cell sorting devices. Beckman Coulter offers high quality technical support by experienced staff and own application laboratories. Our main goal is to guarantee full customer satisfaction with all our product lines. Please contact - support (see page 2) for detailed information regarding integrated solutions or special application protocols.
Figure 9: Automation of the AmpliGrid system

Single cell sorting by flow cytometry The AmpliGrid slides fit perfectly in the Beckman Coulter cell sorter devices (e.g. MoFlo XDP). This enables the user to sort cells for their Immunophenotyp using cell markers and checking their genetic level witin one workflow. The high accuracy of the instrument and the fast sort process on the AmpliGris even allows for high throughput screenings of single cells on a molecular level.

Figure 10: Single Cell sorting with MoFlo XDP

Automation of processes For high throughput analyses and highly precise processing the AmpliGrid can be integrated into liquid handling platforms by either using our slide adapters or integrating the complete system with the AmpliSpeed cycler.

Figure 11: Automation process with AmpliSpeed and Biomek 3000

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05 Order Information

01 Product Information Kit Components Storage Information Recommended equipment Materials required but not provided Precautions before Use 02 AmpliGrid AG480F: Experimental Procedure 03 AmpliGrid AG480E: Experimental Procedure 04 Special Applications 05 Order Information Appendix A: Tested kits and components Appendix B: Troubleshooting

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05 Order Information

05 Order Information
Table 3: Order Information

Product AmpliGrid AG480F

Description DNA-free microliter reaction slide (5 pcs./pck.), 48 reaction sites each, incl. 5 x 500 L sealing solution DNA-free microliter reaction slide (5 pcs./pck.), 48 reaction sites each, incl. 1 x 4 mL sealing solution DNA-free microliter reaction slide (25 pcs./pck.), 48 reaction sites each, incl. 25 x 500 L sealing solution DNA-free microliter reaction slide (25 pcs./pck.), 48 reaction sites each, incl. 4 x 4 mL sealing solution Epoxy coated microliter reaction slide (5 pcs./pck.), 48 reaction sites each, incl. 5 x 500 L sealing solution Epoxy coated microliter reaction slide (5 pcs./pck.), 48 reaction sites each, incl. 1 x 4 mL sealing solution Epoxy coated microliter reaction slide (25 pcs./pck.), 48 reaction sites each, incl. 25 x 500 L sealing solution Epoxy coated microliter reaction slide (25 pcs./pck.), 48 reaction sites each, incl. 4 x 4 mL sealing solution

Order No. OAX04503

AmpliGrid AG480F AmpliGrid AG480F AmpliGrid AG480F AmpliGrid AG480E

OAX04504

OAX04505

OAX04506

OAX04509

AmpliGrid AG480E AmpliGrid AG480E

OAX04510

OAX04511

AmpliGrid AG480E

OAX04512

Sealing Solution

Sealing solution 1 x 500 L ready to OAX04207 use solution

Sealing Solution

Sealing solution 1 x 4 mL ready to use solution

OAX04208

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Appendix A Tested kits and components

01 Product Information Kit Components Storage Information Recommended equipment Materials required but not provided Precautions before Use 02 AmpliGrid AG480F: Experimental Procedure 03 AmpliGrid AG480E: Experimental Procedure 04 Special Applications 05 Order Information Appendix A: Tested kits and components Appendix B: Troubleshooting

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Polymerase AmpliTaq Gold DNA Polymerase (Applied Biosystems; Ordering-No: N8080242) Blue Taq (hot-start Taq) EuroClone EME011500 Applied Biosystems 4335129 Manufacturer Cat. No. Remarks
Table 4:Tested PCR Kits and components

Kit

AmpF/STR Sefiler PCR Amplification Kit

Blue Taq

FluoCycle Probe

Blue Taq (hot-start Taq)

EuroClone

EMQ011100

GeneAmp EZ rTth RNA PCR Kit AmpliTaq Gold DNA Polymerase (Applied Biosystems; Ordering-No: N8080242) AmpliTaq Gold DNA Polymerase (Applied Biosystems;Ordering-No: N8080242) HotStar Taq DNA Polymerase Qiagen 206143 Q-Solution Promega #DC6731 Promega #DC6531

rTth DNA Polymerase

Applied Biosystems

N8080179

RNasin (Promega, OderingNo: N2111) if single cells

PowerPlex 16 System

Appendix A: Tested kits and components

PowerPlex ES System

QIAGEN Multiplex PCR Kit blend of Sensiscript, Omniscript Reverse Transcriptases, HotStar Taq DNA Polymerase HotStar Taq DNA Polymerase Qiagen Qiagen 210210

QIAGEN OneStep RT-PCR

Q-Solution; RNasin (Promega, Ordering-No: N2111) if single cells

Appendix A: Tested kits and components

The AmpliGrid is an open platform and so far no limitations with respect to enzymes or reagent kits are known.

Please contact Beckman Coulter support (see page 2) for detailed information regarding integrated solutions or special application protocols.

Taq DNA Polymerase

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Appendix B Troubleshooting

01 Product Information Kit Components Storage Information Recommended equipment Materials required but not provided Precautions before Use 02 AmpliGrid AG480F: Experimental Procedure 03 AmpliGrid AG480E: Experimental Procedure 04 Special Applications 05 Order Information Appendix A: Tested kits and components Appendix B: Troubleshooting

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Appendix B: Troubleshouting

Appendix B: Troubleshooting
Table 4: Possible causes and solutions

Problem
No detectable amplification product General

Possible causes

Solution

DNA polymerase out of range Primer concentration out of range HotStart Taq activated for at least 10 min Cycling conditions not optimal Pipetting error Mg2+ concentration not optimal Cycle number too low Elongation times too short

Concentration should be 0.5 - 1 U/L Adjust primer concentration to ~ 0.2 pmol/L Change temperature protocol Conditions in ultra-low volumes might have to be adjusted compared to standard volume amplifications Please check proper deposition of reagents and components to the master mix or the AmpliGrid Carry out a dilution series from 1 - 5 mM Increase cycle number Long templates require increased elongation times. Usual processing rate of DNAPolymerase is 1kbp/min Check with a microscope for presence Change temperature profile Include positive controls Decrease amount of DNA Increase amount of DNA. Detection limit of the AmpliGrid slide is in the 10 - 20 pg range Include positive controls Do not dry RNA templates on the AmpliGrid. We recommend including RNA as a component of the master mix

Template single cell

Cell not deposited correctly on hydrophilic reaction site Cell lysis at 95C takes 10 min at the minimum

Template DNA dilution

Template degraded DNA amount too high. PCR process inhibited DNA amount too low

Template RNA dilution

Template degraded

Template from microdissected samples

Residual membranes from laser cutting might interfere with the amplification reaction Dyes like e.g., Hmacolour/ Hmalaun inhibit PCR reactions

Increase initial denaturation to 15 min or more

Change dyes Increase annealing temperature Redesign primers Increase concentration or change stock solution Decrease detergent concentration

Unspecific bands

Annealing temperature too low Primer design not efficient Primers degraded

Oil droplets mix together

Detergent concentration in reaction buffer too high Volume of sealing solution too high (max. 5 L)

Adjust volume to 4.8 - 5 L NOTE: Some aqueous solution may interfere with the sealing solution. It might be necessary to reduce the volume of sealing solution

Volume of aqueous phase too high (max 1 L) Air bubbles in sealing solution Pipetting speed too fast

Decrease volume Use pipette tip to remove air bubbles and decrease pipetting speed

Air bubbles introduced during pipetting

Use electronic multistepper pipets Reduce the heating rate of the first denaturation cycle (e.g. 0.5C/sec)

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Document History
Version 1.0 2.0 3.0 3.1 3.2 3.3 3.4 Date 01.11.2005 01.11.2006 14.03.2007 31.07.2009 13.10.2009 27.09.2010 15.11.2010 Remarks Creation of document Modification CI layout Olympus Change of Logo Modification Layout Change Beckman Coulter Content changes

Sauerbruchstr. 50 D - 81377 Munich Phone +49 89 579 589 - 3540 Fax +49 89 579 589 - 3503

BECKMAN COULTER BIOMEDICAL GMBH