PCR Kit - Imogena
Contents
1. Fig 3 Stage 1 Stage 2 Hold Hold x Temp Secs Optics Temp Secs Optics Temp Secs Optics 37 120 OFF 95 600 OFF Step 3 In the Stage 3 window select 3 Temperature Cycle from the drop down menu Type 45 in the Repeat field Type Temp 95 Secs 10 and Optics OFF into the first line type Temp 64 Secs 60 and Optics ON into the second line type Temp 72 Secs 40 and Optics OFF into the third line Fig 4 Fig 4 Temperature profile setting Stage 1 Stage 2 Stage 3 Hold v Hold v Repeat 45 times Temp Secs Optics Temp Secs Optics 3 Temperature Cycle v 37 120 OFF 95 600 OFF Tama Saree Opice 95 72 40 OFF Set the fluorophore combination in the Dye Set item as FTTC25 At the NC fails if item check Any target criterion is positive Fig 5 In the Analysis Settings tab choose Assay in the Usage column for channel 1 FAM In the Thresh Settings column choose Manual Threshold type 30 into the Manual Threshold column field Enter ON in the Bkgnd Sub column this parameter applies automatically on all used channels For channel 2 TET choose Internal Control in the Usage column In the Thresh Settings column choose Manual Threshold type 30 into the Manual Threshold column field For channels 3 and 4 TxR and Cy5 choose Unused in the Usage column Leave all other parameters unchanged Fig2. Zial SamplelD Assay Result IC Result ICThresh IC FAM wa Run Name Sample ont EndPt Result GeneProof detection 220408 oof PCR A1 Sample 1 Na SPEC 33 2 na ue POS m NA a2 Sample 2 PASS I SPEC 33 5 INA hs am WE O NA TA TET 3 Sample3 FAIL SPEC o NA his 2 NEG O NA temperature JA4 Positive Control Valid NA Gett 325 JNA hs am Vala b223 28 Started Apr 22 2008 06 10 AM Negative Control Valid Pass NC1 328 25 hs Ee Valid O 0 Finished Apr 22 2008 08 04 AM Status Done Notes temperature Assay GeneProof PCR Research Assay Assay Information Lot Number Expiration Date Number of Sites 5 seer Export Report SelectGraphs view Another Run DeleteRuns Update Analysis 10 Detection evaluation Fluorescency Fluorescency Fluorescency Fluorescency FAM PA 2 8 14 20 26 32 38 44 50 No A Cycle number L 2 8 14 20 26 32 38 44 50 H a A e Cycle number 1 0 8 0 6 0 4 0 2 0 2 8 14 20 26 32 38 44 50 Cycle number 1 0 8 0 6 0 4 0 2 0 2 8 14 20 26 32 38 44 50 Cycle number Fluorescency oF H u A Fluorescency TET 2 8 14 20 26 32 38 44 50 Cycle number 0 8 0 6 0 4 0 2 2 8 14 20 26 32 38 44 50 Cycle number Fluorescency oR Nw a Fluorescency 2 8 14 20 26 32 38 44 50 Cycle number
3. 5 Fig 5 Detection parameters settings Analysis Settings Control Settings Probe Check Settings Analysis Research Assay Si Dye Target Usage Curve Fa Manual Auto Auto Min Auto Max Valid Min Valid Max Bkgnd a Name Analysis Thres Thresh Cycle Cycle Cycle Cycle Sub 1 FAM Assay Primary Manual Thresh 30 0 NA 5 Jo 13 0 45 0 JON 2 mr Internal Con JPrimary Manual Thresh 30 0 Ha 5 ho hao en ON 3 TxR Unused Primary Manual Thresh 30 0 NA 15 o 13 0 145 0 Em 4 Cys Unused Primary Manual Thresh 30 0 NA 5 no 13 0 45 0 oN KIE Dye Set FTTC25 v I Require Lot Number Define Result Text NC fails if EI All target criteria are positive Any target criterion is positive Use Patient IDs Convert Assay 5 Description of the results can be set by opening of the Define Result Text button Positive result has the POS parameter in the FAM column and NA parameter in the IC column Negative result has NEG parameter in the FAM column and PASS parameter in the IC column In case of an invalid result the sample has NEG in the FAM column and FAIL in the IC column Fig 6 Fig 6 Result description settings Assay Result Text Settings Assay Result Text Assay Color Light Gray ok Cancer Reset To Defaut 6 Save the created program using the Save Assay
4. 0 8 0 6 0 4 0 2 2 8 14 20 26 32 38 44 50 Cycle number see Troubleshooting p 12 Result Positive Positive Negative Invalid result 11 Troubleshooting Invalid result of a Positive Control analysis FAM TET Result 1 4 g 8 g 08 c 3 o 06 o a 2 P 04 g s S 1 zt 02 be 0 0 2 8 14 20 26 32 38 44 50 2 8 14 20 26 32 38 44 50 Cycle number Cycle number Problem Incorrect programming of the PCR amplification Problem solution 1 Check device programming according to the manual 2 Check correct temperature settings in the individual program blocks Problem Positive control incorrectly held in storage see Storage and transportation conditions Problem solution 1 Check whether kit component is stored according to manufacturer s recommendations 2 Submultiple the Positive control and do not freeze and thaw it Invalid result of a Negative Control analysis Fluorescence Fluorescence FAM TET Result 4 4 o 3 23 2 8 2 1 S1 T 0 0 2 8 14 20 26 32 38 44 50 2 8 14 20 26 32 38 44 50 Cycle number Cycle number 1 o 4 2 08 0 6 3 a D L 04 2 z 0 1 0 0 2 8 14 20 26 32 38 44 50 2 8 14 20 26 32 38 44 50 Cycle number Cycle number 12 Problem PCR reaction contamination Problem solution i Check the process of preparation and pipetting of the PCR mix into tubes 2 Check whether sterile plas
5. window Enter sample description into the Sample ID column for better orientation Fig 8 Fig 8 SE and controls definition User Logs Setup Tools Help EE Define Assays naa Define Graphs DH Run Name Site Table Site Assay Sample ID Notes Sample pod Type A4 GeneProofPOR Positive Control L AS GeneProof PCR Negative Control Assay GeneProof PCR Research Assay Assay Information Lot Number Expiration Date Number of specimens 3 Apply Graphis Temperature S FAM Co Red ba Ge Add Remove Sites StartRun CancelRunSetup RoportRunSetup SelectGraphs 9 Real time imaging of selected values can be set by selecting the Select Graphs button in the bottom menu Use the arrows to select FAM TET and temperature and confirm OK The graph parameters can be changed also in the course of the PCR run after starting the machine Fig 9 Fig 9 Selecting graphs User Logs Setup Tools Help Run Name temperature FAM GeneProof detection YYMMDD TET Notes Temperature Assay GeneProof PCR Research Assay Assay Information mmm Expirati Number of specimens 0 Graph s Cy3 xRed Add Remove Sites gan pa CancetRun Setup SE Se Graphs 10 Start the examina
6. GeneProot PCR Kit This manual is designed for the following kits Mycobacterium tuberculosis PCR kit Borrelia burgdorferi PCR kit Chlamydia trachomatis PCR kit Chlamydia pneumoniae PCR kit Neisseria gonorrhoeae PCR kit Mycoplasma pneumoniae PCR kit CE in vitro Diagnostics Manual for use with the following device SmartCyclet VERZE K1 VERZE01 CZ Contents GENEPROOF PCRURTIT veinisiesnvessdnscdsietssonscednsesevndecedncosenpsesvavavdvasceonsecotntedevaseconvecsntssences duces unteoesnes 3 ISIN AND ISEX VERSIONS OF THE GENEPROOF PCR KIT cccsccesssscssresesseeseseesensnevenees 4 USER MANUAL FOR SMART CYCLER ccccccssssscsssesesseesesseessseeseneeeveseeeseseesenseevesseeseseesenseeoees 5 Reaction preparation and device programming NEEN 5 TR 11 TROUBLESHOOTING ccccsccssssscsssesesseevsssseveseesesseesesseeseseesssseesensessessesseseesensueseseseveseesensnesenees 12 INO RR E 14 GeneProof PCR kit GeneProof PCR kits designed for the detection and quantification of pathogen DNA are based on the principle of amplifying specific target sequences of microorganisms and measuring the amplification product concentration growth in the course of the polymerase chain reaction by means of fluorescence marked probes the probe designated for pathogen detection is marked by the FAM fluorophore The reaction mix includes an Internal Standard IS controlling the possible inhibition of the PCR reaction and the efficie
7. alysis Auto Auto Min Auto Max Valid Min Valid el Bkgnd B Arash Cycle cyela cya Cycle sub NA 10 13 0 5 45 0 ON 5 10 130 45 0 ON 5 10 130 45 0 ON 45 0 ON 5 5 ls 5 Please enter a new name for the assay GeneProof PCR Define Result Text Convert Assay OK Cancel Any target criterion is positive Stage 5 Stage 6 Stage7 Unused Unusea Temp Secs Optics Temp Secs Optics Temp Secs Optics Temp Secs Stage 1 Stage 2 Stage 3 Stage 4 unused Unused Unusea v Unused unusea Temp Secs Optics Temp ges Optics 2 Program the PCR temperature profile according to following instructions Step 1 In the Stage 1 window select Hold from the drop down menu type 37 in the Temp field type 120 into the Secs field and select OFF in the Optics column Fig 2 Fig 2 Stage 1 Stage 2 Stage 3 Hold z Unused v Unused S Temp Secs Optics Temp Secs Optics Temp Secs Optics zm 120 0FF Step 2 In the Stage 2 window select Hold from the drop down menu type 95 in the Temp field type 600 into the Secs field and select OFF in the Optics column Fig 3
8. button in the bottom menu 7 To start a run choose Create Run tab in the main menu Enter the name of the detection kit used in the Run Name field Choose GeneProof PCR program created in the first part of this manual in the Assay drop down menu Fig 7 Be 7 a run Run Name D Site Table GeneProof detection YYMMDD TA Assay Sample ID Notes Sample Type User Logs Setup Tools Help mal Define Assays E Ei Notes Assay GeneProof PCR v Research Assay Assay Information Lot Number Expiration Date Number of specimens mw Graph s FAM TET DR sittin CancelRunSetup RemortRunSetup Select Graphs 8 Type the number of samples to be examined into the Number of specimens field and click Apply count in only the samples without external controls like Positive and Negative control These will be automatically included by the software after the samples to be examined The final number of specimens and their order will be shown in the main window The number of specimen and their order can be modified if necessary by selecting the Add Remove Sites button below the main window Use the arrows to add or remove sample positions In case of redefining the positive or negative control position select PC1 for positive control NC1 for negative control or SPEC for unknown sample in the Sample Type column in the main
9. ncy of DNA isolation process Amplification of IS results in positive signal in TET channel GeneProof PCR kits gt Use the hot start technology minimizing non specific reactions and assuring maximum sensitivity gt Contain uracil DNA glycosylase UDG controlling possible contamination of the PCR reaction by amplification products All PCR kits for pathogen DNA detection can be amplified by means of a universal amplification program Are easy to use the kits always contain one tube with MasterMix and one tube with Positive Control or with an Internal Standard or a set of calibration controls gt Are designed for in vitro diagnostics CE IVD certification ISIN and ISEX versions of the GeneProof PCR Kit All GeneProof PCR kits include an Internal Standard providing for an effective monitoring of eventual inhibition of the PCR amplification and also of the isolation process efficiency The Internal Standard is a precisely defined and quantified construct of a plasmid and insert prepared by methods of genetic engineering GeneProof develops and sells two basic versions of PCR kits with various compositions of the Internal Standard PCR kit ISIN Cat No PCR kit ISIN In this version of the PCR kit the Internal Standard is included in the MasterMix tube This PCR kit version enables PCR inhibition control PCR kit ISEX Cat No PCR kit ISEX In this PCR kit version the Internal Standard is included as an indepe
10. ndent item within the package This PCR kit enables both PCR inhibition control and DNA isolation process efficiency control The Internal Standard should be added into the sample at the beginning of the isolation process as follows 0 1 ul of the Internal Standard per 1 ul of elution volume Elution Volume 25 ul 50 ul 100 ul 200 ul Internal Standard 2 5 ul 5 ul 10 ul 20 ul PCR kit ISIN PCR kit ISEX v v Internal Standard 0 1ul IS per Lu of elution volume DNA isolation SEET 30 ul MasterMix 10 u DNA Cycle number Cycle number PCR inhibition control PCR inhibition and 4 DNA isolation efficiency control User manual for SmartCycler Reaction preparation and device programming Add 15 pl of the MasterMix and 10 pl of the DNA isolate or 10 pl of the Positive Control into a SmartCycler tube The final reaction mix volume will be 25 ul Close the tubes and centrifuge them shortly Insert them into the device and program according to the following procedure 1 Start the SmartCycler Dx software select Define Assays tab in the main menu and select New Assay in the bottom menu New Assay window opens Type in GeneProof PCR and click OK Fig 1 Fig 1 Creating a new PCR program Assay Name wa Done Sranie A nalysis sei Thies 1 FAM lAssay Primary Manual Thresh 30 0 Ch Dye Target Usage E Thresh Setting Name An
11. tics and filtered tips are used 3 Clean the PCR box 4 Ad uracil DNA glycosylase UDG into the reaction Invalid result of an Unknown Sample analysis FAM TET Result o o Se a o 06 S 06 gt 8 04 8 04 kb S S SR 2 8 14 20 26 32 38 44 50 2 8 14 20 26 32 38 44 50 Cycle number Cycle number Problem PCR reaction inhibition PCR kit ISIN and ISEX Problem solution 1 Repeat DNA isolation 2 Check the process of preparation and pipetting of the PCR mix into tubes Problem Invalid process of DNA isolation PCR kit ISEX Problem solution L Repeat DNA isolation 2 Check the process of preparation and pipetting of the Internal Standard at the beginning of the isolation process Problem Incorrect storage of the MasterMix see Storage and transportation conditions Problem solution 3 Check whether MasterMix is stored according to manufacturers recommendations 1 Submultiple the MasterMix and do not freeze and thaw it If you have any questions please contact our Product Support Department at support geneproof com 13 Notes Manufacturet GeneProof a s Vini n 235 615 00 Brno esk Republika Tel Fax 420 543 211 679 e mail sales geneproof com www geneproof com Issue date 8 6 2010 Last Revision Date 8 6 2010 14
12. tion by selecting the Start Run button Detection evaluation 1 After the program finishes select View results tab in the main menu In the Views window select the FAM channel for positive signal control and TET channel for internal standard detection control Fig 10 Fig 10 Results view in the FAM and TET channels User Logs Setup Tools Help Create Run E Check Status Maintenance Run wa Assays Define A Views i Site ID Sample ID Run Name or Results Sample 1 oeneror ce sane jem Sample 3 User Unknown TET Positive Control temperature Negative Control Started Apr 22 2008 06 10 AM Finished Apr 22 2008 08 04 AM Status Done Notes Views l Site ID Sample ID Sample Results 1 Sample 1 GeneProof PCR Sample 2 FAM Samples lt lt Positive Control temperature Negative Control Assay GeneProof PCR Research Assay Information i LotNumber Expiration Date Number of Sites 5 Export Report Select Graphs View Another Run Delete Runs Update Anatysis 2 For evaluating the results select Sample Results in the Views window An automatic evaluation of the results according to preset parameters shows in the Assay Result column Fig 11 11 Detection results evaluation Define Graphs User Logs Setup Tools Help Maintenance
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