User Guide
Contents
1. Optical Caps and Strips Micropipette sets Source Generon or other Lab Suppliers Generon Cat N PGPxxA Generon Cat N ENGOO1 Generon or other Lab Suppliers Generon or other Lab Suppliers 1 Equipment necessary only when ION Force DNA Extractor FAST Cat N EXDO01 is used 2 The assay can be used with Biorad CFX and MiniOpticon Stratagene MxSeries ABI 7300 7500 7900 StepONE StepONE Plus Light Cycler 480 Eppendorf realplex Rotor Gene Q etc The assay is not compatible with Roche Light Cycler and II User guide MODIfinder GMO POTATO Assay Rev 1 25 11 2014 GSeneren 4 Real Time PCR detection 4 1 Reaction setup Allow the reagents to thaw GENERase Mastermix MODIfinder OLIGO MIX Positive Control and Negative Control Vortex tubes when thawed and spin to collect contents at the bottom of the vial Mix 150 ul of MODIfinder OLIGO Mix with 750 ul of GENERase Mastermix to prepare MoODIifinder Working Mastermix WMX Vortex briefly and spin down in order to homogenize the mix Transfer 18 ul of WMX into each well Add 12 ul of Negative Control into wells acting as negative control Add 12 ul of each sample into wells testing the unknown samples Add 12 ul of Positive Control into wells acting as positive control Close wells and ensure no bubbles are present at the bottom of the wells Spin briefly optical PCR tubes or plates 4 2 Instrument setup With GENERase Mastermix set the follo2. min DNA Denaturation 95 15 sec Annealing Extension Plate Reading 60 60 sec The thermal profile presented above was optimized for GENERase Mastermix Cat N ENGOO1 Results analysis If the following conditions are met GMO POTATO TEST FAM Positive Control Negative Control Then the possible results for any sample are GMO POTATO TEST FAM Positive Sample Negative Sample Invalid Sample Inhibited In case of inhibition DNA isolation and purification for the sample need to be improved or you may need to dilute your sample before performing a new test Refer to the Troubleshooting paragraph section 6 in the User Guide for further suggestions Warning and Precaution Please do not interchange components of assays with different lot numbers This assay is designed to be used by laboratory personnel following the common molecular biology precautions GLP Disclaimer Generon s r l guarantees the buyer exclusively concerning the quality of reagents and of the components used to produce the Assay Generon S r l is not responsible and cannot anyway be considered responsible or jointly responsible for possible damages resulting from the utilization of the product by the user The user consciously and under his own responsibilities decides for the utilization purposes of the product and uses it the way he considers most suitable in order to reach his goals and or objectives Generon S r l is not responsible fo
3. Gseneren Advanced Transfer Technologies MODIfinder DETECTION ASSAY GMO POTATO User Guide GSeneren 1 Introduction The introduction of GMO crops in the food chain led to the need to investigate their presence in a wide range of raw materials semi finished and finished products as well as in animal feed The European Commission first introduced thresholds for the accidental unavoidable presence of GM ingredients and according to EU Reg CE 1829 2003 all products food feed GMO derivatives cultures aromas and additives are labeled as to their GMO content Real Time PCR in time established as the gold standard method for GMOs detection and specific ISO norms namely 1SO21568 21569 21570 21571 regulate it s usage The base for GMO POTATO methods is the detection of DNA sequences of genetic control elements such as promoters transcription terminators and markers such as resistance genes Assays performances The MODIfinder GMO POTATO Detection Assay will detect the GMO only if all of the recommended components are stored properly and the recommended protocols are followed When used along with Generon ION Force DNA Extractor FAST Cat N EXD001 the assay shows a Limit of Detection of 0 01 Detection limit is strictly dependent from the matrix and the genome size of the taxa under investigation i e there is a theoretical LOD you cannot go below Assays available parner PC MODIfinder POTATO AMFLORA EH92 527 1 Use
4. ers Thermal Water Bath or Block Generon or other Lab Suppliers Pipette sets Generon or other Lab Suppliers Pipette tips Barrier Generon or other Lab Suppliers Tube rack for 1 5 ml tubes Generon or other Lab Suppliers 2 0 and 1 5 ml micro tubes Generon or other Lab Suppliers Micro centrifuge for 1 5 2 0 ml micro tubes Generon or other Lab Suppliers DNA Extraction VACUUM BOX Vacuum pump or Venturi meter Generon or other Lab Suppliers Each step of sample preparation grinding transferring weighing etc must be done according to GLP so that chance of cross contamination between samples is minimized It is recommended to use disposable equipment when possible If the food samples are not in a powdered or granular form they should be processed grinded or blended before DNA extraction The majority of DNA extraction methods supports from 20 to 50 mg of starting material Generon ION Force DNA Extractor FAST Cat N EXD001 allows processing up to 20 grams of starting material in order to maximize sample s lot representation Once the sample has been pulverized homogenized it can be weighed and the appropriate amount extracted according to DNA extraction method selected Refer to manufacturer user manual for extraction procedure details 3 3 Detection via Real Time PCR Material Equipment Real Time PCR System 2 MODIfinder GMO POTATO Detection Assay GENERase Mastermix Optical Adhesive Seal and Optical reaction plate or
5. ion 6 for further suggestions User guide MODIfinder GMO POTATO Assay Rev 1 25 11 2014 6 GSeneren Advanced Transfer Technologies 6 Troubleshooting Concomitant no target or endo amplification or amplification plots grossly abnormal Possible causes and corrective actions e An excess of DNA in the target might inhibit the reaction and ENDO may be affected due to an excess of DNA and or PCR inhibitors Test samples diluted 1 10 and 1 100 Please use DNase RNase Free Water to prepare dilutions Inadequate sealing of optical caps film caused sample evaporation Redo the analysis using proper tools and proper optical caps film to secure perfect sealing Did not use the proper consumables Redo the analysis and use only optical grade 96 well plates and optical adhesive seal or optical 8 well strips and caps Samples were not properly prepared Remake the sample DNA preps Ensure that the DNA extraction method is properly performed Positive Control reactions failed to amplify but other reactions appear correct e g the endo is amplified Positive Control DNA was not added to the reaction wells If other reactions look normal there may be no need to repeat the run Ill Negative Control reactions are positive e Contamination of the negative control vial or the MODIfinder PCR mix with MODIfinder positive DNA Use more care to prevent contamination while handling assay reagents and setting up assays In case suppor
6. kage is open the user accepts all the conditions without fail if the package is still sealed the product can be returned and the user can be refunded User guide MODIfinder GMO POTATO Assay Rev 1 25 11 2014
7. r guide MODIfinder GMO POTATO Assay Rev 1 25 11 2014 2 GSeneren Advanced Transfer Technologies 2 MODIfinder GMO POTATO Detection Assay When used along with GENERase Mastermix Cat N ENGOO1 this Real Time PCR assay detects a specific DNA sequence in the DNA of target in less than 1 5 hours The amplification of the target sequence is measured by the use of a specific fluorescence labeled probe FAM 2 1 Assay Content Box 50 reactions Box 100 reactions N vials Volume pl N vials Volume pl MODIfinder OLIGO Mix OLIGOS and Probe pre blended mix 1 150 2 150 Positive Control 120 120 Negative Control reagents are supplied with a 5 of extra volume 2 2 Storage amp Expiry information Expiry date see date on the packaging product validity refers to the product kept intact in its original packaging Protect reagents from light exposure as far as OLIGO Mix reagents are photosensitive Store frozen User guide MODIfinder GMO POTATO Assay Rev 1 25 11 2014 3 ENER N Advanced Transfer Technologies 3 Materials and equipments needed 3 1 Extraction Material Equipment Source Extraction Kit Generon ION Force DNA extractor FAST Cat N EXD001 Chemicals n esane Lab Suppliers Tubes 50 ml and 15 ml Generon or other Lab Suppliers DNAse RNAse Free Water Generon or other Lab Suppliers Vortexer Generon or other Lab Suppliers Benchtop Centrifuge for 50 ml Tubes Generon or other Lab Suppli
8. r the data resulting from the use of the products for the utilization that the user independently decides to make of them or for the damages possibly resulting from the disclosure or transmission of the data themselves to third parties under any form or circumstance This clause is automatically accepted by the user when purchasing the products The patent for performing PCR is held by Hoffmann La Roche Authorization to use PCR can be obtained on licence from Hoffmann LaRoche The product equipment and information included in the assay consists of assembled and reagents The licence and licence and authorisation for PCR use are not included in the assay The user is responsible for setting prefixed goals choosing whether or not to perform the PCR reaction and to apply for register his own licence The use assay is designed for the services supply quality control or any other application that is not exclusively an internal company s research and requires a specific licence for PCR use This PCR use licence to supply a service on food analysis field has to be requested directly from Applied Biosystems This assay requires the use of Taq Polymerise enzyme The product was internally tested by our quality control Any responsibility is waivered if the warranty of quality control does not refer to the specific product The user is personally responsible for data that he will obtained and or he will supply to third parties using this assay Once the sealed pac
9. t is needed contact Generon at support generon it 7 Disclaimers The product is intended for research use only Generon makes no warranty of any kind either expressed or implied except that the materials from which its products are made of standard quality If any materials are defective Generon will provide a replacement product Generon shall not be liable for any damages including special or consequential damage or expense arising directly or indirectly from the use of this product Please do not interchange components between assays of different lot numbers This assay is designed to be used by laboratory personnel following the common molecular biology precautions User guide MODIfinder GMO POTATO Assay Rev 1 25 11 2014 7 ENER N Advanced Transfer Technologies Quick Reference Guide Page 1 Product Line MoODIfinder Type qualitative Storage Frozen Execution time about 120 minutes Expiry date see date on the packaging product validity refers to the product kept intact in its original packaging and constantly under suitable temperature conditions as mentioned above Assay Box Content Box 50 reactions Box 100 reactions N vials Volume pl N vials Volume yl MODIfinder OLIGO Mix OLIGOS and Probe pre blended mix 1 150 2 150 Positive Control 1 120 2 120 Negative Control 1 1 1000 All reagents are supplied with a 5 of extra volume Not Provided Article GENERase Mastermix Cat N ENG001 or eq
10. uivalent Reaction Setup Protect reagents from light exposure as far as OLIGO Mix reagents are photosensitive Before setting the analysis we strongly advise to leave the reagents to warm up at room temperature Vortex briefly OLIGO mix afterwards spin to collect contents at the bottom of the vials Spin GENERase Mastermix Cat N ENG001 before opening it Prepare MODIfinder WORKING Mastermix by adding 150 ul of MODIfinder OLIGO Mix into each tube prefilled with 750 ul of GENERase Mastermix Cat N ENGOO1 in order to obtain a single volume of 900 ul of MODIfinder WORKING Mastermix Vortex briefly MODIfinder WORKING Mastermix with the aim of homogenizing the mix and excluding MgCl gradient that could impair the results Spin to collect contents at the bottom of the vial Note label GENERase vials with target name after OLIGO Mix addition Vortex briefly Positive Control and samples before proceeding further spin to collect contents at the bottom of the vial Transfer MODIfinder WORKING Mastermix and samples into the plate as follows Reagents per well Volume Unknown Sample Positive Control Negative Control MODIfinder WORKING Mastermix Final Volume Detector Setup Target Reporter Dye Quencher Dye GMO POTATO FAM BHQ1 NFQ User guide MODIfinder GMO POTATO Assay Rev 1 25 11 2014 ENER N Advanced Transfer Technologies Quick Reference Guide Thermal cycling Step Duration UNG 50 2 min Taq Activation 95 10
11. wing parameters on your thermocycler Total Reaction volume 30 ul Fluorophores Quenchers Target GMO POTATO FAM BHQ1 NFQ Thermal profile Duration UNG 2 min Taq Activation 10 min DNA Denaturation 15 sec Annealing Extension Plate Reading 60 sec User guide MODIfinder GMO POTATO Assay Rev 1 25 11 2014 5 ENER N Advanced Transfer Technologies 5 Data Interpretation Results evaluation must be done according to the analysis software recommended by the Real Time PCR instrument manufacturer After performing PCR each individual sample is analyzed through the instrument software to produce a Cq value quantification cycle for each reporter dye These values are used to determine the presence Qualitative Test of GMO potato into the sample See below an example of the graphics obtained for a positive Fig A control for the GMO potato target amplification blue line Amplification After setting the baseline the analysis outcome should be evaluated following the indications below If the following conditions are met GMO POTATO FAM Positive Control Negative Control Then the possible results for any sample are GMO POTATO FAM Positive Sample Negative Sample Invalid Sample Inhibited In case of inhibition DNA isolation and purification for the sample need to be improved or you may need to dilute your sample before performing a new test Refer to the Troubleshooting paragraph sect
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