InviMag Virus DNA Mini Kit/ IG User manual
Contents
1. Please select the desired assay and recheck the allocated samples that should be processed in this run It is possible to switch between the offered assays by using the two arrow buttons A By default all loaded samples are selected to be processed in this run If samples have to be excluded from the batch exclude them by selecting the corresponding sample and clicking on the Remove from batch button B 23 InviMag Virus DNA Mini Kit IG 0515 Batch checking This screen shows a summary of all checked disposables samples and reagents in one informational screen Please make sure that all required components are loaded correctly In case of any error the problem will be highlighted in by a red font If no errors during the loading steps occurred proceed by pressing the button Batch processing To solve any error click on the red highlighted field and follow the instructions printed on the instrument screen Batch checking Selected assay DVIR E100 S200 i x uL ee NS Batch definition Batch processing Status Ready User service 1 1 1 1 1 1 1 1 1 1 1 21 E pa t Figure 12 Batch checking screen Batch processing After closing the system door the assay can be started by pressing the Start Button A The door will be locked during the run and the system will start with sample processing The door will only be unlocked after a run has been successfully finished or if an error oc2. System 16 Preparing and loading of the InviGenius System 17 Preparing the reagents 17 Preparing the system 17 Sample Loading 18 Reagent Loading 19 Assay Selection 20 Disposable Tip Loading 20 Disposable Sheaths Loading 21 Plate Loading 22 Waste management 23 Batch definition 23 Batch checking 24 Batch processing 24 After the run 24 UV sterilization of the InviGenius 25 Troubleshooting 27 Ordering information 28 2 InviMag Virus DNA Mini Kit IG 0515 Kit contents of InviMag Virus DNA Mini Kit IG Store the MAP Solution B at 4 C Store lyophilized Proteinase K Carrier RNA PKC Tube at 2 8 C Store dissolved Proteinase K Carrier RNA at 20 C Store all other kit components at room temperature RT Catalogue No Lysis Buffer HL PKC IG RNAse free Water Binding Buffer HL MAP Solution B IG Wash Buffer I Wash Buffer II Elution Buffer M Incubation Plate A Working Plate A Elution Plate E Microtube Cap Sheath Box Sealing Foils Incubator Stripe Foils Initial steps 8 x 12 extractions reagents sufficient for 2442120100 30 ml 8 runs for 8 x 800 ul working solution 1 vial per run 15 ml UM 4 runs final volume 32 ml 2 x 2 6 ml 1 vial per 4 run S 8 runs final volume 120 ml l ON 4 runs final volume 2 x 120 ml 30 ml 8 runs 1 8 runs 4 2 runs 1 8 runs 8 1 2 racks 48 sheaths 4 runs per plate 48 pieces 4 2 Add 24 ml of 99 7 Isopropanol to the Binding Buffer HL Mix by inten
3. Wash Buffer R1 o warning H302 312 332 412 EUHOS32 P273 H302 Harmful if swallowed H315 Causes skin irritation H319 Causes serious eye irritation H315 Causes skin irritation H334 May cause allergy or asthma symptoms or breathing difficulties if inhaled H335 May cause respiratory irritation H312 Harmful in contact with skin H332 Harmful if inhaled H412 Harmful to aquatic life with long lasting effects EUHO32 Contact with acids liberates very toxic gas P280 Wear protective gloves protective clothing eye protection face protection P3054 P351 P338 IF IN EYES Rinse cautiously with water for several minutes Remove contact lenses if present Continue rinsing P310 Immediately call a POISON CENTER or doctor physician P405 Store locked up P273 Avoid release to the environment Emergency medical information can be obtained 24 hours a day from infotrac outside of USA 1 352 323 3500 inside of USA 1 800 535 5053 6 InviMag Virus DNA Mini Kit IG 0515 Product characteristics of the InviMag Virus DNA Mini Kit IG The InviMag Virus DNA Mini Kit IG is the ideal tool for an efficient and fully automated viral DNA extraction and purification of fresh or frozen samples using magnetic beads in combination with the InviGenius system Time for preparation Starting material 200 ul serum or plasma 200 ul cell free body fluids 200 ul rinsed liquid from swab depe
4. the InviGenius instrument is performed by use of the touch LCD J located at the top front right side 16 InviMag Virus DNA Mini Kit IG 0515 Preparing and loading of the InviGenius System Preparing the reagents Before you start dissolve one vial of Proteinase K and Carrier RNA with 800 ul DNAse free water respectively Preparing the system Turn on the InviGenius system using the power switch located on the right back side of the instrument The InviGenius software can be started by double clicking the InviGenius icon located on the desktop Keep the door of the InviGenius system closed during initialization After initialization of the InviGenius system a login screen appears Figure 1 Log in with the provided user name and password Login User identifier Password gc o Status Ready Ett ser Figure 1 Login screen of the InviGenius software After login the main screen of the InviGenius software is shown Figure 2 Select Loading to start with loading of the system Select Processing to define and run an assay if the system has already been loaded Main Menu Data Loadin g management Processing Maintenance Shutdown Administration InviGenius Logout Status Ready mm ee User developer Figure 2 Main menu of the InviGenius software 17 InviMag Virus DNA Mini Kit IG 0515 Sample Loading After selecting Loading the sample loading screen app
5. three tip rack positions on the InviGenius system Fig 10 A1 A3 corresponding to Fig 3 D1 D3 Remaining tip numbers are shown in B Tip numbers can be changed by pressing the number field directly Empty tip racks can be unloaded and reloaded by 1 Pressing the Loading Position directly The software will focus this loading position on the main screen 2 Pressing the Unload Button C 3 The loading position can be refilled with a new tip rack by pressing on the corresponding tip rack on D 20 InviMag Virus DNA Mini Kit IG 0515 Each position can be filled either with 50 ul or 1100 ul filter or non filter tips However for the Virus DNA assay only 1100 ul filtered tips will be used Attention It is very important to allocate the type of tips correctly in the software that have been loaded into the instrument In case of false tip allocation overfilling of the tip will irreparably destroy the pipettor head All protocols should be used in combination with filter tips to ensure efficient prevention of sample or reagent cross contaminations STRATEC Molecular will give no guarantee or responsibility if contaminations occur due to the use of non filtered tips Note Disposable tips are not supplied within the kit We recommend the use of validated conductive tips which can be ordered at STRATEC Molecular STRATEC Molecular offers 50 ul conductive tips 10x 96 pieces order no 5011120100 and 1100 ul conductive tips 10x
6. 0 ul filter tips order no 501110040 o Disposable gloves o Distilled H2O o Vortexer O O The InviMag Virus DNA Mini Kit IG is validated with 2 Propanol Rotipuran gt 99 7 p a ACS ISO Order no 6752 from Carl Roth Possible suppliers for Isopropanol Carl Roth Applichem Sigma 2 Propanol 2 Propanol f r die Molekularbiologie 2 Propanol Rotipuran gt 99 7 p a ACS ISO Order no A3928 Order no 59304 1L F Order no 6752 Possible primary tubes manufacturer cat no o BD Vacutainer 6 ml K2E BD 367864 o PS Tube Sarstedt 5 ml 55 476 2445 InviMag Virus DNA Mini Kit IG 0515 Important indications 1 Minimum volume of samples in primary tubes The procedure of the InviMag Virus DNA Mini Kit IG is optimized for the isolation of viral DNA from up to 200 ul cell free body fluids serum plasma CSF and rinsed liquids from swabs It is advised to provide at least 550 ul sample per tube to prevent pipetting distribution errors during sample processing 2 Sample volume smaller than 200 ul For samples of a smaller volume than 200 ul please adjust the volume to 350 ul using PBS or distilled RNase DNase free water 3 Elution volume The final elution volume is 100 ul Prevention of cross contamination To comply with the demanding guidelines of in vitro diagnostics the pipettor of the InviGenius is routed in such a way that possible contamination risks are minimized However we recommend applying the
7. 4 30 Unknown Unknown Unknown Unknown PC108025130300431504 Lysis B HL PC 2015 04 30 Status Ready i User service Figure 5 Reagent loading screen of the InviGenius software Insert all provided reagents into the provided reagent rack of the InviGenius system Take care that the bar code labels face to the right side of the loading bay and decap the bottles and tubes The order of the inserted reagents is not crucial because the type and position of a reagent is identified by the unique bar code However the possible loading positions are limited by the size of the used bottles After rack insertion the loading status of the reagents will be shown In case of unsuccessful reagent allocation remove the rack check the bar code orientation and repeat the procedure slowly 19 InviMag Virus DNA Mini Kit IG 0515 Assay Selection Select the Virus DNA assay DVIR E100 S200 and proceed with disposable tip loading Assay selection Assay description DVIR E100 S200 suitable to loaded No Assay Selected reagents Version 1 Reagent loading Disp tip loading Status Ready i ser service Figure 6 Assay selection screen of the InviGenius software Disposable Tip Loading Disposable tips loading Load Pos 1 Tip Load Position 1 HIHHM HE Tip 1100 ul filter Assay selection Disp sheaths loading Status Ready User developer Figure 7 Disposable tip loading screen There are
8. 96 pieces order no 5011120200 Be sure that conductive tips are used otherwise the tip detection unit installed in the pipetting unit will reject the tips and no run will be possible Disposable Sheaths Loading The sheaths are used as protection devices for the magnetic rods Disposable sheaths loading Sheath Load Position amp eee 4 amp amp amp amp 9 9 e 9 9 9 9 S anes Sheath Type uus Microplates Status Ready THV vTU3Z tI mm User developer Figure 8 Disposable sheaths loading screen The loading procedure of the disposable sheaths works analogous to the disposable tip loading screen For a run always 12 disposable sheaths one row in the sheaths rack are used regardless of the processed sample numbers This is done to assure that the rods are always protected against contaminations In general the number of sheaths supplied within the kit is sufficient for the amount of runs printed on the kit package If you are lacking sheaths they can be ordered separately at STRATEC Molecular 100 pieces bulk order no 5011120300 or 10 x 48 pieces order no 5011120400 Comparable to the disposable tips loading it is possible to define the number of rows left in the tip rack by pressing on the displayed number area Make sure that the disposable sheaths are loaded and displayed consistent to the manually loaded sheaths in the rack to ensure correct sheaths pick up Don t remove sing
9. Elution Buffer M Due to the high purity the eluted viral DNA is ready to use in a broad panel of downstream applications such as o real time PCR quantitative RT PCR like TaqMan und LightCycler technologies o or array technologies For the isolation of DNA from a single serum plasma or blood sample STRATEC Molecular offers the Invisorb Spin Virus DNA Mini Kit or for 8 96 samples the Invisorb Virus DNA HTS 96 Kits for use on a centrifuge vacuum manifold or other robotic workstations For further information please contact phone 49 0 30 9489 2901 2910 in Germany and from foreign countries phone 49 0 30 9489 2903 2907 or ask your local distributor The PCR process is covered by US Patents 4 683 195 and 4 683 202 and foreign equivalents owned by Hoffmann La Roche AG InviMag Virus DNA Mini Kit IG 0515 Example data To show a typical result from an extraction process viral DNA derived from Hepatitis B 200 ul starting material and water samples negative control were isolated in a cross contamination study and analyzed by real time qPCR on a Corbett Rotorgene cycler Quantitation data for Cycling A FAM No Colour Name Ct Rep Ct Rep Ct Std Dev i f for franker mm a let tme mm rmm tmm I 1 mm II wed mem me l me l m re sn bs meh Fig 1 RT qPCR from HBV samples For the RT qPCR 2 ul template was used The temperature prof
10. aboratory use only They must be stored in the laboratory and must not be used for other purposes than intended The product with its contents is not suitable for consumption ss InviMag Virus DNA Mini Kit IG 0515 Safety information When and while working with chemicals always wear a suitable lab coat disposable gloves and protective goggles Avoid skin contact Adhere to the legal requirements for working with biological material For more information please consult the appropriate material safety data sheets MSDS These are available online in convenient and compact PDF format at www stratec com for each STRATEC Molecular product and its components If buffer bottles are damaged or leaking WEAR GLOVES AND PROTECTIVE GOGGLES when discarding the bottles in order to avoid any injuries STRATEC Molecular has not tested the waste generated by the InviMag Virus DNA Mini Kit IG procedures for residual infectious materials Contamination of the waste with residual infectious materials is highly unlikely but cannot be excluded completely Therefore all waste has to be considered infectious and should be handled and discarded accordingly to local safety regulations European Community risk and safety phrases for the components of the InviMag Virus DNA Mini Kit IG to which they apply are listed below as follows Lysis Buffer HL Proteinase K warning danger H302 315 319 P280 305 351 338 H315 319 334 335 P280 305 351 338 310 405
11. ample rack can be processed due to the limited number of wells per row of the plastics For correct identification of the sample tubes the unique bar codes must face to the bar code scanner located at the right side of the loading bay After inserting the sample rack in the very left lane of the loading bay an updated screen will show the identifiers read from the sample bar codes Figure 7 In case of unsuccessful 18 InviMag Virus DNA Mini Kit IG 0515 sample identification remove the rack check the bar code orientation and reinsert the rack slowly It is also possible to rename the samples by selecting the corresponding sample by using the arrow fields followed by the Edit button After a certain time about 5 min the bar code scanner is inactivated In that case the user has to restart the scanner with the START SCANNER button if the loading procedure is not finished After successful loading of the samples proceed with reagent loading by selecting Reagent loading on the bottom right hand side of this screen Reagent Loading The reagent loading process is analogous to the sample loading procedure Reagents loading Barcode Exp date PC410025130900401504 Elution B M PC 2015 04 30 PC211025130100401504 Binding B HL PC 2015 04 30 PC618025302502351504 MAP Sol B PC 2015 04 30 PC306108131800831504 Wash B II PC 2015 04 30 PC707700230228151504 PKC PC 2015 04 30 PC308108131200451504 wash B I PC 2015 0
12. ation procedure to monitor the efficiency of sample preparation Internal control DNA or RNA IC must be combined with Carrier RNA stock solution or with Carrier RNA Proteinase K stock solution in one mixture For each sample the machine transfers a volume of 40 ul of the stock PKC solution to the lysis mix The PKC vials contain 800 ul stock solution so the internal control for 20 samples must be added directly to the PKC tube Example Calculation Per Extraction 4 5 ul of the DNA control would be needed 4 5 ul RXN X 20 RXN 90 ul PKC stock solution has to be made by adding 800 ul 90 ul 710 ul RNAse free water Then add 90 ul control DNA followed by a mixing step Notes If you only have indication of amount per reaction please calculate by using eluate and template volume If the internal control IC is stable in plasma serum CSF urine respiratory samples whole blood stool transport media or on dried swabs e g armored RNA it can alternatively be added to the sample shortly before beginning sample preparation But consider that a bigger amount of internal control is necessary when using bigger volumes of primary sample tubes The minimal required sample volume in the sample tube has to be at least 550 ul If the internal control IC is naked unprotected DNA or RNA and it is unstable in plasma serum CSF urine respiratory samples whole blood stool transport media or on dried swabs the inte
13. curs that requires user interaction Do not try to force open the door during a run This will cause an abort of the run Batch processing Batch identifier 0911191052479620STRATEC BB02E6 User interaction Assay description Basic 01 01 00003 request Actual step E um Ae eee d pe e 00 00 Remaining time 00 Process state Idle Events A Start process b ul Status Ready mmm User developer Figure 13 Batch processing screen At the end of the process the viral DNA containing eluate is located in the appropriate eluate position and can be used for further applications Note The complete process will take approximately 60 minutes 24 InviMag Virus DNA Mini Kit IG 0515 After a run After a run is completed and no additional run shall be started unload all plates and reagents and store them according to GLP guidelines Please keep in mind that the plates could contain infectious material As with all medical clinical and diagnostically equipment all waste liquids tips sheaths and plates should be treated as potentially dangerous bio hazard waste Daily maintenance UV decontamination The InviGenius system is equipped with an internal UV lamp 254 nm wavelength that should be used daily either at the end of the working day or in the morning before a run is started The suggested decontamination time is about 20 min To start the UV decontamination go to the main menu of the InviGeniu
14. device is now decontaminated and can be either switched off or used for sample processing We recommend decontaminating the instrument daily 26 InviMag Virus DNA Mini Kit IG 0515 Troubleshooting Problem pipetting distribution errors low concentration of extracted DNA degraded or sheared DNA no assay selectable eluted DNA is brownish colored Probable cause pipetting of PKC failed samples transfer failed incomplete reagent buffer transfer failed incomplete blood components settled no too much ethanol added to Wash Buffers incorrect storage of starting material old material combination of reagents from different kits missing required reagent Residual magnetic particles are left in eluate Comments and suggestions ensure that the lyophilized PKC is lyophilized with the appropriate volume of water before usage the sample tube must contain at least 400 ul sample ensure that the supplied Wash Buffers Binding Solution is filled up properly with either ethanol or isopropanol do not reuse bottles more often than described in Tab 1 because they will be rejected by the system in case of large sample volumes gt gt 1 ml carefully premix the sample tube before inserting it into the sample rack ensure that the Wash Buffers have been filled up properly with ethanol as indicated in Tab 1 ensure that the storage of starting material is correct avoid multiple freezing and tha
15. e incubator position seven free lanes in the working position and one free lane in the eluate position Please make sure that the depicted lanes on the monitor are consistent with the real lanes in the corresponding positions To avoid contaminations we strongly recommend to not wash reuse disposed plates 22 InviMag Virus DNA Mini Kit IG 0515 Waste management Please make sure that the waste tray s capacity is sufficient for your planned assay If not empty the solid waste Waste management Waste capacity 336 Dropshaft status In place Fill level 0 disposables 0 Number of wasted R disposable sheaths 0 Number of wasted disposable tips Batch definition Status Ready mm ser developer Microplates Figure 10 Waste management screen If waste tray has been emptied please click on the Empty solid waste button A afterwards Batch definition Please select the appropriate assay and check the samples you want to process in this run Batch definition Assay descriptions IA suitable to loaded reagents DVIR_E100_5200 samples SAMPLE 1 SAMPLE 2 SAMPLE 3 SAMPLE 4 SAMPLE 5 SAMPLE 6 SAMPLE 7 SAMPLE 8 SAMPLE 9 SAMPLE 10 SAMPLE 11 SAMPLE 12 Batch checking User service Remove from batch DA Version 1 Aliquots Volume Lae Waste management Status Ready Figure 11 Batch definition screen
16. ears Samples Disposable tips Loading Disposable Reagents Assay Microplates Waste management Status Ready mmm User developer Back Figure 3 Loading screen of the InviGenius software Select Samples to proceed with the sample loading screen Samples loading SAMPLE 1 edited SAMPLE 2 edited SAMPLE 3 edited SAMPLE 4 edited SAMPLE 5 edited SAMPLE 6 edited SAMPLE 7 edited SAMPLE 8 edited SAMPLE 9 edited SAMPLE 10 edited SAMPLE 11 edited SAMPLE 12 edited SSSSOCOOO2e0eeeooo Reagent loading Status Ready ma User Service Figure 4 Sample loading screen of the InviGenius software Please add the samples to the rack Please decap the tubes before transfer to the loading rack If available the primary tubes should be used directly as sample tubes If the samples are not provided in primary tubes please prepare the sample rack with primary tubes that are prefilled with samples from which the viral DNA shall be extracted Sample tubes are not provided with the kit and can be ordered at e g Sarstedt order no 55 476 5 ml tubes 75x12 mm PS or see recommendation at page 10 chapter reagents and equipment to be supplied by user For each reaction a sample volume of 200 ul is processed However it is recommended that the total sample volume filled in the sample tubes should be at least 400 ul to ensure stable processing Please take care that only the first 12 positions of the s
17. ic beads free eluate which is ready to use in different downstream applications like real time PCR quantitative RT PCR like TagMan und LightCycler technologies or array technologies The PCR process is covered by US Patents 4 683 195 and 4 683 202 and foreign equivalents owned by Hoffmann La Roche AG 9 InviMag Virus DNA Mini Kit IG 0515 Yield and quality of viral DNA The amount of purified RNA in the InviMag Virus DNA Mini Kit IG procedure depends on the sample type the virus content sample source transport storage and age Yield and quality of isolated viral DNA is suitable for any molecular diagnostic detection system The diagnostic tests should be performed accordingly to the manufacturers specifications Note f beads are visible in the eluate plate transfer the eluate to a new reaction tube and centrifuge for 1 min at maximum speed e g 13 000 rom Using the Elution Plate E directly for centrifugation is also possible Different amplification systems can vary in efficiency depending on the total amount of nucleic acids present in the reaction Eluates from this kit contain both viral DNA and Carrier RNA In general the amounts of Carrier RNA will greatly exceed the amounts of isolated viral nucleic acids Yields of viral DNA isolated from biological samples are normally less than 1 ug and difficult to determine photometrically due to the very low concentrations Keep in mind that the Carrier RNA 5 ug
18. ices But it is not for in vitro diagnostic use in countries where the EU Directive 98 79 EC on in vitro medical devices is not recognized Product use limitation The kit is validated for viral DNA extraction from cell free body fluids and rinsed liquids specifically for human serum and plasma Related applications will need a separate validation Extraction of eukaryotic DNA from samples has not been evaluated with this kit The included chemicals are only useable once Differing of starting material may lead to inoperability Therefore neither a warranty nor guarantee in this case will be given implied or expressed The user is responsible to validate the performance of the STRATEC Molecular product for any particular use STRATEC Molecular does not provide validations of performance characteristics of the product with respect to specific applications STRATEC Molecular products may be used e g in clinical diagnostic laboratory systems conditioned upon the complete diagnostic system of the laboratory the laboratory has been validated pursuant to CLIA 88 regulations in the U S or equivalents in other countries All products sold by STRATEC Molecular are subject to extensive quality control procedures according to ISO 9001 2000 and ISO EN 13485 and are warranted to perform as described herein Any problems incidents or defects shall be reported to STRATEC Molecular immediately upon detection thereof The chemicals and the plastics are for l
19. ile was 95 C for 10s 60 C for 30s using 45 cycles Figure 1 demonstrates the good conformity and reproducibility of the samples green and the PTC positive control red No amplification products for the water samples blue and the NTC negative control black were detectable demonstrating that no cross contamination occurs during the extraction process Form Flor i i x LE 7 TUR m ERAN PEON XS 5 10 15 ar 25 al J i 45 8 InviMag Virus DNA Mini Kit IG 0515 Sampling and storage of starting material Best results are obtained using freshly extracted samples As long as the samples are not shock frosted with liquid nitrogen or incubated with DNase inhibitors or denaturing reagents the present viral DNA is not stable DNA contained in such deep frozen samples is stable for months However the DNA purification should be performed as soon as possible Serum and plasma After collection serum and plasma derived from blood treated with anticoagulants like EDTA or citrate but not with heparin synovial fluid samples or other cell free body fluids swabs as well as stool samples can be stored at 2 8 C for short time up to 24 h For long term storage we recommend freezing samples in aliquots at 20 C Frozen plasma or serum samples should not be thawed more than once Multiple thawing and freezing cycles before isolating the viral DNA should be avoided This may lead to denaturation and precipitati
20. iologie 2 Propanol Rotipuran gt 99 7 p a ACS ISO Order no A3928 Order no 59304 1L F Order no 6752 28 InviMag Virus DNA Mini Kit IG 0515 Stratecee molecular STRATEC Molecular GmbH Robert Rossle Str 10 13125 Berlin Germany Phone 49 30 94 89 29 01 Fax 49 30 94 89 29 09 E mail info berlin stratec com www stratec com 1G5j02 IG 05 2015
21. ions of the kit reagents To minimize the risk of infections from potentially infectious material we recommend working under laminar air flow o his kit should only be used by trained personnel O O ee 10 InviMag Virus DNA Mini Kit IG 0515 Preparing reagents and buffers Before starting a run equilibrate all reagents at room temperature Where necessary gently mix and redissolve any precipitates by incubation at 30 C Swirl gently to avoid foaming Lysis Buffer HL MAP Solution B and Elution Buffer M are ready to use 8 x 12 viral DNA extractions Add 24 ml of 99 7 isopropanol to the Binding Buffer HL Mix by intensive shaking by inverting for 1 min Resuspend the lyophillized PKC tube by addition of 800 ul RNase free water Mix thoroughly Store dissolved and unused PKC tubes at 20 C Add 60 ml of 96 100 ethanol to the bottle Wash Buffer I Mix thoroughly and always keep the unused bottle s firmly closed Add 84 ml of 96 100 ethanol to each bottle Wash Buffer Il Mix thoroughly and always keep the unused bottle s firmly closed Note Dissolved PKC must be stored at 20 C Avoid multiple freezing and thawing cycles because this may lead to degraded Carrier RNA and reduced kit performance We recommend to prepare only as much tubes reagent as needed for one assay run Reagents and equipment to be supplied by user 96 100 ethanol lsopropanol o Measuring cylinder 250 ml o Conductive pipette tips 1 10
22. le disposable sheaths within a row of the sheaths rack if less than 12 samples are processed within one run because there is a sheaths detection sensor installed in the device If less than 12 sheaths picked up by the instrument a warning will be displayed and all picked up sheath will be discarded into the waste before a next row of sheaths will be picked up for testing To avoid contaminations we strongly recommend to not wash reuse any disposed sheaths 21 InviMag Virus DNA Mini Kit IG 0515 Plate Loading Analogous to the previous loading screens the incubation working and elution plates are loaded within the plate loading screen Figure 9 Eluate Position Free lanes 8 Oo06000000000 Free lanes 8 Plate A dere dnd e dr oe eae a B KA SCA V eb ALE A gt Unload Free lanes amp _Eluate Pos white Disp sheaths Waste loading management Status Ready a User service Figure 9 Plate loading screen In general the Incubation Plate A and Working Plate A identical are used at the incubator and working position whereas at the eluate position the Elution Plate E is used Used plates can be unloaded and reloaded by 1 Pressing the plate position directly A The software will focus at the plate position on the main screen 2 Pressing the Unload button B 3 The plate can be reloaded by pressing on the offered plate in C For a successful run the InviGenius needs one free lane in th
23. nds on the sample 60 min for 12 Source and storage samples The DNA isolation process is based on the interaction of nucleic acids with silica coated magnetic particles at adapted buffer conditions The InviGenius instrument will automatically perform all steps of sample and reagent distribution The DNA purification procedure is performed without any user intervention except the initial loading of the system thus allowing safe handling of potentially infectious samples Sample cross contamination and reagent cross over is effectively eliminated by the automated purification process The use of unique bar codes for samples and reagents avoids unwanted transpositions The InviGenius instrument uses magnetic rods to transport the DNA binding magnetic particles through the various extraction phases lysis binding washing and elution The volume of buffers and other liquids necessary for DNA isolation is reduced to a minimum Eliminating the direct liquid handling and increasing the automation level results in a fast reliable and robust technique After a sample specific lysis using Lysis Buffer HL and PKC optimal binding conditions are adjusted by addition of Binding Buffer HL The viral DNA binds to the simultaneously added magnetic particles and is separated from the solution by the magnetic rods controlled by the InviGenius system Subsequent to three washing steps of the particle bound nucleic acids the DNA is finally eluted in
24. on of proteins resulting in reduced viral titers and yields In addition cryoprecipitates formed during freezing and thawing cycles can cause additional problems If cryoprecipitates are visible centrifuge them at app 6 800 x g for 3 min The cleared supernatant should be aspirated carefully without disturbing the pellet and processed immediately This step will not reduce the viral titer Swabs The protocol works with fresh prepared swabs as well as with dried swabs The protocol has not been validated for isolation of viral DNA from swabs which are stored in special storage buffers of other providers STRATEC Molecular will not take responsibility if other sample types than described above are used or if the sample preparation advices are modified Principle and procedure Lysis Samples are lysed at denaturing conditions in a 2 0 ml Deep Well Plate at elevated temperatures in the presence of 200 ul Lysis Buffer HL and 40 ul PKC Binding of the DNA After addition of 250 ul Binding Buffer HL and 40 ul MAP Solution B to the lysate the DNA is bound to the surface of the magnetic beads Removing residual contaminants Contaminants are efficiently removed using 600 ul Wash Buffer I and twice 650 ul Wash Buffer Il while the nucleic acids remain bound to the magnetic beads Elution The viral DNA is eluted in 100 ul Elution Buffer M A defined aliquot of 50 ul is finally transferred to the Elution Plate The bottom magnets guarantee a magnet
25. ould any product fail to perform the applications as described in the manual STRATEC Molecular will check the lot and if STRATEC Molecular investigates a problem in the lot the product will be replaced free of charge STRATEC Molecular reserves the right to change alter or modify any product to enhance its performance and design at any time In accordance with STRATEC Molecular s ISO 9001 2000 and ISO EN 13485 certified Quality Management System the performance of all components of the InviMag Virus DNA Mini Kit IG have been tested separately against predetermined specifications routinely on lot to lot to ensure consistent product quality If you have any questions or problems regarding any aspects of InviMag Virus DNA Mini Kit IG or other STRATEC Molecular products please do not hesitate to contact us A copy of SIRATEC Molecular s terms and conditions can be obtained upon request or are presented at the STRATEC Molecular webpage For technical support or further information please contact from Germany 49 0 30 9489 2901 2910 from abroad 49 0 30 9489 2907 or contact your local distributor 4 InviMag Virus DNA Mini Kit IG 0515 Intended use The InviMag Virus DNA Mini Kit IG is designed for fully automated extraction and purification of viral DNA from up to 200 ul serum or plasma based samples Up to 12 samples can be processed using a patented magnetic beads system and the InviGenius robotic platform It is advi
26. per 200 ul sample will account for most of the RNA present in the samples The kit is suitable for downstream analysis with nucleic acid techniques like qPCR RT qPCR LAMP LCR Diagnostic assays should be performed accordingly to the manufacturer s instructions Quantitative RT PCR is recommended for determination of viral DNA yield In Gel Electrophoresis and in Capillary Electrophoresis RNA extracted with the provided kit looks like degraded because the kit contains Carrier RNA The Carrier RNA consists of poly A RNA fragments in size of 100 1000 base pairs The kit is not dedicated for applications using this kind of analysis Important notes Important points before starting a protocol Immediately upon arrival of the product inspect the kit and its components as well as the package for any apparent visible damages and correct quantities If there are any unconformities please notify STRATEC Molecular in writing with immediate effect upon inspection thereof If buffer bottles are damaged contact the STRATEC Molecular Technical Services or your local distributor In case of liquid spillage refer to Safety Information see page 6 Do not use damaged kit components since their use may lead to poor kit performance o When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles Discard contaminated gloves immediately Do not combine components of different kits Avoid microbial contaminat
27. rnal control must not be added directly to the samples Refer to the manufacturer s instructions to determine the optimal amount of internal control IC for specific downstream applications Using an amount other than that recommended may lead to wrong quantification results 15 InviMag Virus DNA Mini Kit IG 0515 General overview of the InviGenius System Figure 1 Frontal view of the InviGenius System There are three plate positions available in the InviGenius system which can be loaded with corresponding plates the incubator k A the working B and the eluate position C Lysis is performed at the incubator position A whereas the washing and elution process is performed at the working position B The eluate containing the extracted nucleic acids will be finally transferred to the eluate position Additionally there are three loading positions available for disposable tip trays D1 D3 and one position E for the disposable sheaths The loading bay F is located at the very right side of the instrument The sample rack is loaded into the far left lane whereas the reagent rack is loaded into the right lanes of the loading bay The Magnetic Separation Head MSH G is located on top of the incubator lid parking position The fully automatic pipettor H is installed above the loading bay parking position The disposable waste tray I is located behind the lower cover of the InviGenius Interaction with
28. s software and select Maintenance Main Menu Data management Maintenance Administration InviGenius Status Ready Oa User developer Loading Processing Shutdown Figure 14 Main screen of the InviGenius software When the sub item Maintenance is opened select UV decontamination Periodical maintenance Maintenance UV q decontamination Diagnostics Cleanup procedures Cleanup drop catcher Status Ready mm User service Figure 15 Maintenance screen of the InviGenius software 25 InviMag Virus DNA Mini Kit IG 0515 In the UV decontamination menu adjust the exposure time A and finally press the Start button B During the decontamination process the instrument door will be locked to prevent any UV radiation release in the lab Warning UV radiation is harmful It causes serious burns of the skin and leads to irreparable damage of the eyes and skin Ensure that no lab personnel is submitted to direct UV light Do not try to force open the instrument door during the decontamination process UV decontamination 00 15 00 I HH MM SS Remaining time 00 00 00 Duration UV decontamination status There was no UV decontamination completed N Status Ready m H User Service Figure 16 UV decontamination screen When the decontamination is finished go back to the main menu by using the Back button The
29. sed to provide at least 550 ul sample per tube dead volume to prevent pipetting distribution errors due to the liquid level detection LLD process The final processed sample volume is 200 ul The nucleic acid isolation protocol is suitable for routinely walk away automated preparation of viral DNA from fresh or frozen sample For reproducible and high yields an appropriate sample storage is essential see Sampling and storage of the starting material page 9 Common collection tubes not provided and anticoagulants EDTA and citrate but not heparin can be used to assemble a set of samples All utilities reagents and plastics except conductive tips required for preparation of viral DNA are provided by the InviMag Virus DNA Mini Kit IG THE PRODUCT IS INTENDED FOR USE BY PROFESSIONALS SUCH AS TECHNICIANS PHYSICIANS AND BIOLOGISTS TRAINED IN MOLECULAR BIOLOGICAL TECHNIQUES It is designed to be used with any downstream application employing enzymatic amplification or other enzymatic modifications of RNA followed by signal detection or amplification Any diagnostic results generated by using the sample preparation procedure in conjunction with any downstream diagnostic assay should be interpreted with regard to other clinical or laboratory findings To minimize irregularities in diagnostic results adequate controls for downstream applications should be used The kit is in compliance with EU Directive 98 79 EC on in vitro medical dev
30. sive shaking by inverting for 1 min Shortly before use mix by inverting several times Add 60 ml of 96 100 ethanol to the bottle Wash Buffer I Add 84 ml of 96 100 ethanol to each bottle Wash Buffer Il Resuspend each PKC tube with 800 ul RNase free water per tube 3 InviMag Virus DNA Mini Kit IG 0515 Symbols MM Manufacturer LOT Lot number Catalogue number Expiry date Temperature limitation Do not reuse Consult operating instructions All buffers and kit contents of the InviMag Virus DNA Mini Kit IG except PKC Proteinase K Carrier RNA mixture and MAP Solution B magnetic beads should be stored at room temperature and are stable for at least 12 months at these conditions PKC Tube Lyophilized PKC Tube should be stored at 2 8 C If dissolved in RNase free water storage at 20 C is recommended MAP Solution B The magnetic beads should be stored at 4 C Wash Buffer II Wash Buffer II charged with ethanol should be stored at room temperature and should be appropriately sealed If any precipitates are visible within the provided solutions solve them by carefully warming up to 30 C Room temperature RT is defined as a range from 15 30 C Quality control and product warranty STRATEC Molecular warrants the correct function of the InviMag Virus DNA Mini Kit IG for applications as described in this manual Purchaser must determine the suitability of the product for its particular use Sh
31. stratecee molecular 3 User manual InviMag Virus DNA Mini Kit IG for use on the InviGenius STRATEC Molecular GmbH for automated purification of viral DNA from serum plasma cell free body fluids rinse liquid from swabs with magnetic beads 2442120100 e o STRATEC Molecular GmbH D 13125 Berlin v Instructions for InviMag Virus DNA Mini Kit IG The InviMag Virus DNA Mini Kit IG combines the advantages of the innovative Invisorb technology with easy handling of magnetic particles for a very efficient and reliable isolation of viral DNA with high purity in a fully automated process using the InviGenius platform The viral DNA binding magnetic particles used in the procedure are characterized by a high specific surface area uniform size distribution and good suspension stability Therefore the particles are highly suitable for high throughput processing of nucleic acids The InviMag Virus DNA Mini Kit IG is the ideal tool for walk away isolation and purification of viral DNA from 12 samples of fresh or frozen plasma serum cell free body fluids and rinse liquid from swabs with the InviGenius fully automated pipetting system The interplay of the DNA extraction and purification chemistry provided by the InviMag Virus DNA Mini Kit IG with the InviGenius system was intensely tested C Compliance with EU Directive 98 79 EC on in vitro medical devices Not for in vitro diagnostic use in co
32. supplied well strips and foils beforehand and after a run on the unused and used afterwards wells of the Incubation and Working Plate A 12 InviMag Virus DNA Mini Kit IG 0515 Scheme of the InviMag Virus DNA Mini Kit IG Add the primary tubes in the sample loading rack Add the Buffers in the Buffer loading rack 200 ul sample is mixed with 200 ul Lysis Buffer HL 40 ul PKC An incubation of 15 minutes is performed at 95 C 250 ul Binding Buffer HL follow preparing instructions and 40 ul MAP Solution B are added to the lysate DNA binds to magnetic particles Magnetic separation Washing of the particle fixed viral DNA with 1 x 600 ul Wash Buffer I 2 x 650 ul Wash Buffer II Magnetic separation Elution of viral DNA in 100 ul Elution Buffer M Magnetic separation and removal of MAP Solution B Pure viral DNA 13 InviMag Virus DNA Mini Kit IG 1113 Working Protocol Isolation of viral DNA Please read the instructions carefully and conduct the prepared procedure Important Note The protocol is optimized for the isolation of viral DNA from up to 200 ul of cell free fluid To prevent possible distribution errors we recommend providing at least 400 ul sample in total to ensure stable processing Preparing the samples for processing on the InviGenius system 1 Extraction of viral DNA from serum plasma cell free body fluids This type of sample can be processed directly without any preparations Please ens
33. untries where the EU Directive 98 79 EC on in vitro medical devices is not recognized Trademarks InviGenius Invisorb InviMag Registered marks trademarks etc used in this document even when not specifically marked as such are not to be considered unprotected by law The Invisorb technology is covered by patents and patent applications US 6 110363 US 6 043 354 US 6 037 465 EP 0880535 WO 9728171 WO 9534569 EP 0765335 DE 19506887 DE 10041825 2 WO 0034463 InviGenius InviMag and Invisorb are registered trademarks of STRATEC Biomedical AG The PCR process is covered by US Patents 4 683 195 and 4 683 202 and foreign equivalents owned by Hoffmann La Roche AG 2015 STRATEC Molecular all rights reserved T InviMag Virus DNA Mini Kit IG 0515 Contents Kit contents of InviMag Virus DNA Mini Kit IG 3 Symbols 4 Storage 4 Quality control 4 Intended use 9 Product use limitation 5 Safety information 6 Product characteristics of the InviMag Virus DNA Mini Kit IG 7 Example data 8 Sampling and storage of starting material 9 Principle and procedure 9 Yield and quality of viral DNA 10 Important notes 10 Preparing the reagents and buffers 11 Reagents and equipment to be supplied by user 11 Important indications 12 Scheme of the InviMag Virus DNA Kit IG 13 Working protocol Isolation of viral DNA 14 Preparing of the internal control for the InviGenius System 15 General overview of the InviGenius
34. ure to supply at least 550 ul sample or adjust the volume up to 550 ul using PBS or RNase DNase free water 2 Extraction of viral DNA from swabs Rinse each swab with enough cooled RNase DNase free water or cooled PBS max 500 ul and incubate the swab for 5 min Then squeeze out the swab at the inside of the tube and discard it Finally transfer the liquid into a sample tube and insert it into the sample rack of the InviGenius instrument Keep in mind that the samples have to be labelled with bar codes Otherwise the samples have to be registered manually within the InviGenius software during the sample loading step Prevention of cross contamination To comply with the demanding guidelines of in vitro diagnostics the pipettor of the InviGenius is routed in such a way that possible contamination risks are very unlikely However we recommend applying the supplied well strips and foils beforehand and after a run on the unused and used afterwards wells of the Incubation and Working Plate A Take care not to seal lanes that will be required for a successful run 14 InviMag Virus DNA Mini Kit IG 0515 Preparing of the internal control for the InviGenius system Please read the instructions carefully and conduct the prepared procedure Using an internal control IC Using the InviGenius Virus DNA Kit in combination with a commercially available amplification system may require introducing an internal control IC into the purific
35. wing cycles of the material ensure that the starting material is fresh or stored at appropriate conditions for long time storage at 20 C avoid multiple thawing and freezing cycles of the material old material may contain degraded DNA make sure that only and all reagents belonging to one kit type are used a combination of reagents belonging to different kit types is not supported centrifuge the eluate plate at full speed for 1 min and transfer supernatant to a new plate tube 27 InviMag Virus DNA Mini Kit IG 0515 Ordering information Product Package Size Catalogue No InviMag Virus DNA Mini Kit IG 8 x 12 preparations 2442120100 Related Products Package Size Catalogue No Invisorb Spin Virus RNA Mini Kit 50 preparations 1040300200 Invisorb Spin Virus RNA Mini Kit 250 preparations 1040300300 Invisorb Spin Virus DNA Mini Kit 50 preparations 1040200200 Invisorb Spin Virus DNA Mini Kit 250 preparations 1040200300 InviGenius and consumables InviGenius 1 unit 5011100000 Starting Box I IG 1 box 2400110100 Sheath Box Conductive filter tips 1 ml 2 x 2 rack pack 384 pieces 5 Waste Trays 120 sample tubes Sheath Bundle 10 x 48 pieces 5011100300 Sheaths 1000 pieces 5011100200 Conductive filter tips 1 ml 10 x 96 pieces 5011100400 Waste tray IG 25 pieces 5011100100 InviGenius 1 unit 5011100000 Possible suppliers for Isopropanol Carl Roth Applichem Sigma 2 Propanol 2 Propanol fur die Molekularb
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