User manual - Check
Contents
1. New 4 Color Compensation Set User manual Version 1 2 Issued 29 11 2013 s 4 Color Compensation Set for Check Direct CPE e Re name the new format to Check Points 4 Color Set e Check the boxes of the Excitation Emission for the corresponding Instrument as outlined in Figure 2 Table 1 The five reaction mixes setup Filter Combination Selectio Filter Combination Selectio Emission Emission 610 488 510 610 E E Vv LI LI 2 D ser D er zf S Pm soe D et see OO X P LI Figure 2 Filter Combination Selection for LC 480 I amp II e Change the Quant Factor for all the filter combinations to 10 and the Max Integration Time Sec to 2 as outlined in Figure 3 As a reference also change the name of the filtercombination e g FAM VIC RED CY5 Selected Filter Combination List Excitation Emission Name Melt Quant Max Integration Filter Filter Factor Factor Time Sec a T 1 d LC 480 I Selected Filter Combination List Excitation Emission Name Melt Max Integration Filter Filter Factor Time Sec 1 1 al il LC 480 II Figure 3 Filter Combination List for LC 480 I amp II e Close the Tools window 4 Real time PCR instrument settings e Inthe LC 480 software version 1 5 select New Experiment e In Experiment Setup select Detection Format Check Points 4 Color Set e In Experiment Setup select Customize and make sure all 4 filter combinations ar2. 7 1 Intended use The 4 Color Compensation Set is used to create an application specific color compensation object or file on the Light Cycler 480 system I amp II The 4 Color Compensation Set is to be used in combination with the Check Direct CPE real time PCR diagnostic kit Ref 18 0080 The Check Direct CPE assay requires a color compensation run once a year after the calibration of the optical parts of the LC 480 system I amp II Once the application specific color compensation object has been performed and the data file created it is used to analyze all the data generated with the Check Direct CPE real time PCR diagnostic test Product purpose and function The Check Direct CPE real time PCR diagnostic kit Ref 18 0080 simultaneously detects four different colors on the LC 480 System I amp II Due to the overlap of the emission spectra of organic dyes crosstalk emission between detector channels can occur This phenomenon is described as the overspill of one dye into the next detector channel which may result in the misinterpretation of the data To correct for cross talk emission between detector channels color compensation can be applied when analyzing the data The dye calibrators used in the color compensation set are identical to the dyes used in the Check Direct CPE diagnostic kit During a color compensation run the LC 480 instrument measures the fluorescence of each dye calibrator in all the channels and generates an
3. instrument specific color compensation file or object When analyzing Check Direct CPE experimental data the software uses this color compensation file object data to reassign the fluorescence in each detector channel to the appropriate dye As a result only one dye signal is detected in each channel 4 Color Compensation Set User manual Version 1 2 Issued 29 11 2013 Kit contents for color compensation run 4 Color Compensation Set for Check Direct CPE FAM calibrator 9 0092 1 brown tube green cap 50 ul VIC calibrator 9 0094 1 brown tube yellow cap 50 ul Red calibrator 9 0093 1 brown tube red cap 50 ul 1 brown tube blue cap 50 ul 20 C store in the dark 1 tube green inlay 100 ul 1 tube yellow inlay 100 ul Cy5 calibrator 9 0091 FAM control 9 0096 VIC control 9 0098 1 tube blue inlay 100 1 transparent tube and cap 450 ul Leaflet download from website Cy5 control 9 0095 CPE PCR Mastermix 9 0081 Red control 9 0097 1 tube red inlay 100 ul User manual 9 0099 Storage handling and stability The product is shipped cooled The CPE PCR Mastermix should be stored at 4 C upon receipt All other reagents should be stored at 20 C upon receipt Please visually inspect the box upon initial opening to ensure that its contents are intact Please contact the Check Points office at support check points com if you have any further questions Store kit reagents at i
4. User manual 4 Color Compensation Set for Check Direct CPE Version 1 2 Date of issue 29 11 2013 18 0070 Vi 1 EU 070 05 For use on the Roche Light Cycler 480 system I amp II CE IVD 4 Color Compensation Set User manual Version 1 2 Issued 29 11 2013 s A Color Compensation Set for Check Direct CPE INTENDED USE iraina iE 2 PRODUCT PURPOSE AND EUNCTION ssnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn nnana 2 KIT CONTENTS FOR COLOR COMPENSATION RUN sssnnnsssnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnna 2 STORAGE HANDLING AND STABILITY ccssccccccsssenseessssnnnnssseeneneesseenneersnennnnnnnnasaeenneesseeneneresenaans 2 MATERIALS REQUIRED BUT NOT SUPPLIED WITH THE KIT nsssssnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn 3 GOOD LABORATORY PRACTICES EN 3 PERFORMING THE COLOR COMPENSATION sssssssnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn nnna 4 1 IMPORTANT POINTS BEFORE STARTING cssscsssssssssesssesssesssnssseesseesseesseesseesseesseessaessaeesaeeseesseesseesseessenensensas 4 2 REACTION ET AT 4 3 DETECTION FORMAT ee 4 4 REAL TIME PCR INSTRUMENT SETTINGS ccccccccccsccscsssesssvseveeseceeseusevsevaeseeseeseuseusevaevaesaeseusevsevaevansaesauseneanss 5 BD TINY CT VE 6 FREQUENTLY ASKED QUESTIONS FAQ amp TROUBLESHOOTING cssssstcssssteesssessssssessssteneneaes 6 KEY TO SYMBOLS USED EE 7 TECHNICAL ASSISTANCE E
5. cessary to generate a CC object for every LC 480 instrument A new object has to be created after the optical system has been checked 2 Reaction setup Prepare five reaction mixes one for each calibrator dye and one for the blank The reaction mixes are prepared in order to have enough volume to load 5 replicates of 25 uL for each reaction mixes on the 96 well plate see Table 1 and Figure 1 Prepare the following five reaction mixes according to Table 1 1 Mix Blank add the CPE PCR Mastermix and adjust final volume with MilliQ water 2 Mix FAM prepare the reaction mix with the FAM Calibrator dye solution green cap the FAM Positive Control green inlay and the CPE PCR Mastermix 3 Mix VIC prepare the reaction mix with the VIC Calibrator dye solution yellow cap the VIC Positive Control yellow inlay and the CPE PCR Mastermix 4 Mix Red prepare the reaction mix with the Red Calibrator dye solution red cap the Red Positive Control red inlay and the CPE PCR Mastermix 5 Mix Cy5 prepare the reaction mix with the Cy5 Calibrator dye solution blue cap the Cy5 Positive Control blue inlay and the CPE PCR Mastermix 3 Detection Format A new Detection Format needs to be generated when using the 4 Color Compensation Set for the first time If the format has already been generated continue to step 4 e Goto open tools in the LC 480 Software e Select Detection Formats and click on
6. cycler is carried out in the post PCR room Never transfer items from the post PCR room to the pre PCR room 4 Color Compensation Set User manual Version 1 2 Issued 29 11 2013 4 Color Compensation Set for Check Direct CPE To keep laboratory free of PCR product contamination Use pipettes with hydrophobic filter tips Make sure to always use a new pipette tip when adding solutions test samples and controls to wells of a 96 well plate Follow proper pipette dispensing techniques to prevent aerosols Wear clean disposable gloves and clean lab coats for the different steps of the test Change gloves whenever you suspect that they are contaminated Keep the tubes of all kit components and samples closed as much as possible Clean the lab benches and all equipment regularly with a 0 5 sodium hypochlorite solution Users are strongly advised to read the full protocol before starting the test Performing the color compensation 1 Important points before starting e Follow the manual instructions to create the color compensation object For further information refer to the LC 480 Instruments Operator s Manual Software version 1 5 section Advanced Software Functionalities Color Compensation Analysis e On the LC 480 System I amp II the color compensation reactions can be run in parallel to experimental samples e g Check Direct CPE real time PCR assay e Color compensation CC is instrument specific thus it is ne
7. e active and the Integration Time Mode is set to Dynamic e In Experiment Setup enter Reaction Volume 25 e In Experiment Programs create the color compensation PCR program as presented in Table 2 4 Color Compensation Set User manual Version 1 2 Issued 29 11 2013 s A Color Compensation Set Table 2 Color compensation program setup Acquisition Hold Mode hh mm ss 00 02 00 00 10 00 Acquisition Hold Mode hh mm ss Acquisition Mode hh mm ss for Check Direct CPE Analysis Mode none Acquisition per C Acquisition per C None Im 220 ojojo Data analysis Important points before starting For a detailed description of how to operate your real time PCR instrument and how to analyze data please refer to the real time PCR instrument s instruction manual 1 In Sample Editor select work flow Color Comp 2 Define the properties of the samples Enter the Dominant Channel corresponding to the sample calibrator loaded on the plate for the color compensation As described in Figure 4 for samples in position A1 to E1 select Dominant Channel Water for samples in position A3 to E3 select Dominant Channel FAM for samples in position A5 to E5 select Dominant Channel VIC for samples in position A7 to E7 select Dominant Channel Red for samples in position A9 to E9 select Dominant Channel Cy5 Select Analysis Select Color Compensation f
8. ndicated temperature until expiration date Materials required but not supplied with the kit PCR grade water e g Milli Q Disposable laboratory powder free gloves Lab coat Pipettes amp disposable sterile filter tips for volumes of 1 to 1000 ul 1 5 ml tubes Eppendorf tubes LightCycler 480 multiwell plate 96 Product no 04729692001 PCR plate seal LightCycler 480 Real time PCR system I II Roche CH Vortex mixer Plate centrifuge Mini centrifuge Good laboratory practices The quality of the results depends on strict compliance with the following good laboratory practices especially The test must be performed by adequately trained personnel Do not use reagents after their expiration date Before use thaw frozen reagents completely at room temperature and vortex briefly to obtain a homogeneous solution After vortexing briefly spin down the solution to avoid contamination when opening the lid Follow recommendations for storage handling and freeze thaw cycles to preserve the quality of the kit s reagents Protect reagents from light to avoid photo bleaching of the dyes Periodically verify the accuracy and precision of pipettes as well as correct functioning of the instruments Prevention of contaminations Use separate rooms a pre PCR room and a post PCR room The preparation of the amplification reactions are carried out in the pre PCR room Incubation in the real time PCR thermo
9. o Diagnostic Use RED Red calibrator Catalog number C VIC calibrator LOT Batch code ONTROL CY5 Cy5 control IFU number ONTROL FAM FAM control Y5 Cy5 calibrator ONTROL RED Red control FAM calibrator CONTROL VIC VIC control PCR Mastermix CPE CPE PCR Mastermix Temperature limitation Use before YYYY MM Contains sufficient for lt n gt tests mal Manufacturer Technical assistance support check points com 31 317 453 908 Despite the utmost care in the development and preparation of the protocol Check Points cannot take any responsibility for errors omissions and or future changes herein Notice to Purchaser This product is sold under license from PHRI Properties and may be used under PHRI Properties patent rights only for human in vitro diagnostics food testing veterinary testing or research Trademarks Cy5 is a registered trademark of Amersham Biosciences Ltd FAM and VIC are registered trademarks of Applera Corporation or its subsidiaries in the US and or certain other countries LightCycler and LC 480 are registered trademarks of Roche Diagnostics GmbH Check Points Health BV Tel 31 317 453 908 ES Binnenhaven 5 Fax 31 317 210 147 6709 PD Wageningen info check points com Chec k Points The Netherlands www check points com rapid molecular detection 4 Color Compensation Set User manual Version 1 2 Issued 29 11 2013
10. rom the Analysis menu Select the subset of samples corresponding to the color compensation experiment Select Calculate Select Save CC Object The stored Color Compensation Object should be used for the analysis of runs conducted with the following product Check Direct CPE Real time PCR kit Ref 18 0080 a ZE SE E Step 1 Select Workfl p Select Filter Combination Abs Quan HR 465 510 IV 533 580 IY 533 610 IV 618 660 Units 4 i4 e Del R io Ls Rep Of Sample Name Dominant Ch Abs Quant Rel Quant Scanning Color Comp Tm Melt Geno Endpt Geno z H annel VIM NDM vim NDM vIM NDM VIM NDM VIM NDM Blank Blank Blank Blank Blank Water FAM E vic RED EI Cen Figure 4 Screen shot of LC 480 Software version 1 5 Sample editor selection for the A color compensation sample set with LC 480 4 Color Compensation Set User manual Version 1 2 Issued 29 11 2013 Frequently asked questions FAQ amp Troubleshooting The real time PCR gives an error message Refer to the real time PCR instrument user manual or contact the local technical support of the real time PCR instrument company left Solutions out of the 20 C 4 F storage These reagents must be stored at 20 C 4 F for proper performance of the test The performance of the product cannot be fully guaranteed if these solutions were left out of 20 C 4 F for more than 24 hours Real time res
11. ults show no detection or very low fluorescent signals detection for all calibrators and in all detector channels Possible causes and troubleshooting e The Calibrator dye solutions containing the fluorescent probes and primers are degraded Please check expiration date the number of thaw freezing cycles that CPE solution tube have undergone and if the kit was stored correctly e The real time PCR system may be responsible for these results Please refer to instrument User s manual or contact your real time PCR instrument local representative Check Direct CPE results show cross talk emission signals in one or more detector channels e The Calibrator dye solutions containing the fluorescent probes and primers is degraded Please check expiration date the number of thaw freezing cycles that CPE solution tube have undergone and if the kit was stored correctly e The color compensation run was not performed well repeat the assay e The CPE PCR Mastermix did not perform well Check expiration date and if the solution was stored correctly Data Analysis and Interpretation If you encounter difficulties with the data analysis and interpretation please refer to LC 480 Instruments Operator s Manual Software version 1 5 section Advanced Software Functionalities Chapter 7 Color Compensation Analysis pp 248 256 Alternatively contact Check Points Technical Support at support check points com Key to symbols used IVD For In Vitr
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