Novallele™ Genotyping Assay User Manual For Novallele
Contents
1. All Novallele Genotyping Assays Novallele Genotyping Mastermix and 2x Novallele Oligo Dilution Buffer are For Research Use Only Not for use in diagnostic procedures Product Warranty Canon BioMedical warrants that each Novallele Genotyping Assay Novallele Genotyping Mastermix or 2x Novallele Oligo Dilution Buffer each a Novallele Product sold to the Customer will substantially perform in accordance with the written specifications therefor set forth in the applicable package insert or user manual originally included by Canon BioMedical with each Novallele Product delivered to the Customer until the instructed use by date set forth on the Novallele Product packaging Canon BioMedical s sole liability for and the Customer s sole remedy under this limited warranty will be for Canon BioMedical to replace the nonconforming Novallele Product with a conforming Novallele Product provided that in each case Canon BioMedical is notified and returned by the Customer during the warranty period of the nonconforming Novallele Product as instructed by an authorized Canon BioMedical employee The warranty period on any replacement Novallele Product will be the period from the shipment date until the replacement Novallele Product use by date A copy of the Canon BioMedical Inc standard terms and conditions applicable to the Novallele Genotyping Assays Novallele Genotyping Mastermix and 2x Novallele Oligo Dilution Buffer can be obtained2. QIAGEN MagnaPure Compact automated system Roche with large and small Nucleic Acid Isolation kits and QlIACube automated system QIAGEN with QlAamp Blood DNA Mini Kit and QlAprep Spin Mini Prep Kit For Research Use Only Not for use in diagnostic procedures HRMA is sensitive to changes in the salt and buffer components in samples Therefore Canon BioMedical strongly recommends that all samples experimental and control be prepared using the same extraction method to reduce variability in the subsequent analysis DNA Quality All DNA samples should show consistent quality by UV spectrophotometric measurements or related methods DNA purity after extraction may be determined at the same time the DNA concentration is measured Concentration as measured by A260 should be greater than or equal to 20 ng ul Samples with a concentration of less than 20 ng ul should be either excluded from analysis or concentrated by using ethanol precipitation Samples with a higher concentration should be diluted to 20 ng ul by using nuclease free water The acceptable A260 A280 for use is 1 7 2 1 For Research Use Only Not for use in diagnostic procedures Novallele Genotyping Assay Protocol This protocol is for use with Novallele Genotyping Assays and Novallele Genotyping Mastermix together with a HRMA compatible thermocycler For the latest information on HRMA compatible thermocyclers please contact Canon BioMedical
3. also found on the Novallele Genotyping Assay s webpage at www canon biomedical com 8 Place the PCR plate in the thermocycler Use a compression pad with the optical film sealed plate disc formats if recommended by the manufacturer Start the run 9 After the run is finished dispose of the PCR plate according to your institution organization guidelines 10 In order to analyze the data please refer to the thermocycler manufacturer s instructions on analyzing HRM results If you have questions please contact Canon BioMedical Support at 1 844 CANON BIO Note Visually inspect the plate after the run for any signs of evaporation from any of the wells If evaporation is observed note which wells are affected as this may affect the results of data analysis Note Do not open any previously processed plate Removing the strips or film releases PCR product into the air where it may contaminate and affect the results of future experiments For Research Use Only Not for use in diagnostic procedures Data Analysis Canon BioMedical recommends that users analyze data using the instrument software For additional recommendations please contact Canon BioMedical Support at 1 844 CANON BIO Appendix Using Novallele Reagents with User designed Assays Novallele Genotyping Mastermix is a laboratory optimized fast PCR chemistry formulation that supports HRMA Users with previously designed HRM assays may increase sample throughput a
4. packaging 600 ul in one vial Documentation included Product Specification Sheet with assay specific protocol Product Name 2x Novallele Oligo Dilution Buffer Catalog Number 40017 Number of reactions Makes enough Assay for 166 10ul reactions Volume of reagent and packaging 300 ul in one vial Documentation included Product Specification Sheet Shipping and Storage Novallele Genotyping Assays are shipped under environmental conditions Novallele Genotyping Assays should be stored at 2 8 C When stored correctly this product is stable for six 6 months from the date of receipt Novallele Genotyping Mastermix is shipped under environmental conditions Novallele Genotyping Mastermix should be stored at 2 8 C When stored correctly this product is stable for six 6 months from the date of receipt 2x Novallele Oligo Dilution Buffer is shipped under environmental conditions 2x Novallele Oligo Dilution Buffer should be stored at 2 8 C When stored correctly this product is stable for six 6 months from the date of receipt If the products arrive damaged or without cold ice packs please call Canon BioMedical Support at 1 844 CANON BIO For Research Use Only Not for use in diagnostic procedures All of the Novallele reagents mentioned above can be stored during test preparation for up to two 2 hours at room temperature 15 25 C Intended Use RESEARCH USE ONLY
5. BioRad CFX Other HRMA platforms such as Roche LC480 ThermoFisher QuantStudio Rotor GeneQ Roche LightCycler 2 0 and Biofire LS32 may be compatible instruments however testing has not been performed by Canon BioMedical For other platforms listed above verification or validation of suitability is recommended please contact Canon BioMedical Support at 1 844 CANON BIO for additional questions or concerns What types of source DNA can be used for the Novallele assays Canon BioMedical has tested genomic DNA extracted from cell lines and blood as well as synthetic samples including plasmids and double stranded linear segments What DNA extraction methods are suitable for the Novallele assays Canon BioMedical has tested DNA extracted by many methods with comparable results Any method that yields suitable concentration purity and integrity is acceptable for use but it is essential that all samples be prepared using the same method to generate optimal genotyping results How many samples can be tested with a single vial of the Novallele Genotyping Assay Canon BioMedical recommends using 3 6uL of Novallele Genotyping Assay per reaction and thus sufficient mix is provided for approximately 166 assays 600 uL 3 6 uL 166 per single vial Please be advised that pipetting errors may reduce the number of possible assays What control s should use with my Novallele Genotyping Assay Canon BioMedical routinely uses both negative and pos
6. ENERAL PATENT OR OTHER LICENSE OF ANY KIND OTHER THAN THIS SPECIFIC RIGHT OF USE FROM PURCHASE IS GRANTED HEREBY Copyrights 2015 Canon BioMedical Inc All Rights Reserved 50583 Rev 0 Last revised September 8 2015 For Research Use Only Not for use in diagnostic procedures Canon BioMedical Inc One Canon Park Melville NY 11747 USA USA 1 844 CANON BIO www canon biomedical com For Research Use Only Not for use in diagnostic procedures
7. Novallele Genotyping Assay User Manual For Novallele Genotyping Assays Novallele Genotyping Mastermix and 2x Novallele Oligo Dilution Buffer For Research Use Only Not for use in diagnostic procedures Canon BioMedical Inc One Canon Park Melville NY 11747 USA 1 844 CANON BIO www canon biomedical com Company Info Canon BioMedical Inc is focused on empowering the biomedical research and healthcare communities by developing manufacturing and marketing innovative technologies and solutions The technologies and solutions developed will help enable clinicians and scientists to improve our health and advance science Canon BioMedical will continue to pursue innovative solutions in line with Canon s Kyosei philosophy through the use of existing and emerging Canon technology as well as strategic partnerships Kyosei defined as all people regardless of race religion or culture harmoniously living and working together into the future For Research Use Only Not for use in diagnostic procedures Table of Contents Contents of Products Shipping and Storage Intended Use Product Warranty Technical Assistance Safety Information Introduction Principles and Procedure Equipment and Reagents to be supplied by User Important Notes General DNA Extraction DNA Quality Novallele Genotyping Assay Protocol Important points before starting Procedure Data Analysis Appendix l Using Novallele Reagents with User designed As
8. Support at 1 844 CANON BIO Important points before starting Familiarize yourself with the HRMA compatible thermocycler before starting this protocol Refer to the thermocycler user manual and follow the manufacturer s instructions for proper operation and maintenance Pipetting accuracy and precision affects the consistency of results Be sure that all pipets and instruments have been checked and calibrated according to the manufacturer s recommendations Make sure that no bubbles are introduced into the wells of the PCR plate during pipetting If bubbles form remove them by centrifuging the plate at 1000 rpm for 1 min 96 well plate 2000 rpm for 2 min 384 well plate This step is not necessary for all thermocyclers Do not use DEPC treated water Use high quality nuclease free water Procedure 1 Briefly invert or vortex all of the Novallele Genotyping Mastermix and the Novallele Genotyping Assays to mix the contents of the tube 2 Briefly centrifuge the Novallele Genotyping Mastermix and the Novallele Genotyping Assays 10 15s to bring the contents to the bottom of the tube Note The Novallele Genotyping Mastermix is only active after heat activation therefore reactions can be prepared and maintained at room temperature 15 25 C for up to two 2 hours 3 Prepare a reaction mix for each sample according to Table 1 Recommendation Prepare a reaction mix for a minimum of
9. The importance of these small variations in our genomes stems from their ability to influence disease risk response to therapeutics and even predict how we look and behave A Single Nucleotide Polymorphism SNP is a type of variation within the genome occurring commonly within a population in which a single nucleotide A T C or G in the genome differs between members of a biological species or paired chromosomes SNPs also commonly known as mutations are one of the most important types of genetic changes influencing common diseases typical genetic variations include insertions deletions and copy number variations CNVs A diverse set of genotyping technologies including sequencing microarrays in situ hybridization and PCR based methods are used to detect mutations Such variety of methods enables researchers to match the correct technology with not only their experimental question but also user preferences for data analysis cost and turnaround time Principles and Procedure Novallele Genotyping Assays in combination with Novallele Genotyping Mastermix contain all the reagents needed to identify specific SNPs or mutations within a given human genomic DNA sample The Novallele Genotyping Assays and Novallele Genotyping Mastermix perform rapid PCR HRMA of the PCR products follows the completion of rapid PCR First DNA samples provided by the end user are mixed with the Novallele Genotyping Mastermix and Novallele Genotyping A
10. amplicon HRM assays and for unlabeled probe HRM assays If you have any questions regarding which table you should follow please contact Canon BioMedical Support at 1 844 CANON BIO For Research Use Only Not for use in diagnostic procedures Table 2 Preparation of a short amplicon DIY Assay Mixture Concentration Volume 2x Novallele Oligo Dilution Buffer 2x 300 ul Forward Primer 100 uM 18 ul Reverse Primer 100 uM 18 ul Nuclease free Water 264 ul Final Volume 600 ul This mixture is the equivalent of a Novallele Genotyping Assay Note Forward and Reverse Primers are designed and supplied by the user Table 3 Preparation of an unlabeled probe DIY Assay Mixture Concentration Volume 2x Novallele Oligo Dilution Buffer 2x 300 ul Limited Primer 100 uM 3 6 ul Excess Primer 100 uM 18 ul Unlabeled Probe 100 uM 18 ul Nuclease free Water 260 4 ul Final Volume 600 ul This mixture is the equivalent of a Novallele Genotyping Assay Note Limited Excess Primers and Unlabeled Probes are designed and supplied by the user Note Table 3 describes a 1 5 ratio of limited to excess primer users may need to adjust this ratio based on assay design For guidance please contact Canon BioMedical Support at 1 844 CANON BIO Using DIY Assay Mixtures in genotyping experiments After preparing a DIY Assay mixture users should follow the Novallele Genotyping Assay Protocol as d
11. escribed above starting on pg substituting the DIY Assay mixture for the Novallele Genotyping Assay in the protocol While the conversion of a DIY Assay for use with the Novallele Genotyping Mastermix is a simple procedure the user may need to optimize cycling parameters to obtain optimal results For guidance please contact Canon BioMedical support at 1 844 CANON BIO Table 4 Preparation of reaction mix Single test 1 test well per sample Technical replicates in triplicate 3 test wells per sample Novallele Genotyping Mastermix 3 6 ul 11 ul DIY Assay mixture 3 6 ul 11 ul DNA 66 6 ng 220 ng Nuclease free Water Up to Final volume Up to Final volume Final Volume 11 ul 33 pl For Research Use Only Not for use in diagnostic procedures Troubleshooting and FAQs This troubleshooting and FAQs section supports users by answering Frequently Asked Questions and providing helpful hints from the scientists that developed the Novallele Products For updated information see also the Frequently Asked Questions page at www canon biomedical com If this section does not address your questions or concerns please contact Canon BioMedical Support at 1 844 CANON BIO On what types of instruments can these be run Canon BioMedical has established performance of the Novallele Genotyping Assays Novallale Genotyping Mastermix and 2x Novallele Oligo Dilution buffer on the following instrument platform
12. itive controls for all of our assays Canon BioMedical recommends using a no template control negative to rule out any environmental contamination and a homozygous wild type control positive for each run To the extent possible the inclusion of additional controls such as heterozygous or homozygous variants is recommended but not required For Research Use Only Not for use in diagnostic procedures Where can find the recommended reaction conditions The applicable product specification sheet contains the detailed assay conditions for each Novallele Genotyping Assay The specification sheets are included with the Novallele Genotyping Assay as well as available at www canon biomedical com Is there a recommended analysis method for the assays Please refer to the user manual of your PCR instrument for the analysis software Generally both small amplicon and probe based assays are analyzed using the first derivative of the melting curve known as the derivative plot There is an additional bump hump shoulder on one of my samples what does this mean Extra features on the melt curve can originate from many sources Additional melt curve features may simply be a consequence of the magnitude of smoothing used for data analysis but could also be an indicator of an additional sequence variation within the region being analyzed or contamination Another possibility is that the additional melt feature is an artifact caused by an instrumen
13. nd decrease reagent costs by converting to Novallele Genotyping Mastermix and 2x Novallele Oligo Dilution Buffer The section below describes how a user should prepare their primers and optional unlabeled probes for use Important notes before starting The use of Novallele Genotyping Mastermix and 2x Novallele Oligo Dilution Buffer together is essential for a successful reaction Do not replace the 2x Novallele Oligo Dilution Buffer with other commercial counterparts The use of the Novallele Genotyping Assays and the Novallele Genotyping Mastermix is essential for a successful reaction Pipetting accuracy and precision affects the consistency of results Be sure that all pipets and instruments have been checked and calibrated according to the manufacturer s recommendations Use high quality nuclease free water Generate Design it Yourself DIY Assay Mixtures The use of a self designed HRM assay with the Novallele Genotyping Mastermix and the 2x Novallele Oligo Dilution Buffer is a simple process that begins with creating an assay mixture that mimics the Novallele Genotyping Assay s chemistry Users can create a working stock of their assays by diluting the primers probes in the 2x Novallele Oligo Dilution Buffer 2x Novallele Oligo Dilution Buffer contains all of the necessary chemistry for use DIY assays with the Novallele Genotyping Mastermix The two tables below outline DIY Assay Mixtures for short
14. on request If you have questions about product specifications or performance please call Canon BioMedical Support at 1 844 CANON BIO or visit www canon biomedical com Technical Assistance At Canon BioMedical we strive to deliver the highest quality technical support Our support team is composed of experienced scientists with extensive academic and industry laboratory expertise in genotyping and PCR technologies If you have any questions about Canon BioMedical products please do not hesitate to contact us For technical assistance and more information please visit our website at www canon biomedical com or call Canon BioMedical at 1 844 CANON BIO SAFETY INFORMATION When working in a laboratory use personal protective equipment including lab coat disposable gloves and protective eyewear Follow your local institutional policies and procedures regarding the appropriate handling of biological materials and lab reagents For more information please consult the appropriate safety data sheets SDSs To obtain a SDS for Canon BioMedical products please contact Canon BioMedical Support at 1 844 CANON BIO For Research Use Only Not for use in diagnostic procedures Introduction Humans share almost the same sequence of 3 billion DNA bases within our 23 pairs of chromosomes Current estimates indicate that up to 0 1 of human genome DNA may vary meaning any two unrelated individuals may differ at fewer than 3 million DNA positions
15. rification page X Spectrophotometer and associated consumables cuvettes etc if required Nuclease free water HRM compatible PCR cycler see Table 5 in the Troubleshooting and FAQ section Single and or multichannel pipets with nuclease free tips Nuclease free microcentrifuge tubes Centrifuge and rotor for PCR plate Instruments should be checked and calibrated according to the manufacturer s recommendations Important Notes General Canon BioMedical recommends the following guidelines Use sterile aerosol barrier pipet tips designed for PCR applications Users should setup two physically separate workspaces the first for PCR setup and the second for PCR and post PCR processing operations This physical separation will minimize amplicon contamination that may impact data interpretation Decontaminate the PCR workspace and labware pipettes tube racks etc with UV light or with 10 bleach followed by wiping with isopropanol Thaw frozen genomic DNA samples thoroughly at room temperature 15 25 C or on ice before starting to prepare reactions DNA Extraction There are no special limitations or assurances given for using DNA extracted with any commercial kits The following kits and extraction methods have been used during testing by Canon BioMedical Gentra Puregene Blood Kit for manual DNA extraction QIAGEN Genomic DNA Buffer Kit
16. says Important points before starting Generate Design it Yourself DIY Assay Mixtures Using Design it Yourself DIY Assay Mixtures in genotyping experiments Troubleshooting and Frequently Asked Questions References Ordering Information Notes Trademarks Limited License Agreements Limited License Statements Copyrights For Research Use Only Not for use in diagnostic procedures Contents of Products This user manual applies to three types of products as defined below that are ordered independently The tables below describe the contents of each product package The Novallele Genotyping Mastermix a PCR chemistry formulation allowing rapid PCR followed by High Resolution Melt Analysis HRMA may be used with either Novallele Genotyping Assays or with user designed assays that will require the use of 2x Novallele Oligo Dilution Buffer The use of Novallele Genotyping Mastermix is essential for successful function of the Novallele Genotyping Assays Do not replace this reagent with other commercial products Product Name Novallele Genotyping Mastermix Catalog Number 40018 Number of reactions 166 10ul reactions Volume of reagent and packaging 600 ul in one vial Documentation included Product Specification Sheet Product Name Novallele Genotyping Assays Catalog Number Depends on the human SNP selected Number of reactions 166 10ul reactions Volume of reagent and
17. ssay in either a microcentrifuge tube or the well of a PCR plate Subsequent amplification of DNA by PCR and amplicon melting is performed rapidly Finally changes in the fluorescence intensity of the amplicons are captured by the optical system within the user s HRMA compatible thermocycler HRMA is a method for detecting changes in DNA sequence It uses a fluorescent dye at saturating concentrations that will bind to double stranded DNA during the PCR reaction Once the PCR is complete the reaction temperature is raised double stranded DNA denatures and the bound dye is released decreasing the fluorescence The exact fluorescence change with increasing temperature will depend on the DNA sequence of the amplicon A shift in melting temperature Tm or a change in the shape of the melt curve is observed when a variation is detected in a sample DNA compared to reference DNA sample Equipment and Reagents to be supplied by User In addition to the Novallele Genotyping Assays or the 2x Novallele Oligo Dilution Buffer and Novallele Genotyping Mastermix the following reagents and equipment are required For Research Use Only Not for use in diagnostic procedures 96 well or 384 well PCR plate with Optical Thin Wall 8 Cap Strips or Optical Adhesive Film or Rotor Disc with Rotor Disc Heat Sealing Film or the appropriate thermocycler compliant reaction vessels Such as PCR tubes or capillaries Purified DNA samples see DNA pu
18. t error or particulate contamination that disrupted the imaging process Alternatively the DNA quality may be compromised thus Canon BioMedical recommends analyzing the sample DNA to confirm an A260 A280 ratio within the range of 1 7 2 1 Canon BioMedical recommends repeating the assay on this DNA sample if possible References Canon BioMedical maintains a database of scientific publications utilizing Canon BioMedical products For a list of references please contact Canon BioMedical Support at 1 844 CANON BIO Ordering Information Product Catalog Number Description Novallele Genotyping Mastermix 40018 Rapid PCR chemistry formulation for High Resolution Melting Analysis Novallele Genotyping Assays Assay dependent Genetic variation specific PCR assay 2x Novallele Oligo Dilution Buffer 40017 Primer Probe dilution buffer for use with Novallele Genotyping Mastermix Notes This will create blank pages to facilitate correct page numbers for printing Completed after formatting For Research Use Only Not for use in diagnostic procedures Trademarks Novallele is a trademark of Canon BioMedical Inc All other referenced marks are trademarks of their respective owners and are hereby acknowledged Limited License Statements THE PURCHASE OF THIS PRODUCT GRANTS THE PURCHASER RIGHTS UNDER CERTAIN ROCHE PATENTS TO USE IT SOLELY FOR INVESTIGATIONAL RESEARCH OR PROVIDING HUMAN IN VITRO DIAGNOSTIC SERVICES NO G
19. three 3 replicate reactions for each sample Prepare ten percent 10 more mix than is required to allow for imprecision in pipetting Recommendation For each experiment a No Template Control NTC and a positive homozygous wild type control are recommended For the NTC and positive control sample substitute nuclease free water for the DNA in Table 1 to prepare the reaction mixture For Research Use Only Not for use in diagnostic procedures Table 1 Preparation of reaction mix Single test Technical replicates in 1 test well per sample triplicate 3 test wells per sample Novallele Genotyping Mastermix 3 6 ul 11 ul Novallele Genotyping Assay 3 6 ul 11 ul DNA 66 6 ng 220 ng Nuclease free Water Up to final volume Up to final volume Final Volume 11 ul 33 pl 4 Add 10 ul of the reaction mixture to each well of either a 96 well plate or a 384 well plate using a single channel pipet Note Change pipet tips between each sample to avoid any cross contamination between the wells 5 Tightly seal the PCR plate as directed by the manufacturer 6 Remove any air bubbles in any of the well plate by centrifuging the plate at 1000 rpm for 1 min 96 well plate 2000 rpm for 2 min 384 well plate This step is not necessary for QIAGEN Rotor Gene 7 Program the HRM compatible thermocycler according to the PCR and HRM conditions provided on the Novallele Genotyping Assay Product Specification Sheet which is
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