System IntroductionApplied Biosystems 7300 Real-Time PCR System

Applied Biosystems 7500 & 7500 Fast Real-Time PCR System

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Applied Biosystems 7300 &7500 Real-Time PCR System, 7500 Fast Real-Time PCR System

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7300 & 7500 hardware

� Excitation source : Halogen- Tungsten lamp� 7300 :

– 1 excitation filter & 4 emission filters� 7500 :

– 5 excitation filters & 5 emission filters (matched to Ex)� The emission is measured by a CCD camera

� 7500 and 7500 Fast differ by block and electronics

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Multicolor Detection 7500

Dyes:

SYBR® Green 1 528 nm

FAM™ 530 nm

VIC® 554 nm

JOE™ 554 nm

NED™ 576 nm

TAMRA™, Cy3 ™ 582 nm

ROX™, TEXAS RED ® 610 nm

Cy5 ™ 650 nm

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Spectral Overlap

500 nm 660 nm

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FAM™ VIC® TAMRA™ ROX™ (dyes)

First cycle

Last cycle,Increase of fluorescence signal in filter A (Fam or SYBR Green)

Spectra View – Raw Data7300 instrument

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Component View : Matrix CalculationFrom spectra, using Background and Pure Spectra files, a matrix calculation is done giving the contributions of the different dyes

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Semilog or linear view

Amplification Plot ViewReporter Signal

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� Single Tube, Dual Probe Detection � Represents a plate read after a PCR reaction

Fluo

resc

ence

Inte

nsity

Dye

I

Fluorescence Intensity Dye II

Allelic Discrimination View

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• Step for data collection can be selected• Annealing/extension step recommended • Keep extension step ≥ 32 seconds

Data Collection

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• Data acquisition in all 96 wells• On-line monitoring • Auto-save function after run is completed• Settings for baseline and threshold as well as results

are saved in an .sds file • Template files can be created as „.sdt“

Analysis parameters can be included • Setup of 4 standard curves per run is possible

SDS SoftwareGeneral information

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• Run on Windows XP SP1• Relative quantitation (RQ) analysis as add-on for the 7300

system or included in the 7500 system• Incl. 5 network user licences or

unlimited licences for unlinked computers

SDS Software

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Latest Software Version

� SDS v1.4 software for 7300 and 7500 instruments

� SDS v1.4 software to assist with 21CFRp11

– For 7500/7500Fast systems only

– Including a new firmware version

�SDS v1.4 is not being distributed to the existing

7300 / 7500 / 7500 Fast systems install base

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– Runs on Windows XP SP1

– Extend cycles during run

• Adds cycles before the run is complete

• One extension per run possible

– Fast thermal cycling mode for 7500 Fast System

– Not compatible with 7000 instruments The 7000 System software will remain at SDS Software v1.2.3

SDS Software v1.4

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- Extend cycles during run

SDS Software v1.4

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Expert Mode for the 7500 Fast System only� Provides the capability

– to reduce run times to less than 30 min

– for users to select only those filters for which they wish to collect data

� Only available when selecting “Absolute” or “RQ” documents with “Fast Cycling” Mode

� Mandatoty for HRM analysis

� Delta Rn is expected to be slightly lower when using Expert Mode with default extension time

� Obtain UB through Documents on Demand p/n 4367499A

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� The default extension time for Expert Mode is reduced to 20 sec instead of 30 sec

Filter Selection

Expert Mode for the 7500 Fast System only

� Minimum time requirement depending on the number of selected filters

Disposables

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� Optical 96-well Reaction Plate (with or without barcode)– supported volume 25-100 µl

� MicroAmp® Optical Adhesive Covers

Note:– Use the correct Precision Plate Holder– Do not use a compression pad

Disposables96-well plates

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� Optical 96-well Fast Thermal Cycling Plate– supported volume 10-30 µl

� MicroAmp® Optical Adhesive Covers

Note: – Centrifugation of 96-well Fast plates at 900xg, 1 min is

recommended– Use the correct Precision Plate Holder– Do not use a compression pad

Disposables96-well Fast plates only for the 7500 Fast instrument

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DisposablesTube Strips

� MicroAmp® Optical Caps, 8 flat Caps / Strip

� MicroAmp® Optical Tubes, 8 Tubes / Strip are closed with caps

� MicroAmp® Fast 8-Tube Strips

Note:

� Use the correct Precision Plate Holder

� Optical Tube Strips or single Tubes are available for the 7500 Fast instrument if using the correct Plate holder

� Do not use the MicroAmpTM 96-well Tray / Retainer in the instrument

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• AB 7300 or 7500 instruments have two different PPHs each:• One for the 96-well Optical Reaction Plates • One for MicroAmp� Optical Tubes

• AB 7500 FAST have two different PPHs NOW• One for Optical 96-well Fast Thermal Cycling Plate

• One for MicroAmp� Fast Optical Tubes

Precision Plate Holder

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„Help“ Function in SDS Software

MaintenanceApplied Biosystems 7300 Real-Time PCR SystemApplied Biosystems 7500 & 7500 Fast Real-Time PCR System

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� System Start: a. Computerb. Real-Time PCR Systemc. SDS Software

� Restart the computer daily

� Switch the instrument off when not in use

� Do not use other software while data are collected

General Recommendations

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• Keep distance to the wall to allow efficient cooling

• Keep instrument under controlled temperature conditions

• Avoid direct sunlight

• Full functionality up to 30°C

General RecommendationsInstrument Location

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If the pixel intensity shows a bright spot in the ROI Inspector view, and background raw data exceeds 72.000 rfu, check the thermal cycler block for contamination:

• Leave the block empty

• Open ROI Inspector

• Block up

• Take Snapshots

Monthly Maintenance

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2048 ms

Filter A

Snapshot

Block up

�If any well shows a bright spot, run a background run (see following slides)

• Open a new document

• Instrument > Calibrate

ROI Inspector

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• Use Water HPLC grade or EtOH p.a a.) Cleaning with

cotton budsb.) Cleaning with lint- and fluorescence-free Kimwipes®

Block Cleaning

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Instrument → Lamp Status/Replacement

Note: Lamp automatically stops 4h after run is completed (if nobody touches the computer)

Lamp Status /Replacement

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• Run ROI calibration • using the „ROI Calibration Plate“ from the „Spectral

calibration Kit“ for 7300 & 7500 systems(using the Green Tray for 7000 system)

• Run and extract a background

• using the „Background Plate“ from the „Spectral Calibration Kit“ or any 96 well plate filled with water.Signal should be:

< 72.000 for 7300 / 7500 systems Filter A-D< 90.000 for filter E (only 7500 system)(< 1.200 for the 7000 system)

After a Lamp Change

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Check ROI Mask after Lamp Change1) Open a new Document 2) Instrument > Calibrate, select „Block up“3) Select first Filter A, take „Snapshot“4) Click „Generate calibration“ green circles appear5) Save calibration6) Run calibration for each filter

- 1024 ms- Block up- Select Filters

A to D or E- Take Snapshot

�Please follow the guidelines of the „Installation and Maintenance“ Guide

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Instrument Log

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• Perform a System Function Test: select „ALL Tests“

Never select „Download Firmware“

Function Test

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• 1) Generate and extract a new background file using a water plate

• 2) Defragment computer hard disk after backing up data!

Start → Programs → Accessories → System Tools →Disk Defragmenter

Bi-annual Maintenance

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• Calibrate the instrument using instrument specific calibration kits • Applied Biosystems 7300 Real Time PCR Systems

Spectral Calibration Kit

• Applied Biosystems 7500 Real Time PCR Systems Spectral Calibration Kit I

Note: Back-up calibration documents on an external disk

Annual Maintenance

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And see Getting Started Guide for „Installation and Maintenance“

Use Online Help

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System Maintenanceby Applied Biosystems Service Team

• We offer a wide selection of service options that can help you to keep your system on a high performance level

• Regular Planned Maintenance by Service engineers

• Assure highest priority response for repair

• Keep instrument up-to-date (software and hardware)

• Increase reliability & data accuracy

• Maximize instrument uptime, performance and lifetime

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Legal Statements � For Research Use Only. Not for use in diagnostic procedures.

� NOTICE TO PURCHASER:

� PLEASE REFER TO THE USER'S GUIDE OR PACKAGE INSERT OF THE PRODUCTS NAMED HEREIN FOR LIMITED LABEL LICENSE OR DISCLAIMER INFORMATION.

� Applera, Applied Biosystems, AB (Design), ABI PRISM, MicroAmp, and VIC are registered trademarks and FAM, JOE, NED, ROX and TAMRA are trademarks of Applera Corporation or its subsidiaries in the US and/or certain other countries.

� SYBR Green is a registered trademark of Molecular Probes, Inc.

� All other trademarks are the sole property of their respective owners.

� © 2008 Applied Biosystems. All rights reserved.