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AG480F / E

1 L Assays

USER GUIDE

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of 23 AmpliGrid AG480 User Guide M10002 Version 3.4

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 exist ing artwork or images from thisguide that you may want to include in your project may be protected under copyright law. The unauthorised incorporation of such material into yournew work could be a violation of the rights of the copyright owner. Please be sure to obtain any permission required from the copyright owner. Anyreferences 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 GmbHSauerbruchstrae 50

D-81377 MunichPhone: +49-(0)89-579589-3540Fax: +49-(0)89-579589-3503

E-Mail: [email protected]

For all other regions go towww.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 storage temperature expiry date

refer to manual REF LOTordering information Lot-number

15C

25C

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AmpliGrid AG480 User Guide M10002 Version 3.4 3of 23

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

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User Guide - AmpliGrid AG480F

Intended Use

The AmpliGrid slides have been designed by Beckman Coulter tocarry out a wide range of biological and biochemical reactions ina 1 L volume. This includes exponential amplication, isothermal

amplication, whole genome amplication, 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 InformationKit ComponentsStorage InformationRecommended equipmentMaterials required but not

providedPrecautions before Use

02 AmpliGrid AG480F: Experi-

mental Procedure03 AmpliGrid AG480E: Experi-

mental Procedure04 Special Applications05 Order Information

Appendix A: Tested kits andcomponents



Kit Components

Storage Information

Recommended Equipment

Materials required but not

provided


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The AmpliGrid slides (g. 1) comprise 48 lithographically dened

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 bystructured chemical surfaces which form reaction sites surroundedby hydrophilic and hydrophobic circles (g. 2). Components for

biological or chemical reactions can be pre-deposited either as amaster mix or sequentially with intermediate air-drying.

All AmpliGrid products are manufactured and packed in state-of-

the-art class 100 clean rooms to ensure superior product quality.

The AmpliGrid line substrate uses ultra-at and extra-lowuorescence 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 center-to-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 anautomated liquid handling robot or uorescence activated cellsorters.

The unique optical quality allows for superior control of e.g. cell

deposition as well as image acquisition using uorescent scanners.

Beckman Coulter offers two different formats of the AmpliGridsystem:

AmpliGrid AG480F

Features: 100% DNA-free with an inactivated surface to ensurelowest binding efciency for biomolecules like polymerases ornucleic acids.

Applications: 1 L amplication research assays (e.g. PCR / RT-PCR),

single cell deposition using ow cytometry instruments, long termstorage of DNA and cells (no covalent immobilisation), target for

microdissection.


Figure 1: Dimensions [mm] of the AmpliGrid slide

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

site

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AmpliGrid AG480E

Features: Highly reactive epoxy coated surface (g. 3) for covalentimmobilisation of both amino-modied and unmodied long oligos,cDNAs, proteins, cells and tissues.

Research Applications: 1 L amplication and/or hybridisation assays(microarray based), solid phase PCR, immobilising biomolecules fordownstream analysis, target for microdissection.

Kit ComponentsTable 1: Kit components

Order Number AmpliGrid slides Sealing Solution Product Manual

OAX 04503/04509 1x 5 5x 500 L 1

OAX 04505/04511 1x 25 25x 500 L 1

OAX 04504/04510 1x 5 1x 4 mL 1

OAX 04506/04512 1x 25 4x 4 mL 1

Storage Information

To ensure constant quality, the AmpliGrid slides and the sealingsolution 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 usedimmediately or can be stored at room temperature in a dark, dry

and dust-free place. Do not use any of the components after expirydate.

Recommended Equipment Thermal Cycler

All AmpliGrid experiments have been optimised with AmpliSpeed

ASC200D slide cycler (g. 4; Beckman Coulter, Order No. :OAX04102).

Electronic Multi-step pipettes (e.g. Eppendorf Multipette

Stream or Rainin AutoRep E). Manual multi-step pipettes mightcause 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 oragarose gel electrophoresis, UV-spectrometer).

Figure 3: Cross section of AG480E epoxy coated microliter

reaction site


Figure 4: AmpliSpeed ASC200D slide cycler

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Materials required but not provided

Powder free gloves.

Template (DNA / single cells / tissues).

Amplication reagents (master mix, single components,polymerase).

Note: Some commercially available DNA polymerases havenot been designed for use in low volumes. Please refer to theBeckman Coulter support (see page 2) or Appendix A for a

list of tested enzymes and kits.


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, sothat you can read the engraved markings.

Amplication of human DNA (e.g. forensic strains) is subjectto contaminations by very small amounts of non-template

human DNA. Extreme care and good laboratory practice isnecessary to avoid cross-contamination throughout all stepsof the experiment.

Each reaction drop should be covered with sealing solution

as soon as possible. The loaded reaction volume (1 L perreaction site) may dry depending on room temperature,humidity etc.

Inadequate volumes will compromise the interpretation of results.

A divided workow is advisable.

Wear powder free gloves and use aerosol resistant pipettetips.

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

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0of 23 AmpliGrid AG480 User Guide M10002 Version 3.4


1. Put the AmpliGrid slide on a dark surface to ensure goodvisualisation of the engraved circles marking the hydrophilicregions of the slide.

2. Optional: Up to 1 L of template solution e.g. primers, DNAdilutions or other sample material such as single cells, can betransferred onto each reaction site (g. 5) and subsequently air-

dried at room temperature, then the reaction mix can be addedas described in step 3.

3. Transfer 1 L of reaction mix onto each reaction site of the

AmpliGrid AG480F. Due to the hydrophilic structure of thereaction sites, aqueous liquids will automatically move to thecenter 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 mixbefore covering with the sealing solution.


5. Incubate the AmpliGrid on the AmpliSpeed slide cycler accordingto your required thermal cycling prole specications.

It might be necessary to adapt established thermal prolesdue to the superior performance of the AmpliSpeedinstrument.

6. Retrieving sample from the 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).

7. Sample is ready for analysis (capillary electrophoresis, PAGE, etc.).

Figure 5: Workow AmpliGrid AG480F


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3

4

Sorter

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User Guide


03 AmpliGrid AG480E:

Experimental Procedure








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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 spottingparameters, 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 CoulterBiomedical GmbH, Ref: OAX05205) for depositing DNAmolecules on the AmpliGrid surface. This ensures high quality spotmorphology and binding efciency.

Possible applications: DNA-Microarrays with integratedamplication, SNP-typing, antibiotic resistance screening ofmicroorganisms.

Inactivation of the slide surface and immobilisation of capturemolecules.

Incubate AmpliGrid AG480E slides over night at 42C in ahumidity chamber with a saturated water atmosphere.

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.

Air-dry or spin-dry the slides using the AdvaTube AT400

slide holder (Beckman Coulter, Order No.: OAX05216) ina 50 mL Falcon size swing-out bucket (3 min @ 500xg).

1. Put the AmpliGrid slide on a dark surface to ensure goodvisualisation 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-driedat room temperature. Then the reaction mix can be added asdescribed above (g. 6).

3.Transfer 1 L of reaction mix onto each reaction site of theAmpliGrid AG480E. Due to the hydrophilic structure of thereaction sites, aqueous liquids will automatically move to thecenter on contact with the surface.

Figure 6: Workow AmpliGrid AG480E


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4. Cover each droplet with 5 L of sealing solution.

NOTE: Ensure that there is no evaporation of the reaction mixbefore covering with the sealing solution.


5. Incubate the AmpliGrid on the AmpliSpeed slide cycler accordingto your required thermal prole specications.

It might be necessary to adapt established thermal proles due tothe 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 solutiononto a suitable column (e.g. Sephadex).

Proceed with the protocol for direct image analysis (below).

6b. Direct Image Analysis

Pre-soak slides thoroughly in a 1 % SDS / 2 x SSC solution toremove most of the sealing solution.

Subsequent washing steps should be performed according tostandard microarray washing protocols. NOTE: Do not useSDS-based washing solutions after the rst step as this mightresult in a higher background.

Use of AdvaWash AW400 automated slide washing station(g. 8; Beckman Coulter, Order No.: OAX05107) includingthe AdvaTube slide holder is recommended for reliable andreproducible washing conditions (tab. 2). Please refer to the

corresponding manual for further information.

Table 2: Washing conditions for AG480E in AdvaWash

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.


Reagent Soak time

2 x SSC / 0.1 % Triton X-100 5 min (Pulsing:Y)

1 x SSC 40 s

1 x SSC 4 min 20 s

0.2 x SSC 5 min

Figure 7: Magnication of pipetted AmpliGrid slide

Figure 8: AdvaWash AW400 automated slide washing station

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Remove the slides from the AdvaTube and blow-off excesssealing solution with compressed nitrogen to reduceuorescence background.

Detection of uorescent-labelled amplication 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 20g/mL and 1 mg/mL.

Pipet 1 L of protein solution onto the AmpliGrid reaction

site.

Incubate AmpliGrid over night in a humidity chamber. Ensurethat droplets do not evaporate.

Remove AmpliGrid slides from humidity chamber and allowthe droplets to dry at room temperature.

NOTE: Slides can be stored for several weeks at 4 C untiluse.

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 proteinslides. For high reproducibility we recommend using the AdvaWash

AW400 instrument (Beckman Coulter, Ref: OAX05107). Forfast and efcient drying of the slides we recommend using theAdvaTube slide holder (Beckman Coulter , Ref: OAX05216) in a

centrifuge with standard 50 mL Falcon size swing-out buckets at500xg for 2-3 min.


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User Guide










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Reagents and template material can be dispensed onto theAmpliGrid by standard liquid handling platforms (g. 9) or cellsorting devices.

Beckman Coulter offers high quality technical support by

experienced staff and own application laboratories. Our main goal isto guarantee full customer satisfaction with all our product lines.

Please contact - support (see page 2) for detailed informationregarding integrated solutions or special application protocols.

Single cell sorting by ow cytometry

The AmpliGrid slides t perfectly in the Beckman Coulter cellsorter devices (e.g. MoFlo XDP). This enables the user to sort cellsfor their Immunophenotyp using cell markers and checking theirgenetic level witin one workow.

The high accuracy of the instrument and the fast sort process onthe AmpliGris even allows for high throughput screenings of singlecells on a molecular level.

Automation of processes

For high throughput analyses and highly precise processing theAmpliGrid can be integrated into liquid handling platforms by eitherusing our slide adapters or integrating the complete system with

the AmpliSpeed cycler.

Figure 9: Automation of the AmpliGrid system

Figure 10: Single Cell sorting with MoFlo XDP

Figure 11: Automation process with AmpliSpeed and

Biomek 3000

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User Guide










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Table 3: Order Information

Product Description Order No.

AmpliGridAG480F

DNA-free microliter reaction slide(5 pcs./pck.), 48 reaction sites each,incl. 5 x 500 L sealing solution

OAX04503

AmpliGrid

AG480F

DNA-free microliter reaction slide

(5 pcs./pck.), 48 reaction sites each,incl. 1 x 4 mL sealing solution

OAX04504

AmpliGridAG480F

DNA-free microliter reaction slide(25 pcs./pck.), 48 reaction sites each,incl. 25 x 500 L sealing solution

OAX04505

AmpliGridAG480F DNA-free microliter reaction slide(25 pcs./pck.), 48 reaction siteseach, incl. 4 x 4 mL sealing solution

OAX04506

AmpliGridAG480E

Epoxy coated microliter reactionslide (5 pcs./pck.), 48 reaction

sites each, incl. 5 x 500 L sealingsolution

OAX04509

AmpliGridAG480E

Epoxy coated microliter reactionslide (5 pcs./pck.), 48 reaction siteseach, incl. 1 x 4 mL sealing solution

OAX04510

AmpliGridAG480E

Epoxy coated microliter reactionslide (25 pcs./pck.), 48 reaction

sites each, incl. 25 x 500 L sealingsolution

OAX04511

AmpliGrid

AG480E

Epoxy coated microliter reaction

slide (25 pcs./pck.), 48 reaction siteseach, incl. 4 x 4 mL sealing solution

OAX04512

Sealing

Solution

Sealing solution 1 x 500 L ready to

use solution

OAX04207

SealingSolution

Sealing solution 1 x 4 mL ready touse solution

OAX04208


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User Guide


Appendix A

Tested kits and components








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

Table 4: Tested PCR Kits and components

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

special application protocols.

K

it

Polymerase

Manufacturer

Cat.No

.

Remarks

A

mpF/STRSefler

PCRAmplifcationKit

AmpliTaqGold

DNA

Polymerase

(AppliedBiosystems;O

rdering-No:

N8080242)

Applied

Biosystems

4335129

B

lueTaq

BlueTaq

(hot-startTa

q)

EuroClone

EME011500

FluoCycle

Probe

BlueTaq

(hot-startTa

q)

EuroClone

EMQ011100

G

eneAmp

EZrTth

R

NAPCRKit

rTthDNAPolymerase

Applied

Biosystems

N8080179

RNasin

(Promega,Odering-

No:N2111)ifsinglecells

PowerPlex

16System

AmpliTaqGold

DNA

Polymerase

(AppliedBiosystems;O

rdering-No:

N8080242)

Promega

#DC653

1

PowerPlex

ESSystem

AmpliTaqGold

DNA

Polymerase

(AppliedBiosystems;Ordering-No:

N8080242)

Promega

#DC673

1

Q

IAGEN

Multiplex

PCRKit

HotStarTaq

DNAPo

lymerase

Qiagen

206143

Q-Solution

Q

IAGEN

OneStep

RT-PCR

blendofSensiscript,O

mniscript

ReverseTranscriptases,HotStarTaq

DNAPolymerase

Qiagen

210210

Q-Solution;RNasin

(Promega,Ordering-No:

N2111)ifsinglecells

TaqDNAPolymerase

HotStarTaq

DNAPo

lymerase

Qiagen


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User Guide


Appendix B

Troubleshooting








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Appendix B: Troubleshouting


Table 4: Possible causes and solutions

Problem Possible causes Solution

No detectableamplication product

General DNA polymerase out of range Concentration should be 0.5 - 1 U/L

Primer concentration out of range Adjust primer concentration to ~ 0.2 pmol/L

HotStart Taq activated for at least10 min

Change temperature protocol

Cycling conditions not optimal Conditions in ultra-low volumes might have to be adjusted compared to standardvolume amplications

Pipetting error Please check proper deposition of reagents and components to the master mix or theAmpliGrid

Mg2+ concentration not optimal Carry out a dilution series from 1 - 5 mM

Cycle number too low Increase cycle number

Elongation times too short Long templates require increased elongation times. Usual processing rate of DNA-Polymerase is 1kbp/min

Template single cell Cell not deposited correctly onhydrophilic reaction site

Check with a microscope for presence

Cell lysis at 95C takes 10 min atthe minimum

Change temperature prole

Template DNA dilution Template degraded Include positive controls

DNA amount too high. PCRprocess inhibited

Decrease amount of DNA

DNA amount too low Increase amount of DNA. Detection limit of the AmpliGrid sl ide is in the 10 - 20 pgrange

Template RNA dilution Template degraded Include positive controls

Do not dry RNA templates on the AmpliGrid. We recommend including RNA as acomponent of the master mix

Template frommicrodissected samples

Residual membranes from lasercutting might interfere with theamplication reaction

Increase initial denaturation to 15 min or more

Dyes like e.g., Hmacolour/Hmalaun inhibit PCR reactions

Change dyes

Unspecic bands Annealing temperature too low Increase annealing temperature

Primer design not efcient Redesign primers

Primers degraded Increase concentration or change stock solution

Oil droplets mixtogether

Detergent concentration inreaction buffer too high

Decrease detergent concentration

Volume of sealing solution toohigh (max. 5 L)

Adjust volume to 4.8 - 5 L

NOTE: Some aqueous solution may interfere with the sealing solution. It might benecessary to reduce the volume of sealing solution

Volume of aqueous phase too high(max 1 L)

Decrease volume

Air bubbles in sealingsolution

Pipett ing speed too fast Use pipette t ip to remove air bubbles and decrease pipett ing speed

Air bubbles introduced duringpipetting

Use electronic multistepper pipets

Reduce the heating rate of the rst denaturation cycle (e.g. 0.5C/sec)

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BECKMAN COULTER BIOMEDICAL GMBHSauerbruchstr. 50 D - 81377 Munich Phone +49 89 579 589 - 3540 Fax +49 89 579 589 - 3503

Document History

Version Date Remarks

1.0 01.11.2005 Creation of document

2.0 01.11.2006 Modication

3.0 14.03.2007 CI layout Olympus

3.1 31.07.2009 Change of Logo

3.2 13.10.2009 Modication

3.3 27.09.2010 Layout Change Beckman Coulter

3.4 15.11.2010 Content changes

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