System Introduction Applied Biosystems 7300 &7500 Real ... - CRN2M

System IntroductionApplied Biosystems 7300Real-Time PCR SystemApplied Biosystems 7500 &7500 Fast Real-Time PCR SystemApplied Biosystems 7300 &7500 Real-Time PCRSystem, 7500 Fast Real-Time PCR System2¢¡¤£ £ ¥§¦©¨ ¨

7300 & 7500 hardwareExcitation 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 camera7500 and 7500 Fast differ by block and electronics3¢¡¤£ £ ¥§¦©¨ ¨ Multicolor Detection 7500Dyes:SYBR ® Green 1FAMVIC ®JOENEDTAMRA, Cy3 ROX, TEXAS RED ®Cy5 528 nm530 nm554 nm554 nm576 nm582 nm610 nm650 nm5¢¡¤£ £ ¥§¦©¨ ¨

Spectral Overlap500 nm 660 nm6¢¡¤£ £ ¥§¦©¨ ¨ Spectra View – Raw Data7300 instrumentFAM VIC ® TAMRA ROX (dyes)First cycleLast cycle,Increase offluorescence signalin filter A (Fam orSYBR Green)7¢¡¤£ £ ¥§¦©¨ ¨

Component View : Matrix CalculationFrom spectra, using Background and Pure Spectra files,a matrix calculation is done giving the contributions of thedifferent dyes8¢¡¤£ £ ¥§¦©¨ ¨ Amplification Plot ViewReporter SignalSemilog or linear view9¢¡¤£ £ ¥§¦©¨ ¨

Allelic Discrimination View Single Tube, Dual Probe Detection Represents a plate read after a PCR reactionFluorescence Intensity Dye I10Fluorescence Intensity Dye II¢¡¤£ £ ¥§¦©¨ ¨ Data Collection• Step for data collectioncan be selected• Annealing/extension steprecommended• Keep extension step≥ 32 seconds11¢¡¤£ £ ¥§¦©¨ ¨

SDS SoftwareGeneral information• Data acquisition in all 96 wells• On-line monitoring• Auto-save function after run is completed• Settings for baseline and threshold as well as resultsare 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 possible12¢¡¤£ £ ¥§¦©¨ ¨ SDS Software• Run on Windows® XP SP1• Relative quantitation (RQ) analysis as add-on for the 7300system or included in the 7500 system• Incl. 5 network user licences orunlimited licences for unlinked computers13¢¡¤£ £ ¥§¦©¨ ¨

Latest Software VersionSDS v1.4 software for 7300 and 7500 instrumentsSDS v1.4 software to assist with 21CFRp11– For 7500/7500Fast systems only– Including a new firmware versionSDS v1.4 is not being distributed to the existing7300 / 7500 / 7500 Fast systems install base14¢¡¤£ £ ¥§¦©¨ ¨ SDS Software v1.4– 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 instrumentsThe 7000 System software will remain at SDS Software v1.2.315¢¡¤£ £ ¥§¦©¨ ¨

SDS Software v1.4- Extend cycles during run16¢¡¤£ £ ¥§¦©¨ ¨ Expert Mode for the 7500 Fast System onlyProvides the capability– to reduce run times to less than 30 min– for users to select only those filters for which they wish to collect dataOnly available when selecting “Absolute” or “RQ” documents with “FastCycling” ModeMandatoty for HRM analysis Delta Rn is expected to be slightly lower when using Expert Mode withdefault extension timeObtain UB through Documents onDemand p/n 4367499A17¢¡¤£ £ ¥§¦©¨ ¨

Disposables96-well platesOptical 96-well Reaction Plate (with or without barcode)– supported volume 25-100 µlMicroAmp ® Optical Adhesive CoversNote:– Use the correct Precision Plate Holder– Do not use a compression pad35¢¡¤£ £ ¥§¦©¨ ¨ Disposables96-well Fast platesonly for the 7500 Fast instrumentOptical 96-well Fast Thermal Cycling Plate– supported volume 10-30 µlMicroAmp ® Optical Adhesive Covers36Note:– Centrifugation of 96-well Fast plates at 900xg, 1 min isrecommended– Use the correct Precision Plate Holder– Do not use a compression pad¢¡¤£ £ ¥§¦©¨ ¨

DisposablesTube StripsMicroAmp ® Optical Caps, 8 flat Caps / StripMicroAmp ® Optical Tubes, 8 Tubes / Strip are closed with capsMicroAmp® Fast 8-Tube StripsNote: Use the correct Precision Plate Holder Optical Tube Strips or single Tubes are available for the 7500 Fast instrument if usingthe correct Plate holder Do not use the MicroAmp TM 96-well Tray / Retainer in the instrument37¢¡¤£ £ ¥§¦©¨ ¨ Precision Plate Holder• 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 Tubes38¢¡¤£ £ ¥§¦©¨ ¨

„Help“ Function in SDS Software39¢¡¤£ £ ¥§¦©¨ ¨ MaintenanceApplied Biosystems 7300 Real-Time PCR SystemApplied Biosystems 7500 & 7500 Fast Real-Time PCR System

General Recommendations 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 collected41¢¡¤£ £ ¥§¦©¨ ¨ General RecommendationsInstrument Location• Keep distance to the wall to allow efficient cooling• Keep instrument under controlled temperatureconditions• Avoid direct sunlight• Full functionality up to 30°C42¢¡¤£ £ ¥§¦©¨ ¨

Monthly MaintenanceIf the pixel intensity shows a bright spot in the ROI Inspectorview, and background raw data exceeds 72.000 rfu, check thethermal cycler block for contamination:• Leave the block empty• Open ROI Inspector• Block up• Take Snapshots43¢¡¤£ £ ¥§¦©¨ ¨ ROI Inspector• Open a new document• Instrument > Calibrate2048 msBlock upFilter ASnapshot44If any well shows a bright spot, run abackground run (see following slides)¢¡¤£ £ ¥§¦©¨ ¨

Block Cleaning• Use Water HPLC grade or EtOH p.aa.) Cleaning withcotton budsb.) Cleaning with lint- andfluorescence-free Kimwipes ®45¢¡¤£ £ ¥§¦©¨ ¨ Lamp Status /ReplacementInstrument → Lamp Status/ReplacementNote: Lamp automatically stops 4h after run is completed(if nobody touches the computer)46¢¡¤£ £ ¥§¦©¨ ¨

After a Lamp Change• Run ROI calibration• using the „ROI Calibration Plate“ from the „Spectralcalibration Kit“ for 7300 & 7500 systems(using the Green Tray for 7000 system)• Run and extract a background• using the „Background Plate“ from the „SpectralCalibration 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)¢¡¤£ £ ¥§¦©¨ ¨ 47Check ROI Mask after Lamp Change1) Open a new Document2) 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 FiltersA to D or E- Take Snapshot48Please follow the guidelines of the£ ¥§¦©¨ ¨ ¢¡¤£„Installation and Maintenance“ Guide

Instrument Log49¢¡¤£ £ ¥§¦©¨ ¨ Function Test• Perform a System Function Test: select „ALL Tests“Never select„Download Firmware“50¢¡¤£ £ ¥§¦©¨ ¨

Bi-annual Maintenance• 1) Generate and extract a new background file using awater plate• 2) Defragment computer hard disk after backing updata!Start → Programs → Accessories → System Tools →Disk Defragmenter51¢¡¤£ £ ¥§¦©¨ ¨ Annual Maintenance• Calibrate the instrument using instrument specificcalibration kits• Applied Biosystems 7300 Real Time PCR SystemsSpectral Calibration Kit• Applied Biosystems 7500 Real Time PCR SystemsSpectral Calibration Kit INote: Back-up calibration documents on an external disk52¢¡¤£ £ ¥§¦©¨ ¨

Legal StatementsFor 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 LICENSEOR DISCLAIMER INFORMATION.Applera, Applied Biosystems, AB (Design), ABI PRISM, MicroAmp, and VIC are registered trademarks and FAM, JOE, NED, ROX andTAMRA 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.55¢¡¤£ £ ¥§¦©¨ ¨