PRODUCT INSERT - Immucor, Inc.
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1. Barrier filter tips Pipettors multichannel pipettors and tips 1 20 L 20 200 L 1000 L MATCH IT RBC Software Supplied Lifecodes Cat No 888729 MATCH IT RBC Lot Information Update CD Supplied Lifecodes Cat No 888728 Instruments and Related Ancillaries Luminex Instrument and XY Platform Lifecodes Cat No 888300 Luminex Sheath Fluid Lifecodes Cat No 628005 Luminex Calibration beads o Cal 1 Lifecodes Cat No 628006 o Cal 2 Lifecodes Cat No 628007 o Con 1 Lifecodes Cat No 628008 o Con 2 Lifecodes Cat No 628009 Thermal cycler Required Specifications heated lid adjustable ramp times minimal thermal range 2 to 100 C accuracy within 0 5 C Vortex mixer Bath sonicator Fisher Scientific Cat No FS15 or FS20 Microcentrifuge Heat Block Fisher Scientific Standard Heatblock Cat No 13259 030 Rubber mat or metal plate Page 3 of 6 LC1129 4 11 11 REAGENT STORAGE AND USE 1 Refer to kit component packaging label for proper storage temperatures 2 If salts have precipitated out of solution during shipping or storage resolubilize completely prior to use by vortexing at room temperature 18 to 30 C 3 Probe mixes and R Phycoerythrin Conjugated Streptavidin are light sensitive Keep away from light Do not freeze these reagents 4 Do not use R Phycoerythrin Conjugated Streptavidin that has been frozen during shipment or2. 1 Turn on the Luminex Instrument between 30 minutes and 4 hours before assaying the samples 2 Import the lot specific IDT template provided on the RBC Lot Information Update CD using the Luminex IS User s Manual Please note that the template versions are lot number specific and correspond to the kit lot numbers 3 Prior to analyzing the samples on the Luminex Instrument set up a Batch Run by which the samples will be analyzed For example if analyzing an RBC run add a Batch Run for RBC a Select Create a New Batch from the File menu b Follow the stepwise instructions that appear on the screen for creating batches c When naming the batch do not include commas in the name information located after a comma will be lost in data export d For further instructions on creating batches and multibatches refer to the Luminex IS User s Manual 4 Click the Eject icon to eject the plate holder Place the 96 well thermal cycler plate containing the samples in the XYP heater block present on the plate holder 5 Click the Retract icon The samples are now ready to be analyzed An instrument priming step should be performed before starting the run 6 After the samples have been run through the instrument a sanitization step with 70 Isopropanol or 20 household bleach should be performed followed by two wash steps The instrument can be turned off at this point if it is not going to be used for the remainder of the day 7 After a batch is compl
3. Page 1 of 6 LC1129 4 11 11 Gen Probe Transplant Diagnostics Inc 550 West Avenue Stamford CT 06902 Tel 203 328 9500 or 888 329 0255 Fax 203 328 9599 WWW GEN PROBE COM LIFECODES RBC R TYPING KIT for use with Luminex For Research Use Only Not for In Vitro Diagnostic Use TABLE OF CONTENTS SECTION PAGE NO Definition of Symbols 1 Intended Use 2 Reagents by Product Number 2 Materials Reagents and Equipment Required but not Provided 2 Reagent Storage and Use 3 Genomic DNA Preparation and Use 3 Warnings and Precautions 3 Procedure 4 A Genomic DNA Purification 4 B Amplification 4 C Hybridization 4 D Analysis using Luminex Instrument 5 E Analysis using MATCH IT RBC Software 5 Troubleshooting 6 DEFINITION OF SYMBOLS Product Labels and Supplemental Documents Lot Number Catalog Number REF Temperature limitations Upper limit of temperature Expiration Date exp Keep away from light Sufficient for N tests Do Not Freeze Caution See instructions for use See instructions for use Manufacturer P R O D U C T I N S E R T Page 2 of 6 LC1129 4 11 11 PRODUCT DETAILS LIFECODES RBC R Typing Kit The LIFECODES RBC R assay uses single nucleotide polymorphism SNP detection by the Polymerase Chain Reaction Reverse Sequence Specific Oligonucleotide PCR RSSO method This multiplex assay has been designed to genotype the antigens of t
4. anual Sample flow path blocked Remove and sonicate needle Perform backflush Call Lifecodes Technical Support if problem persists 888 329 0255 Failed sample Sample failed to amplify or amplified poorly Low DNA Check DNA concentration and purity see Genomic DNA Preparation and Use Salts in Master Mix are out of solution Heat Master Mix at 37 C for 5 minutes vortex gently and spin down briefly 2 to 5 secs Poor Taq Polymerase Use only Recombinant Taq Lifecodes Cat No 167075 is recommended Amplification conditions not within specific parameters Run thermal profile on thermal cycler to verify parameters are within specified parameters Adjust if necessary Low Median Fluorescent Intensity MFI value Ensure Dilution Solution is stored at room temperature 18 to 30 C Warm Dilution Solution at 45 C for 5 minutes before use and vortex Replace R Phycoerythrin Conjugated Streptavidin Evaporation during hybridization Ensure the plate is sealed tightly If not using an entire plate leave one row empty on each side of samples to be assayed to assist in obtaining a tight plate seal SPECIFIC PERFOMANCE CHARACTERISTICS Specific performance characteristics have not been determined MANUFACTURER Manufacturer Gen Probe Transplant Diagnostics Inc 550 West Avenue Stamford CT 06902 Phone 203 328 9500 888 329 0255 Fax 203 328 9599 European Technical Service Phone 32 3 385 4791
5. ardous compounds Avoid contact with skin and eyes and dispose of accordingly See Material Safety Data Sheets for additional information www gen probe com Page 4 of 6 LC1129 4 11 11 PROCEDURE A Genomic DNA Purification 1 See Genomic DNA Preparation and Use for important information 2 Purify DNA using method of choice 3 Final concentration of genomic DNA should be 10 to 200 ng ul 4 Adjust DNA concentrations if necessary using nuclease free water Keep all samples at similar concentrations B Amplification 1 Bring Master Mix kit component to room temperature 2 Prepare Master Mix reaction solution using the following table Prepare immediately prior to use Do not store Total volume per reaction is 25 L It is recommended that one water blank be run with each test 3 Aliquot DNA 100ng optimal 40 to 200ng acceptable into PCR tubes 4 Aliquot Master Mix reaction solution into tubes Discard any remaining Master Mix reaction solution 5 Place samples in the thermal cycler and run amplification program The program shown below has yielded optimal results in internal testing Conditions may need to be altered in order to optimize thermal profiles for individual thermal cyclers used C Hybridization 1 Warm Probe Mix at 55 to 60 C for 5 to 10 minutes 2 Sonicate and vortex Probe Mix briefly to thoroughly resuspend the beads 3 Aliquot 15 L of Probe Mix into each well of a Costar plate vortex Probe Mix after every 8 sam
6. ete the data are exported as a Comma Separated Values csv file These files are named OUTPUT CSV and saved in a folder with the Batch Name entered into the Luminex IS These data are then available for analysis using the MATCH IT RBC software E Analysis Using Lifecodes MATCH IT RBC Software 1 Import the lot specific EDS file provided on the RBC Lot Information Update CD using the Lot Data Set Import tab 2 Import the csv file obtained from Luminex analysis using the Batch Data Import tab 3 Analyze the batch using the User s Manual for MATCH IT RBC Software 4 See User s Manual for MATCH IT RBC Software User s Manual for a description of possible run results a Indeterminate results are noted by an ID label in the software report b Indeterminate ID results can occur due to low amplification probe value too close to the cut off or an unexpected rare result In these cases the sample should be repeated or typed using another red cell typing technology of Samples Dilution Solution DS SA PE 1 170 L 3 4 L 5 850 L 17 0 L 10 1700 L 34 0 L 20 3400 L 68 0 L 50 8500 L 170 0 L Page 6 of 6 LC1129 4 11 11 TROUBLESHOOTING PROBLEM POSSIBLE CAUSE SOLUTION Low Bead Count Probe Mix not well suspended Prewarm sonicate and vortex probe mix and repeat assay Instrument not functioning properly Out of Calibration Calibrate instrument refer to Luminex IS User s M
7. he following rare blood groups Colton Coa Cob Dombrock Hy Joa Doa Dob Scianna Sc1 Sc2 Lutheran Lua Lub Diego Dia Dib Wra Wrb Landsteiner Weiner LWa LWb Cartwright Yta Ytb Knops Kna Knb McCa McCb SI1 SI2 Cromer Cra REAGENTS BY PRODUCT NUMBER RBC R LIFECODES RBC R Typing Kit for use with Luminex Product 728200 50 Reagent Product Number Fill volume Storage 50 Sufficient for 50 samples MX RBC R LIFECODES RBC R Master Mix 728202 580 L 2 to 8 C BM RBC R LIFECODES RBC R Probe Mix 728201 810 L 2 to 8 C Protect From Light DS Dilution Solution 628515 19 7 mL 18 to 30 C Probe Mixes are light sensitive keep away from light CAUTION Do not freeze components and Do not use components past their expiration dates MATERIALS REAGENTS AND EQUIPMENT REQUIRED BUT NOT PROVIDED as listed or equivalent Reagents or Chemicals or Kits R Phycoerythrin Conjugated Streptavidin SA PE 1mg mL Lifecodes Cat No 628511 Recombinant Taq Polymerase Lifecodes Cat No 167075 70 Isopropanol or 20 household bleach Ancillaries PCR tubes and caps Corning 6542 VWR 29442 354 0 2mL 8 strip tubes and caps OR ABGene ThermoFast 96 well PCR plates skirted cat AB 1000 Natural 50 plates box Thermal cycler PCR 96 well plates Costar Cat No 6509 Microseal Film Bio Rad Cat No MSA 5001 or Tape Costar Cat No 6524
8. ples 4 Aliquot 5 L of PCR product into each well 5 Seal plate with polyethylene tape or film 6 Place in thermal cycler and run hybridization program Use a rubber mat or metal plate to prevent evaporation Component Amount per PCR sample reaction Master Mix 10 L Genomic DNA Total of 100 ng Taq polymerase 0 25 L 1 25 U Nuclease free water To 25 L final volume 95 C for 4 min Number of cycles 1 95 C for 30 sec 51 C for 45 sec 65 C for 2 min 30 sec Number of cycles 45 65 C for 5 min IMPORTANT NOTES Pre warm Probe Mix Keep beads protected from light Vortex beads Dilute all hybridized samples at 56 C before removing tray 56 C for 22 min 56 C HOLD Page 5 of 6 LC1129 4 11 11 7 Prepare a mixture of Dilution Solution and PE Streptavidin 1mg ml using the table shown following Protect mixture from light 8 At the 56 C HOLD step dilute each sample with 170 L of the Dilution Solution PE Streptavidin mixture NOTE Do not remove the tray from the thermal cycler until all samples have been diluted 9 Remove the sample tray from the thermal cycler and immediately place in the Luminex instrument for analysis NOTE For best results acquisition should begin within 5 minutes after hybridization program ends D Analysis Using Luminex Instrument refer to Luminex IS User s Manual for instrument operation including daily startup calibration maintenance and shutdown procedures
9. storage 5 Do not use components past their expiration dates GENOMIC DNA PREPARATION AND USE 1 DNA can be prepared using any preferred method The assay has been tested on genomic DNA from whole blood CAUTION All biological and blood samples should be treated as potentially infectious Use Universal Precautions when handling 2 DNA extracted from blood preserved in heparin cannot be used in this assay 3 The presence of alcohol detergents or salts may adversely affect DNA amplification 4 Absorbance measurements of the DNA sample should yield a 260nm to 280nm ratio ranging from 1 65 to 2 0 5 The isolated DNA should be resuspended and stored in 10mM TRIS pH 8 0 to 9 0 or in nuclease free water If a chelating agent such as EDTA is present the final concentration of the chelating agent should not exceed 0 5mM 6 Final DNA concentration should be 10 to 200 ng L 7 DNA can be used immediately after isolation or stored at 20 C for up to 1 year Repeated freeze thawing should be avoided since this can result in DNA degradation WARNING AND PRECAUTIONS 1 For Research Use Only Not for in vitro diagnostic use 2 Separate pipettes should be designated for Pre PCR manipulations as well as for Post PCR manipulations 3 All biological and blood samples should be treated as potentially infectious Use Universal Precautions when handling 4 Dilution Solution Probe mixes Taq Polymerase and R Phycoerythrin Conjugated Streptavidin contain haz
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