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1. 222222222222222222 21 11 Notice to Purchaser 0 2 ccc c cc ccccccccccccccecceececcecceccecceececaeeceenceneeteecseeseess 22 12 Contact Information 22 2 0 o oo cece cece cece ence cenccncecceecetseeseeseeseeeeseeees 23 Devyser KRAS BRAF CE IVD 7 A021 EN v1 2012 Page 3 of 23 Devyser AB 2012 1 Introduction to Devyser KRAS BRAF Intended use The Devyser KRAS BRAF kit is an in vitro diagnostic kit for identification of mutations in the KRAS and BRAF genes Devyser KRAS BRAF is not intended to diagnose cancer The results from Devyser KRAS BRAF are intended to be used as an aid to the selection of appropriate drug therapy treatment for patients with colorectal cancer Clinical decisions for patient treatment should not be made based on the results of Devyser KRAS BRAF alone Included in the kit The Devyser KRAS BRAF kit contains ready to use reagents for PCR amplification of genetic markers Test procedure DNA extraction The Devyser KRAS BRAF kit has been validated using the QlAamp DNA FFPE Tis sue Kit Qiagen cat 56404 for extraction of DNA from formalin fixed paraffin embedded FFPE tissue samples RNase A treatment during DNA extraction is essential for optimal results Amplification The Devyser KRAS BRAF kit has been validated using ABI GeneAm p Systems 9700 Detection Applied Biosystems Genetic Analyzers ABI PRISM 310 3100 3130 3500 3730 that supp2. 7 3 Detection eree aee be d bes ibede rent Idee NN SINNE eh ehe rr KSS 14 Sample preparation 2 2 2 0 0 20 ecco ccc ccc ccc n cece cncceccececcceceeceeseceeeseteeesesceneeseees 14 Sample Preparation for capillary electrophoresis 14 Instrument Preparation 2 2 2 2 20 22 ce cece eee ence cece cece cece ee ceeeceeeceeeseeseeeseees 15 Run Modules NII 15 8 Results and Analysis 22 22 20 2 20 20 2 occ ccc cc cece cece cece cece ececcececceceecececceceeceseeeeee 16 Pre analytic amplification control PAC 0 20 20 0020 2 c cece ccc cececceccececcececseceses 16 Devyser KRAS BRAF CE IVD 7 A021 EN v1 2012 Page 2 of 23 Devyser AB 2012 KRAS and BRAF mutation analysis 2mmmmmmeee nm ss sr rr neon nn nana 17 Table 2 Summary of PCR fragments detected 18 une II ua Aa KA AA WA AA WAA ehe eant dest ce AA secas iA ER CRE Lon AS Be ROMA RA ce AZAA 18 9 Performance Characteristics 22222 2222 20 Sensitivity of the Devyser KRAS BRAF kit 0 0 ccccccecceccececceeceeceeceecceaetaeees 20 Cross reactivity 2 2 20 2 c cece cece ence eee e cece PE SANNES eeeeeetseeeseeees 20 Clinical performance 00 2 0 cccecceccecceceeceeceeceeceecceaeeeeaceccenceccecceesecensenaes 20 10 Procedural Limitations
3. Devyser AR Devyser KRAS BRAF Art No 8 A050 For in vitro Diagnostic Use Instructions for Use CE Devyser KRAS BRAF CE IVD 7 A021 EN v1 2012 Page 10f 23 Devyser AB 2012 Table of Contents Table of Contents mmmammmmaa ec eee eee e cee sese esse esse esse sse spei 2 1 Introduction to Devyser KRAS BRAF 22 20 0222 0 2 20 cece ec ccc ececcececcececceceececeece 4 Intended USE mor AA eee ee 4 Included in the kit cceccecccccececceececeteseesceseeettetseeteeesees 4 Test procedure 2 0 o oo ccc cece cece cece cee ceccceceecenceeeeneeeseeseeseeeteaeeetsseseeneenee 4 Background cecceccecceceeceeceeceeseeseeceeaseeeeetstnsetseteteeteneess 4 Principle of the Procedure 0 20 2 2 cece cece cece ec cccceccecceceeceeseeecesensetsteseeseeaes 4 2 Warnings and Precautions 22 22 20 20 2 coe eee cece cece cece cee cececcececeececceceececeee 6 3 Symbols used on Labels 22 2 2 0 0 0 e cece cece cece cece ec eee ec ee ceccececcececcececeececeeees 7 4 Required Material 2 22 2 0 0 20 00 c cece cece cc cece cece cee cee ec cee cecececceccececeececeeees 8 4 1 Included in the Devyser KRAS BRAF kit 88 A050 22 222 2222222222 8 Configuration RR 8 Components ua IA 8 4 2 Required but Not Provided 2 2 0 2 00 00 20 ccc ccc ccc cecceccecceccecceceeceeeeeseesseseeseeseensess 8 Re
4. negative or false positive results D Devyser KRAS BRAF kit should be used only for the detection of specific mutations in the KRAS and BRAF genes according to the instructions for use The assay has not been validated for diag nosis of cancer Results obtained with Devyser KRAS BRAF kit can only be directly applied to the tissue or specific sample material tested Devyser KRAS BRAF CE IVD 7 A021 EN v1 2012 Page 21 of 23 Devyser AB 2012 11 Notice to Purchaser Results from Devyser KRAS BRAF kit should be interpreted with consideration of the overall pic ture obtained from clinical and laboratory findings Devyser AB will not accept responsibility for any clinical decisions taken Purchase of this product does not provide a license to perform PCR under patents owned by any third party Devyser KRAS BRAF CE IVD 7 A021 EN v1 2012 Page 22 of 23 Devyser AB 2012 12 Contact Information Devyser AB Instrumentvagen 19 SE 126 53 Hagersten SWEDEN Phone 46 8 562 15 850 Homepage www devyser com Technical Support Phone 46 8 562 15 850 E mail techsupport devyser com Devyser KRAS BRAF CE IVD 7 A021 EN v1 2012 Devyser AB 2012 Page 23 of 23
5. tubes may be stored at 2 to 8 C for at least 7 days and at below 18 C for at least 90 days Avoid repeated freeze thawing C Dispose of unused reagents and waste in accordance with country federal state and local reg ulations D Do not mix reagents from different kit lot numbers Devyser KRAS BRAF CE IVD 7 A021 EN v1 2012 Page 10 of 23 Devyser AB 2012 6 Sample Reguirements Clinical Samples The Devyser KRAS BRAF kit is for use with human genomic DNA extracted from formalin fixed paraffin embedded FFPE tissue samples RNase A treatment during DNA extraction is essential for optimal results Use three to five sections 104 M of the FFPE tissue sample for DNA extraction Procedure amp Storage According to manufacturer instructions for use Controls It is recommended that suitable controls such as normal DNA and negative control no DNA are included in each run Devyser KRAS BRAF CE IVD 7 A021 EN v1 2012 Page 11 of 23 Devyser AB 2012 7 Instructions for Use Run Sizes Each Devyser KRAS BRAF kit Art 8 A050 contains reagents for 25 samples It is recommended that the activated reaction mix is dispensed into appropriate PCR reaction tubes after preparation Before dispensing ensure that the activated reaction mix is properly mixed see section 7 1 Dispense in 20 ul aliquots and store at below 18 C To avoid contamination always use un opened tubes Any reagents left in opened tube
6. Endogenous Control KRAS Codon 13 RRAS Codon 12 100 110 120 130 140 150 180 170 2800 f 2400 2000 1600 4200 800 400 o a poe a Control 115 67 rai 3 KRAS BRAF im m KRASOMA BRAF V600E _ 100 410 420 130 140 150 160 170 2800 2400 2000 4600 1200 800 400 A B M veooE 140 02 298 Devyser KRAS BRAF CE IVD 7 A021 EN v1 2012 Page 19 of 23 Devyser AB 2012 9 Performance Characteristics Sensitivity of the Devyser KRAS BRAF kit The limit of detection of the Devyser KRAS BRAF kit has been determined to be lt 3 mutant sequence in a background of wildtype DNA sequence Cross reactivity The Devyser KRAS BRAF kit has been shown not to give any false positive mutant peaks when 100 ng wild type cell line DNA non formamide treated is tested It has been observed that a G13C positive sample may also give a G13D peak It has been observed that a G12V positive sample may also give a G13R peak A G12F positive sample may result in a G12V peak No other cross reactivity has been observed Clinical performance A total of 28 clinical samples of known KRAS and BRAF genotype determined by real time PCR Sanger sequencing Pyrosequencing or allele specific PCR have been tested with the Devyser KRAS BRAF kit Nine samples were provided as DNA extracted from FFPE materials Nineteen samples were provided as FFPE material and were extracted by Devyser using the Qiagen FFPE Tissue Kit including RNase A treatment I
7. NA concentration and DNA purity are important factors for successful testing using the Devyser KRAS BRAF kit DNA should be free from contaminating RNAs proteins and salts Poor quality DNA may result in increased background or amplification failure Addition of too much or too little DNA to the PCR reaction can cause amplification failure When using DNA extracted from FFPE tissue samples a pre evaluation of the DNA quality with the PAC is recommended see below RNAse treatment of all samples during DNA extraction is strongly recommended in order to obtain optimal results It is recommended that alternative DNA extraction methods and sample materials are thor oughly evaluated with the Devyser KRAS BRAF kit prior to the results being used for diagnostic use Addition of Sample Samples should be added in a dedicated area separated from reagent preparation ampli fication and detection areas 1 Add 5 ul of clinical sample to dedicated PCR reaction tubes containing activated reaction mix from step 7 1 2 Cap the tubes and centrifuge briefly to collect the content Amplification Turn on the Thermal Cycler at least 30 minutes prior to amplification For ABI GeneAm p System 9700 set ramp speed to 9600 mode For use of alternative thermal cyclers the following ramping rates must be applied heating Q 0 8 C s cooling 1 6 C s Amplification Area Program the Thermal Cycler for amplification according to the followi
8. See section 4 3 for details Sample Preparation for capillary electrophoresis 1 Prepare a loading cocktail by combining and mixing 2 uL of the size standard e g 560 SIZER ORANGE with 100 uL Hi Di Formamide sufficient mix for 6 wells tubes 2 Vortex for 15 seconds 3 Dispense 15 uL of the loading cocktail into the required number of wells of a micro well plate or into individual tubes ABI310 to be placed on the Genetic Analyzer 4 Add 1 5 uL of the sample PCR product to the corresponding well tube containing loading cocktail 5 Seal the plate tubes Devyser KRAS BRAF CE IVD 7 A021 EN v1 2012 Page 14 of 23 Devyser AB 2012 Instrument Preparation Create a sample sheet using the data collection software with the following settings e Sample ID e Dye Set DEV 5 or D e Recommended run Module See below for different polymers and instruments Run Modules ABI 310 Run Parameters POP 4 ABI 310 ABI 3100 3130 ABI 3500 Run Parameters Capillary length Run temperature Injection voltage Injection time Run voltage Run time The amount of PCR product injected into the capillaries can be adjusted by increas ing decreasing the injection voltage and or injection time Devyser KRAS BRAF CE IVD 7 A021 EN v1 2012 Page 15 of 23 Devyser AB 2012 8 Results and Analysis Pre analytic amplification control PAC A spectrophotometric analysis of the DNA extracted from FFPE tissue may not giv
9. agent Preparation _ 2 0 220 c ccc cece cence cece cence eee cece eee eceeeeeeeeeeeeeeee 8 DINNA EXEFaCLIOTIS m o tee sue hee es Rit eee eeu eee 8 Amplification cece ccccecceccccceceeceeceesceseeececeetetesetsetseeseesteses 8 Detection RR RT 8 siz Standard serco tit WA 8 4 3 Dye Set Calibration 2 20 20 0 20020 ccc cece ccc ccc ec cnc ce ccccececccccetceeseesetsetcesetseeseeees 9 5 Storage and Handling Requirements 22 22 2020 0 22 2 c cece cece eee c cece ceceececeeceees 10 6 Sample Requirements 22 22 20 2 cece cece eee eee ccc e cece ec cece cececcececceceeeeceeesees 11 Clinical Samples ee 11 Procedure amp Storage sssssssssssssssssssssssssl sss lel selIelRelllrrllll2lllll2lz 222222 11 CONTOS cn c mi cac Lad D D LC DU We T A 11 7 Instructions for Use _ 2 2 2 2 0 20 0 e cece cece ccc eee cece cece ese esse sse smell le sese lli 12 RUNS NZS AER RM ea os eee AA Rn 12 7 1 Workflow Devyser KRAS BRAF kit Art 8 A050 0000 00 00 0 ooo ccc eee eee cece 12 7 2 Sample Preparation and PCR Amplification 12 DNA Extraction ss eos eU MEMMIUS ORE e Ite esas 12 Addition of Sample III IrlRrlerlellrlltell2lz22 13 Amplification 220 00 0 00 20 o ccc cece cece ccc cnc e E a aE 13 Amplification Area ss seeeooo LDD LLDD L Lo rrna 13
10. agent tubes The use of sterile disposable aerosol barrier pipette tips is recommended C Do not pool reagents from different lots or from different tubes of the same lot D Do not use a kit after its expiry date E Do not use opened or damaged kit reagent tubes F Workflow in the laboratory should proceed in a unidirectional manner beginning in the reagent preparation area and moving to the DNA extraction area and then to the amplification area and finally to the detection area Pre amplification activities should begin with reagent prep aration and proceed to DNA extraction Reagent preparation activities and DNA extraction activ ities should be performed in separate areas Supplies and equipment should be dedicated to each activity and not used for other activities or moved between areas Gloves should be worn in each area and should be changed before leaving that area Equipment and supplies used for reagent preparation should not be used for DNA extraction activities or for pipetting or proc essing amplified DNA or other sources of target DNA Amplification and detection supplies and equipment should remain in the amplification and detection area at all times G Handling of kit components and samples their use storage and disposal should be in accord ance with the procedures defined by national biohazard safety guidelines or regulations H Wear powder free disposable gloves laboratory coats and eye protection
11. e reliable results for the assessment of the amount of DNA accessible for PCR amplification DNA cross linking and fragmentation effects during formalin fikation may limit purification and ampli fication efficiency RNA remaining after DNA extraction may also affect spectrophotometric analysis Sample analysis using the Devyser PAC Mix helps to determine the degree of DNA degradation and accessibility for PCR amplification The Devyser PAC Mix amplifies regions of the KRAS gene to generate fragments of different lengths figure 1 The product sizes are listed in table 1 The height of the peaks indicate the amount of amplifiable DNA in the sample the larger the peak height the more amplifiable DNA is present It is recommended to use PAC analysis to assess the optimal amount of DNA to be used in the Devyser KRAS BRAF analysis The PAC and KRAS BRAF assays are designed to give similar frag ment peak heights for the 97 bp fragment of the PAC analysis and the 115 bp endogenous con trol fragment of the KRAS BRAF analysis Optimal sensitivity when using the Devyser KRAS BRAF kit can be expected when the height of the 97 bp PAC peak is between 4000 and 7000 relative fluorescent units rfu using an ABI 310 3100 or 3130 Genetic Analyzer and between 10000 and 25000 rfu using an ABI 3500 Genetic Analyzer Samples that result in signals exceeding these ranges may lead to false positive results and should therefore be diluted The relative peak height
12. in the size range between 110 bp and 120 bp figures 2 3 and 4 Optimal sensitivity when using the Devyser KRAS BRAF kit can be expected when the height of the endogenous control peak is between 4000 and 7000 relative fluorescent units rfu using an ABI 310 3100 or 3130 Genetic Analyzer and between 10000 and 25000 rfu using an ABI 3500 Genetic Analyzer Samples that result in signals exceeding these ranges may lead to false pos itive results and should therefore be diluted KRAS mutations in codons 12 and 13 A KRAS gene mutation in codons 12 or 13 will be detected in the blue window as a peak in the size range between 135 bp and 165 bp table 2 and figure 3 KRAS mutations in codon 61 A KRAS gene mutation in codon 61 will be detected in the green window as a peak in the size range between 115 bp and 125 bp see table 2 for details Devyser KRAS BRAF CE IVD 7 A021 EN v1 2012 Page 17 of 23 Devyser AB 2012 BRAF V6OOE mutation A BRAF V600E mutation will be detected in the green window as a peak in the size range between 135 bp and 145 bp table 2 and figure 4 Table 2 Summary of PCR fragments detected Estimated fragment Window B h eme codon mutation Basechange WA bp KIE Endogenous control fragment p Gym GIZA GGT gt CeT esac 1575 blue kiwa 612c ecrster esset 163 blue Gly124sp 6120 GGT gt GAT casa 2495 blue Gia GIZA GGT gt GCT e3sec 16 blue H aa Gwsse
13. ion Applied Biosystems Genetic Analyzer ABI 310 3100 3130 3500 3730 Performance optimized polymers POP 4 or POP 7 Hi Di Formamide Genetic Analysis Grade 1x Genetic Analyzer Buffer Micropipette multipipette dispenser with aerosol barrier tips or displacement tips 1 5 uL 15 uL Size Standard Devyser Dye Set DEV 5 560 SIZER ORANGE Devyser cat 8 A402 Devyser KRAS BRAF CE IVD 7 A021 EN v1 2012 Page 80f 23 Devyser AB 2012 ABI Dye Set D Gene Scan 500 ROX Size Standard ABI cat H401734 H4310366 4 3 Dye Set Calibration ABI 3100 3130 3730 Use DEV 5 Dye Set MultiCap kit Devyser cat 8 A401 in the Any5Dye dye set or DS 30 Matrix Standard Kit ABI cat 4345827 in Dye set D ABI 3500 Use DEV 5 Dye Set MultiCap kit Devyser cat 8 A401 and generate the DEV 5 dye set or use DS 30 Matrix Standard Kit ABI cat 4345827 and generate Dye set D ABI 310 Matrix file generation Use DEV 5 Dye Set SingleCap kit Devyser cat 8 A400 Run with module file GS STR POP4 1 mL G5 md5 ABI Dye Set D 6FAM HEX ROX ABI cat 401546 and NED ABI cat 4 402996 Run with module file GS STR POP4 1 mL D md4 Devyser KRAS BRAF CE IVD 7 A021 EN v1 2012 Page 9 of 23 Devyser AB 2012 5 Storage and Handling Reguirements A Store all components below 18 C B The activated reaction mixes prepared by addition of Devyser PAC Mix and Devyser KRAS BRAF Mix to separate PCR Activator
14. n 24 out of 28 samples the results obtained using the Devyser KRAS BRAF kit were consistent with the previously identified genotypes Devyser KRAS BRAF kit results from four DNA samples were inconsistent with the previously reported results Further confirmatory testing of the four discrepant samples was performed using pyrosequencing The confirmatory method produced the same result as Devyser KRAS BRAF in 3 out of 4 discrepant samples The fourth discrepant sample was determined as G12V GTT using Devyser KRAS BRAF G12C TGT and G12V GTT using allele specific PCR and G12F TTT using pyroseguencing Devyser KRAS BRAF CE IVD 7 A021 EN v1 2012 Page 20 of 23 Devyser AB 2012 10 Procedural Limitations A Use of this product should be limited to personnel trained in the techniques of PCR and capillary electrophoresis B The Devyser KRAS BRAF kit has been validated using the ABI Thermal Cycler GeneAm p 9700 It is recommended that alternative thermo cycler instruments are thoroughly evaluated with the Devyser KRAS BRAF kit prior to the results being used for diagnostic use C The Devyser KRAS BRAF kit has been validated using the AlAamp DNA FFPE Tissue Kit Qiagen cat 56404 for extraction of DNA from FFPE tissue samples RNase A treatment Qiagen art No 19101 during DNA extraction is essential for optimal results Performance with other sam ple materials and DNA extraction kits has not been validated and may result in false
15. ng Thermo Profile con sult the User s Manual for additional information on programming and operation of the ther mal cycler 95 C 15 min 94 C 30 sec 60 C 30 sec 72 C 30 sec for 2 cycles 94 C 30 sec 66 C 60 sec 72 C 60 sec for 32 cycles 72 C 15 min 4 C FOREVER Devyser KRAS BRAF CE IVD 7 A021 EN v1 2012 Page 13 of 23 Devyser AB 2012 Pre Denaturation Cycling 1 Cycling 2 Final Extension TB an 94 C I 15min RR i 30sec 30sec 1 i 72 C 72 C 72 C iv 15mi 3 30sec i 60 sec min i H 66 C H 90 60 sec 30 sec Ambient Noa Temperature 6e us 2cycles 32 cycles j 1 Set reaction volume to 25 ul 2 Start the amplification duration approximately 2 5 hrs 3 Following amplification remove the tubes containing completed PCR amplification reaction from the thermal cycler and place into a suitable holder Centrifuge briefly to collect the con tent Remove the caps carefully to avoid aerosol contamination Do not bring amplified mate rial into the pre amplification areas Amplified material should be restricted to amplification and detection areas 7 3 Detection Sample preparation Refer to the respective ABI PRISM Genetic Analyzers User Manual for instructions on main tenance and handling Prior to running the Devyser KRAS BRAF kit the instrument must be spec trally calibrated to support detection of the proper dye set with the polymer used
16. ort the detection of Devyser Dye Set DEV 5 or ABI Dye Set D DS 30 Background Mutations in KRAS and BRAF genes can indicate prognosis and might prevent the therapeutic success of anti EGFR therapies since tumors carrying mutant forms of the KRAS and BRAF genes are less likely to respond to anti EGFR therapy Testing for KRAS and BRAF mutations can facilitate the identification of tumors that will not respond to anti EGFR drugs and shortening the time for identifying alternative treatment options Principle of the Procedure The method employed by the Devyser KRAS BRAF kit uses multiplex allele specific PCR ampli fication for detection of point mutations in the KRAS and BRAF genes The PCR products gen erated are analysed by capillary electrophoresis CE and fluorescent detection on a CE instrument Devyser KRAS BRAF CE IVD 7 A021 EN v1 2012 Page 4 of 23 Devyser AB 2012 The kit also includes a pre analytic amplification control PAC It is recommended that the PAC is used to confirm the presence of amplifiable DNA in a clinical sample before performing the KRAS and BRAF mutation analysis Devyser KRAS BRAF CE IVD 7 A021 EN v1 2012 Page 5 of 23 Devyser AB 2012 2 Warnings and Precautions A Devyser KRAS BRAF has been validated using a total PCR reaction volume of 25 ul Changing the reaction volume will compromise the kit performance B Avoid microbial contamination of reagents when removing aliquots from re
17. r GissoGOGc esrara 1425 blue Gyism GisR GGOCGc esc 14 bee Gwisos ensc seoTec esros wo bee Brar 600 vel cochwveoor GreeGAG tispA 1395 green Based on observed fragment lengths basepair bp using ABI 3130 and POP7 Example data Figure 2 Typical results obtained from a sample without mutations indogenous Control RAS Codon 13 RAS Codon 12 100 110 120 130 140 150 160 470 6000 5000 o ra T Control 115 76 6565 KRAS BRAF KRAS Q61H BRAF V600E 100 110 120 130 140 150 460 470 6000 5000 3000 2000 1000 Devyser KRAS BRAF CE IVD 7 A021 EN v1 2012 Page 18 of 23 Devyser AB 2012 Figure 3 Typical results obtained from a sample containing a KRAS G12D mutation Endogenous Control KRAS Codon 13 Codon 12 100 410 120 130 140 150 180 170 2800 2400 2000 1600 1200 800 A o a a av Control 6120 115 75 140 25 208 823 gt KRAS BRAF m m ERASO 7 BRAF V600E 1 100 110 120 130 140 150 160 170 2800 2400 2000 4600 1200 800 400 Figure 4 Typical results obtained from a sample containing a BRAF V600E mutation
18. s of the different PAC fragments in a sample is an indication of the degree of DNA degradation If the 250 bp fragment peak height is significantly lower than the 97 bp and 160 bp fragment peak heights the DNA sample is degraded but the Devyser KRAS BRAF kit can be used to detect mutations in the KRAS and BRAF genes If the 160 bp fragment peak height is significantly lower than the 97 bp fragment peak height the DNA sample is severely degraded Detection of KRAS or BRAF mutations might still be pos sible in severely degraded DNA samples if the sample contains a high percentage of mutated tumor material In this case it is recommended to analyze a new freshly prepared DNA sample instead Devyser KRAS BRAF CE IVD 7 A021 EN v1 2012 Page 16 of 23 Devyser AB 2012 If the 97 bp fragment peak height is very low or absent no KRAS or BRAF mutation can be detected In this case it is recommended to analyze a new freshly prepared DNA sample Table 1 Wind 1 97 bp KRAS e 2 160 bp PAC3 O 250 bp Based on observed fragment lengths basepair bp using ABI 3130 and POP7 ius 1 Typical results from PAC amplification and detection on an ABI 3130 do OS e 5r PAS a KRAS and BRAF mutation analysis The KRAS BRAF mix amplifies an endogenous control fragment that should be detectable in all samples The endogenous control fragment should be detected as a peak in the blue window
19. s should be discarded 7 1 Workflow Devyser KRAS BRAF kit Art 8 A050 The activated reaction mixes should be prepared before preparing the samples if the complete process is performed in one day Only if the samples are prepared the day before amplification or earlier the opposite order is advisable The activated reaction mixes are prepared by adding the Devyser KRAS BRAF and Devyser PAC Mixes to separate PCR Activator tubes The Devyser KRAS BRAF kit has been validated using a total PCR reaction volume of 25 ul Changing the reaction volume will compromise the kit performance 1 Centrifuge each tube briefly to collect the content Do not vortex the tubes at this step 2 Carefully add 500 ul of the Devyser PAC Mix and Devyser KRAS BRAF Mix to separate PCR Activator tubes 3 Mix manually by pipetting 300 500 ul several times from the bottom of each tube 4 Vortex the activated reaction mix tubes and centrifuge briefly to collect the content 5 Add 20 ul of the activated reaction mixes to separate PCR reaction tubes 6 Cap the reaction tubes and centrifuge briefly to collect the contents The activated reaction mixes are stable at 2 8 C for at least 7 days and at below 18 C for at least 90 days Avoid repeated freeze thawing 7 2 Sample Preparation and PCR Amplification DNA Extraction According to manufacturer s instructions for use Devyser KRAS BRAF CE IVD 7 A021 EN v1 2012 Page 12 of 23 Devyser AB 2012 D
20. when handling spec imens and kit reagents Wash hands thoroughly after handling specimens and kit reagents Devyser KRAS BRAF CE IVD 7 A021 EN v1 2012 Page 6 of 23 Devyser AB 2012 3 Symbols used on Labels LOT Lot or batch number Expiry date Manufacturer Number of tests In vitro diagnostic device gt 5 EK r3 Store below temperature shown Devyser KRAS BRAF CE IVD 7 A021 EN v1 2012 Devyser AB 2012 Page 7 of 23 4 Reguired Material 4 1 Included in the Devyser KRAS BRAF kit 8 A050 Configuration The Devyser KRAS BRAF kit contains reagents for analysis of makimum 25 samples Components PCR Activator 4 A018 2x 25 Tests Devyser KRAS BRAF Mix 4 A132 1x25 Tests Dewyser PAC Mix 4 A141 1x 25 Tests 4 2 Required but Not Provided Reagent Preparation Consumables for the Thermal Cycler Micropipette dispenser with aerosol barrier tips or displacement tips 500 ul Disposable protective gloves powder free DNA Extraction Reagents and equipment according to manufacturer instructions for use RNase A Qiagen art No 19101 Micropipette multipipette with aerosol barrier tips Amplification Thermal Cycler ABI GeneAmp PCR System 9700 using 9600 mode For use of alternative ther mal cyclers the following ramping rates must be applied heating 9 0 8 C s cooling Q 1 6 C s Micropipette dispenser with aerosol barrier tips or displacement tips 5 20 ul Detect
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