AmpliGrid AG480F

Reaction slide for 1 L Assays & Single Cell Analysis.

Order Information and Kit Components

Ref.-No. oaX04503 oaX04504 oaX04505 Product ag480f ag480f ag480f Description dna-free microliter slide (5 pcs./pck), 48 reaction sites each, incl. 5x500 l sealing solution dna-free microliter slide (5 pcs./pck), 48 reaction sites each, incl. 1x4 ml sealing solution dna-free microliter slide (25 pcs./pck), 48 reaction sites each, incl. 25x500 l sealing solution dna-free microliter slide (25 pcs./pck), 48 reaction sites each, incl. 4x4 ml sealing solution

Product Information
the ampligrid is a superior amplification platform for single cell applications, based on a chemically structured microscope slide. With this innovative system, sensitivity, efficiency and reproducibility are reliable constants for single cell analysis, low-copy number approaches and ultra low volume amplification techniques. the ampligrid slides comprise 48 chemically defined reaction sites, on their surface each holding 1 l of reaction solution. evaporation is prevented by covering the aqueous phase with a special sealing solution (see fig. 1). all liquid components are safely contained so that the loaded slide can be transferred safely on the amplispeed slide cycler. components for biological or chemical reactions can be pre-deposited either as a mix or sequentially with intermediate air-drying. due to the glass substrate of the slide no binding of biological molecultes as nucleic acids takes place. everythin added to the slide takes part in the reation. the ampligrid 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 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 an automated liquid handling robot or fluorescence activated cell sorters. the perfect flatness of the glass substrate which allows for precise optical control of the whole workflow using a microscope or a slide scanner and the unique optical quality allows for superior control of e.g. cell deposition as well as image acquisition using fluorescence scanners.

oaX04506

ag480f

contact us via e-mail at singelcell@beckman.com, visit on-line at www.advalytix.com or contact your local representative

Storage Conditions
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.

Quality Control
all ampligrid products are manufactured and packed in a state-of-theart cleanroom to ensure superior product quality. the reaction sites are inactivated so that there is no binding of biomolecules e.g. nucleic acids or proteins. furthermore, ampligrid ag480f slides are certified rna and dna free. to ensure constant quality the ampligrid slides and the sealing solution are packed under inert gas in light-protective aluminum foil. each lot is quality controlled for physical and biological performance.

Applications
the ampligrid system provides workflows to guide researchers through single cell analysis (e.g. Pcr/rt-Pcr), no matter whether single cells are captured via flow sorting, micromanipulation or laser capture microscopy, or whether downstream analysis involves qPcr, Page, ce or sequencing. due to the structured, two-dimensional layout of the ampligrid, single cells can be easily deposited on the slide surface by a flow cytometry instrument (moflo), rendering a variety of single cell applications possible as well as providing full access to the one-cell genome. one big advantage over other amplification platforms is the following applications have been successfully established on ampligrid system: analysis of the genetic heterogeneity of tumor stem cells or circulating tumor cells increasing sensitivity and saving costly reagents of standard human identification kits in forensics or paternity testing single cell Pcr, rt-Pcr, rt-qPcr or sequencing for molecular analysis of cells after high throughput cell sorting Pre-amplifying single cells with the ampligrid system followed by standard qPCR combines existing workflows with superior single cell sensitivity and allows measurement of the mrna expression High throughput cell sorting on the ampligrid slides for adding single cell molecular analysis with the Beckman coulter moflo and other sorters using our flow cytometry adaptor and calibration slide

Customer Support
Beckman Coulter Biomedical GmbH Sauerbruchstrae 50 81377 Munich - Germany Telephone +49 (0) 89 579 589 3540 Fax +49 (0) 89 579 589 3503 E-Mail singlecell@beckmancoulter.com
1 Figure 1: Cross section of an AmpliGrid reactionsite.

Productinformation amPligrid ag480f #Pi10004 Version 1.0

User Guide
Materials required but not provided
Powder free gloves. template (single cells / dna / rna / tissues). amplification reagents (master mix, polymerase, single components). note: some commercially available dna polymerases have not been designed for use in low volumes. Please refer to the Beckman coulter support or visit our application section at www.advalytix.com for information about tested enzymes and kits. amplispeed slide cycler instrument. electronic multi-step pipettes (e.g., eppendorf multipette stream or Biohit eline). note: 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., qPcr system, capillary electrophoresis, polyacrylamide or agarose gel electrophoresis, uV-spectrometer).

6.

retrieving sample from the ampligrid: remove the ampligrid from the amplispeed slide cycler. gel analysis: add loading dye by gently touching the top of the sealing solution with the drop of loading dye (e.g. 4 l). the loading dye will move through the sealing solution and merge with the Pcr sample. retrieve the liquid by piercing the pipette through the sealing solution into the aqueous phase, aspirate the complete aqueous phase (e.g. 4 l loading dye + 1 l sample) and load the gel. other analyses: Pipette the aqueous phase into a new repository by piercing through the sealing solution and aspirating the aqueous phase (1 l sample). add buffers for downstream processing and continue with analysis (e.g., ce sequencing, qPcr). note: alternatively add 4 l of buffers for downstream processing or nuclease free water to the sample as described for the gel analysis procedure. By increasing the volume before retrieving the sample from the ag pipetting gets easier and pipetting errors are minimized.

2 Figure2: AmpliGrid AG480F

Precautions before Use

slide surface must not be touched. Please work under 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. a mplification of human dna (e.g., forensic stains) is subject to contamination by very small amounts of non-template human dna. extreme care and good laboratory practice is necessary to avoid crosscontamination 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.

Product use Limitations, Warranty, Disclaimer

Beckman coulter manufactures products for a number of intended uses. Please refer to the product label for the intended use statements for specific products. Beckman coulter products contain chemicals which may be harmful if misused. due care should be exercised with all Beckman coulter products to prevent direct human contact. each Beckman coulter product is shipped with documentations stating specifications and other technical information. Beckman coulter products are warranted to meet or exceed the stated specifications. Beckman coulters sole obligation and the customers sole remedy is limited to replacement of products free of charge in the event products fail to perform as warranted. Beckman coulter makes no other warranty of any kind whatsoever and sPecificallY disclaims and eXcludes all otHer Warranties of anY Kind or nature WHatsoeVer, directlY or indirectlY, eXPress or imPlied, including WitHout limitation as to tHe suitaBilitY, ProductiVitY, duraBilitY, fitness for a Particular PurPose or use, mercHantiBilitY, condition, or anY otHer matter WitH resPect to Beckman coulter Products . in no event shall Beckman coulter be liable for claims for any other damages, whether direct, incidental, foreseeable, consequential or special (inducing but not limited to loss of use, revenue or profit), whether based upon warranty, contract, tort (including negligence) or strict liability arising in connection with the sale or the failure of Beckman coulter products to perform in accordance with the stated specifications.
eppendorf multipette is a registered trademark of eppendorf ag, germany. Biohit eline is a registered trademark of Biohit, deutschland gmbH, germany

Experimental Procedure
1. 2. Place the ampligrid slide on a dark surface to ensure good visualization of the engraved circles marking inner spot of the reaction site. up to 1 l of template solution e.g., primers, dna dilutions or other sample material such as single cells, can be deposited onto each reaction site and subsequently air-dried at room temperature - then the process goes on as described in step 3.

3. transfer 1 l of reaction mix onto each reaction site tu be used of the ampligrid ag480f. due to the hydrophilic structure of the reaction sites aqueous liquids will self-acting move to the center when 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.

5. transfer the ampligrid on the amplispeed slide cycler start your required thermal cycling profile specifications. it might be necessary to adapt established thermal profiles due to the superior performance of the amplispeed instrument.

For research use only. Not for use in diagnostic procedures.

BeCkman CoulteR BiomediCal gmBh Sauerbruchstrae 50, 81377 munich germany tel: +49 (0)89 579589-0, Fax: +49 (0)89 579589-3503 e-mail: singlecell@beckman.com www.advalytix.com Worldwide Biomedical Research Division Offices: australia (61) 2 98446000 Canada (905) 819-1234 China (86) 10 6515 6028 Eastern Europe, Middle East, North Africa (41) 22 994 07 07 France 01 49 90 90 00 Hong Kong (852) 2814 7431/2814 0481 Italy 02-953921 Japan 03-5404-8359 Mexico (55) 560-57770 Netherlands 0297-230630 Singapore (65) 63393633 South-Africa, Sub-Saharan Africa (27) 11-805-2014/5 Spain 91 3836080 Sweden 08-564 85 900 Switzerland 0800 850 810 Taiwan (886) 2 2378 3456 Turkey 90 216 309 1900 U.K. 01494 441181 U.S.A 1-800-526-3821