InviMag Blood DNA Maxi Kit/ IG User manual
Contents
1. User Figure 2 Log in screen of the InviGenius software After logged in the main screen of the InviGenius software is displayed Figure 3 Select Loading A to proceed with loading of the system or select Processing B to define and run an assay if the system has been loaded properly Main Menu management Processing InviGenius Status Ready es User service Figure 3 Main menu of the InviGenius software 42 InviMag Blood DNA Maxi Kit IG 0515 Sample Loading After Loading was selected the sample loading screen is shown Select Samples A to proceed with the sample loading process Loading Reagents Assay selection Disposable sheaths Disposable tips Mi Waste management Figure 4 Loading submenu of the InviGenius software Status Ready User service Samples loading SAMPLE 1 edited SAMPLE 2 edited SAMPLE 3 edited SAMPLE 4 edited SAMPLE 5 edited edited edited edited edited edited SAMPLE 11 edited SAMPLE 12 edited 20 9999oooooooe AP gt 4 y VS Reagent loading Status Ready EE User service Figure 5 Sample loading screen of the InviGenius software Prepare the sample rack with primary sample tubes that are prefilled with blood from which the gDNA shall be extracted Sample tubes are not provided within the kit The operator must either use primary tubes provided from the blood collecti2. InviMag Blood DNA Maxi Kit IG stratecee molecular User manual for use on the InviGenius STRATEC Molecular GmbH for automated purification of DNA from 2 ml whole blood samples with magnetic beads 2431320X00 aaan STRATEC Molecular GmbH D 13125 Berlin v 4 Instructions for InviMag Blood DNA Maxi Kit IG The InviMag Blood DNA Maxi Kit IG combines the advantages of the innovative Invisorb technology with easy handling of magnetic particles in combination with the InviGenius robotic platform for an efficient and reliable isolation of nucleic acids with a high purity The InviMag Blood DNA Maxi Kit IG is the ideal tool for walk away automated isolation and purification of highly pure total genomic and mitochondrial DNA from 2 ml whole blood samples stabilized with EDTA or citrate but not heparin The kit is designed for use on the InviGenius workstation The interplay of the DNA extraction and purification chemistry provided by the InviMag Blood DNA Maxi Kit IG was intensely tested and validated The DNA binding magnetic particles are characterized by a high surface area uniform size distribution and good suspension stability and therefore are highly suitable for high throughput processing Due to the high purity the isolated DNA is ready to use in a broad panel of downstream applications or can alternatively be stored at 20 C for subsequent use The kit is neither validated for the isolation
3. and product warranty STRATEC Molecular warrants the correct function of the InviMag Blood DNA Maxi Kit IG for applications as described in this manual Purchaser must determine the suitability of the product for its particular use Should any product fail to perform the applications as described in the manual STRATEC Molecular will check the lot and if a lot connected problem is determined the product will be replaced free of charge SIRATEC 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 Blood DNA Maxi Kit IG have been tested separately against predetermined specifications routinely on lot to lot to ensure consistent product quality In case of questions or problems regarding any aspects of InviMag Blood DNA Maxi Kit IG or other STRATEC Molecular products please do not hesitate to contact us A copy of STRATEC 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 2903 2907 or contact your local distributor 4 InviMag Blood DNA Maxi Kit IG 0515 Intended use The InviMag Blood DNA Maxi Kit IG is designed for fully a
4. of genomic DNA from cell cultures tissues blood cards dried blood stains nor urine The application of the kit for isolation and purification of viral DNA has neither been tested nor evaluated For research use only Trademarks InviMag Invisorb InviGenius 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 InviMag Invisorb InviGenius 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 ds InviMag Blood DNA Maxi Kit IG 0515 Table of Content Kit contents of the InviMag Blood DNA Maxi Kit IG oymbols Storage Quality Control Intended use Product use limitation Safety information Product characteristics of the InviMag Blood DNA Maxi Kit IG Sampling and storage of starting material Principle and procedure Yield and quality of genomic DNA Important notes Preparing reagents and buffers Reagents and equipment to be supplied by user Important indications General overview of the InviGenius system Preparing and loading of the InviGenius system PREPARING THE REAGENT
5. of the InviGenius software When the sub item Maintenance is opened select UV decontamination 20 InviMag Blood DNA Maxi Kit IG 0515 Maintenance q UV Periodical decontamination maintenance Cleanup procedures Cleanup drop catcher Back Status Ready m User service Figure 16 Maintenance screen of the InviGenius software 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 eyes and can lead to irreparable damage Ensure that no lab personnel are submitted to direct UV light Do not try to force open the instrument door during the decontamination process UV decontamination Duration HH MM SS Remaining time 00 00 00 UV decontamination status There was no UV decontamination completed N Status Ready EEE User service Figure 17 UV decontamination screen When the decontamination is finished go back to the main menu by using the Back button The device is now decontaminated and can be either switched off or used for sample processing We recommend decontaminating the instrument daily Pj InviMag Blood DNA Maxi Kit IG 0515 General notes on handling DNA Nature of DNA The length and delicate physical nature of DNA
6. possible to rename barcode recognized samples too Select the corresponding sample using the arrow buttons followed by the Edit button and enter the desired sample name After a short time period about 5 min the bar code scanner will be deactivated if unused In that case the operator has to restart the scanner by pressing the START SCANNER button After all samples have been loaded recognized and or renamed 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 Unknown Unknown PG410055130900441505 Elution B M PG 2015 05 31 PG618034301302931505 MAP Sol B PG 2015 05 31 PG306108131800771505 Wash B ll PG 2015 05 31 PG706024340102531505 Proteinase K PG 2015 05 31 PG218098100000621505 Binding Sol PG 2015 05 31 Unknown Unknown PG124078130700521505 Lysis B HLT PG 2015 05 31 2O00200200000 MS t e DX A Unknown Unknown Status Ready mms User Service Figure 6 Reagent loading screen of the InviGenius software Decap all reagents before inserting them into the reagent rack Ensure that the bar code labels face to the right side of the loading bay The order of inserted reagents sharing a same bottle size is not important because the type and position of each reagent is identified by the unique r
7. the installed tip allocation module of the pipettor it is required that only filter tips are used Non filter tips will be rejected by the system To avoid contaminations never wash or reuse consumed tips Continue with Disp Sheaths loading Disposable Sheaths Loading The sheaths function as protection devices for the magnetic rods and are picked up automatically during the run Disposable sheaths loading Sheath Load Position eee 4 OKOROKOROKORORKOROKORORKO anes e e eme e ee eeeee Unload Sheath Type R Microplates Status Ready m ser service Figure 9 Disposable sheaths loading screen The loading procedure of the disposable sheaths works analogous to the disposable tip loading screen For each run always 12 disposable sheaths one row of the sheaths rack are picked up regardless of the sample number processed This is done to ensure that all rods are protected against contaminations at all time In general the number of sheaths supplied within the kit is sufficient for the amount of runs imprinted on the kit package In case of low or not enough available sheaths a reorder at SIRATEC Molecular 100 pieces bulk order nr 5011120300 or 10x 48 pieces order no 5011120400 is possible Comparable to the disposable tips loading procedure the number of rows left in the tip rack can be defined by clicking on the number area Please ensure that the disposable sheaths
8. Keep unused bottles firmly closed Hesuspend each lyophilized Proteinase K tube with 2 4 ml dd H2O and mix thoroughly Add 84 ml of 96 100 ethanol to each bottle Wash Buffer mix thoroughly and always keep unused bottles firmly closed One run is defined as a run using 12 samples Please keep in mind that partially used bottles may be used in other runs The InviGenius tracks the sample numbers and runs that were extracted with each bottle MAP Solution B and Proteinase K may be only used in two runs for quality mixing 3 InviMag Blood DNA Maxi Kit IG 0515 Symbols Manufacturer E O Lot number Catalogue number Expiry date Consult operating instructions Temperature limitation e BER Do not reuse Storage All buffers of the InviMag Blood DNA Maxi Kit IG except dissolved Proteinase K and MAP Solution B magnetic beads should be stored at room temperature and are stable for 12 months at this condition Proteinase K IG Dissolved Proteinase K must be stored at 20 C MAP Solution B IG The magnetic beads should be stored at 2 8 C Wash Buffer and Binding Buffer Wash Buffer and Binding Buffer charged with either ethanol or isopropanol should be stored at room temperature and must be appropriately sealed If there are any precipitates 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
9. Mag Blood DNA Maxi Kit IG 0515 Ordering information Product Package size Catalogue No InviMag Blood DNA Maxi Kit IG 8 x 12 preps 2431320100 Related products InviMag Blood DNA Mini Kit IG 8 x 12 preps 2431120100 InviMag Blood DNA Giga Kit IG 8 x 12 preps 2431320400 Invisorb Spin Blood Mini Kit 50 preparations 1031100200 Invisorb Spin Blood Mini Kit 250 preparations 1031100300 Invisorb Spin Blood Midi Kit 50 preparations 1031110300 Invisorb Spin Blood Midi Kit 250 preparations 1031110500 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 Possible suppliers for Isopropanol Carl Roth Applichem Sigma 2 Propanol 2 Propanol fur die Molekularbiologie 2 Propanol Rotipuran gt 99 7 p a ACS ISO Order no A3928 Order no 59304 1L F Order no 6752 24 InviMag Blood DNA Maxi 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 1G3a03 1IG 05 2015
10. S PREPARING THE SYSTEM SAMPLE LOADING REAGENT LOADING ASSAY SELECTION DISPOSABLE TIP LOADING DISPOSABLE SHEATHS LOADING PLATE LOADING WASTE MANAGEMENT BATCH DEFINITION BATCH CHECKING BATCH PROCESSING AFTER A RUN DAILY MAINTENANCE UV DECONTAMINATION General notes on handling DNA Troubleshooting Ordering Information oo dq O CO N OA oa fF A A C 11 12 12 12 13 14 15 15 16 17 18 18 19 19 19 20 22 23 24 InviMag Blood DNA Maxi Kit IG 0515 Kit contents of the InviMag Blood DNA Maxi Kit IG Store the MAP Solution B at 2 8 C Dissolved Proteinase K must be stored at 20 C otore all other kit components at room temperature RT Component 8 x 12 preps Catalogue No 2431320100 Lysis Buffer HLT 4 x 90 ml 2 runs per bottle Proteinase K IG for 8 x 2 4 ml working solution 1 vial per run MAP Solution B IG 8 x 3 5 ml 1 vial per run Binding Solution 4 empty bottles fill with 99 796 Isopropanol final volume 4 x 110 ml 2 runs per bottle 4 x 36 ml Wash Buffer final volume 4 x 120 ml 2 runs per bottle Elution Buffer M 4 x 60 ml 2 runs per bottle Incubation Plate D 2x4 1 run per plate Working Plate A 2x4 1 run per plate Elution Plate E 1 8 runs per plate Microtube Cap 8 Sheath Box 1 2 racks 48 sheaths 4 runs per rack Initial steps Add 110 ml Isopropanol molecular biologic grade undenaturated to each empty bottle labeled with Binding oolution
11. are loaded and displayed consistent to the manually entered sheath rows in the rack to guarantee correct sheaths pick up Don t remove single disposable sheaths within a row of the sheaths rack if less than 12 samples are processed within one run There is a sheaths detection sensor installed in the instrument If less than 12 sheaths are picked an error message is displayed and all picked up sheaths will be discarded into the waste container before the next row of sheaths are picked up for verification To avoid contaminations never wash or reuse consumed sheaths 16 InviMag Blood DNA Maxi Kit IG 0515 Plate Loading Analogous to the previous loading screens the incubation working and eluate plates are loaded Free lanes 1 Disp sheaths rt Waste Back loading management am Status Ready u ser service m Figure 10 Plate loading screen In general an Incubation Plate D Nerbe Plus 12 well reservoir DWP is used at the incubator position a Working Plate A 2 ml Sarstedt DWP is used at the working position and an Elution Plate E SLG stripes is used at the eluate position Plates can be unloaded reloaded by 1 Pressing the plate position directly A The software will focus the plate position on the main screen 2 Pressing the Unload button B 3 The plate can be reloaded by pres
12. e Tip Loading Disposable tips loading Tip Load Position 3 ack Disp sheaths loading m User service Status Ready Figure 8 Disposable tip loading screen There are three tip rack positions available in the InviGenius system Fig 8 A1 A3 that can be loaded with 1100 ul conductive filtered tips All provided assay files will work only with filtered tips to guarantee efficient prevention of cross contaminations of samples and reagents Remaining tip numbers in the rack are shown in field B Tip numbers can be changed by pressing the number field B and manually enter the number of available tips Empty tip racks can be unloaded and reloaded by 15 InviMag Blood DNA Maxi Kit IG 0515 1 Press the Loading Position directly The software will focus the loading position on the main screen 2 Press the Unload Button C 3 The loading position can then be refiled with a new tip rack by pressing on the corresponding tip rack and then selecting the tip type D Note Disposable tips are not supplied within the kit We recommend the use of validated conductive tips which can be ordered at STRATEC Molecular Offered tips are 50 ul conductive tips 10x 96 pieces order no 5011120100 and 1100 ul conductive tips 10x 96 pieces order no 5011120200 Ensure that conductive tips are used otherwise the liquid level detection LLD will not work which will lead to an error and thus an abort of the run Due to
13. eagent bar code and the bar code at the corresponding loading position of the reagent rack However the possible loading positions are limited by the size of useable bottles In total the reagent rack offers loading positions for 3 x 120 ml bottles 2x 30 ml bottles a 60 ml bottle and three 3 5 ml reagent tubes After rack insertion the loading status of the reagents is shown In case of unsuccessful reagent identification remove the rack check the bar code orientation and repeat the loading process slowly If all reagents are recognized properly continue with Assay selection 14 InviMag Blood DNA Maxi Kit IG 0515 Assay Selection Assay selection Assay description suitable to loaded reagents DBLD E400 S2000 No Assay Selected Version 1 Reagent loading Disp tip loading Status Ready mam User service Figure 7 Assay Selection screen of the InviGenius software Select the assay file DLBD E400 S2000 and ensure that the field suitable to loaded reagents is checked in order to list only assay files that are compatible with the loaded reagents If no assay is visible in the list there is either an error related to reagent loading blocked barcode label or at least one reagent container has been loaded that was already in use and does not contain enough reagent to run with the selected amount of loaded samples If the assay selection was successful proceed with Disposable tip loading Disposabl
14. en After the waste container was emptied click on Empty solid waste to reset the filling level status Continue with Batch checking Batch definition Batch definition Assay descriptions suitable to loaded reagents DBLD E400 S2000 Samples SAMPLE 1 SAMPLE 2 SAMPLE 3 SAMPLE 4 SAMPLE 5 SAMPLE B SAMPLE 7 Remove SAMPLE 8 from batch SAMPLE 9 SAMPLE 10 SAMPLE 11 SAMPLE 12 Version 1 Aliquots Volume 2000 EEA 2000 ja management Status Ready User service Figure 12 Batch definition screen This screen allows de selection of the desired assay and sample numbers in one informational screen The assay can be switched by using the two arrow buttons A If samples have to be excluded from the batch use the arrows B to switch between samples and click on the Remove from batch button field B If deselected samples have to be added to the process list the button Remove from batch will be changed to Add to batch By default all loaded and recognized samples are selected Continue with Batch checking 18 InviMag Blood DNA Maxi Kit IG 0515 Batch checking This screen shows a summary of all loaded disposables samples and buffers in one screen and functions as a final security check In case of any error the problem position is highlighted in red color To clear a displayed error click directly on the red highlighted field and foll
15. er sources like stool samples tissues bacteria fungi or viruses was neither tested nor validated No guarantee in operability is issued if other samples than whole blood are used The included chemicals are only useable once Differing of starting material can 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 in clinical diagnostic laboratory systems conditioned upon the complete diagnostic system of the laboratory after the lab 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 should be reported to STRATEC Molecular immediately upon detection thereof The chemicals and plastics are for laboratory 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 9 InviMag Blood DNA Maxi Kit IG 0515 Safety information When and w
16. er than 2 ml For samples with a volume s 2 ml please adjust the final volume with either distilled water or 1x PBS to 2 5 ml 3 Elution volume The final processed elution volume is 400 ul After the elution step an aliquot of about 380 yl is transferred to the elution plate Typically 400 ul eluate will contain about 15 50 ug of purified gDNA 4 Residual beads in eluate Due to the high DNA concentration present in the eluate it can happen that a very small of residual beads are visible within the eluates In that transfer the eluate to a new tube and centrifuge them for 1 min in at 13000rpm e g in a table centrifuge Transfer the clear supernatant into a new tube without disturbing the pelleted beads at the tube bottom 10 InviMag Blood DNA Maxi Kit IG 0515 General overview of the InviGenius system Figure 1 Layout of the InviGenius system There are three plate positions available in the InviGenius system which can be loaded with corresponding plates the incubation A working B and eluate position C The lysis is performed at the incubation position A whereas the washing and elution process is performed at the working position B The eluate containing the extracted nucleic acids is finally transferred to the eluate position C There are three loading positions for disposable tip trays D1 D3 and one position E for the disposable sheaths available The loading bay F is located at the ve
17. ever if a whole blood sample is mixed with anticoagulant containing buffer solutions the overall leukocyte concentration decreases and the yield of the gDNA extraction procedure is reduced slightly Important notes Important points before starting a protocol After receiving the kit check all components for visible damage If buffer bottles are damaged or leaking contact the STRATEC Molecular Technical Services or your local distributor In case of liquid spillage refer to Safety information page 6 Do not use damaged kit components because their use may lead to poor kit performance o When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles o Discard contaminated gloves immediately o Do not combine reagents from different kits even if the lot numbers are identical because the use of unique barcodes will not allow merging different kit components o Avoid microbial contamination of the kit reagents o To minimize the risk of diseases from potentially infectious material we recommend working under laminar air flow for all steps performed outside the InviGenius platform o his kit should only be used by trained personnel Preparing reagents and buffers Before starting a run equilibrate all reagents at room temperature Where necessary gently mix and redissolve any precipitates by incubating at 30 C Swirl gently to avoid foaming Lysis Buffer HLT and Elution Buffer M are ready to u
18. hile 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 Blood DNA Maxi Kit IG procedures for residual infectious materials Contaminations of the waste with residual infectious materials is highly unlikely but cannot be excluded completely Therefore all generated waste has to be considered infectious and should be handled and discarded accordingly to local safety regulations Below European Community risk and safety phrases for the components of the InviMag Blood DNA Maxi Kit IG are listed Lysis Buffer HLT Wash Buffer HLT Proteinase K contains guanidine hydrochloride H302 315 319 P280 305 351 338 H315 319 334 335 P280 305 351 338 310 405 H315 Causes skin irritation H319 Causes serious eye irritation H334 May cause allergy or asthma symptoms or breathing difficulties if inhaled H335 May cause respiratory irritation P280 Wear protective gloves protective clot
19. hing eye protection face protection P305 P351 P338 If in eyes Rinse cautiously with water for several minutes Remove contact lenses and continue rinsing P310 Immediately call a POISON CENTER or doctor physician P405 Store locked up 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 Blood DNA Maxi Kit IG 0515 Product characteristics of the InviMag Blood DNA Maxi Kit IG The InviMag Blood DNA Maxi Kit IG is the ideal tool for an efficient and fully automated DNA extraction and purification from fresh or frozen whole blood samples using magnetic beads in combination with the InviGenius robotic platform preparation 2 ml fresh or frozen whole blood 15 50 ug depending on about 200 min stabilized with EDTA or citrate but not the blood sample 12 samples heparin transport storage age and source 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 the whole DNA purification procedure including sample and reagent distribution without any user intervention except the initial loading of the system This allows safe handling of potentially infectious samples whereas cross contaminations and reagent cross overs are effectively eliminated by the automated pu
20. imination of ethanol Elution of pure gDNA O O O x After lysis the gDNA binds to the added magnetic particles whereas contaminations and enzyme inhibitors are efficiently removed during the following seven washing steps At the end highly purified gDNA is eluted Lysis Whole blood samples are lysed in a Deep Well Reservoir Plate at elevated temperatures in the presence of Lysis Buffer HLT and Proteinase K Binding After addition of Binding Solution and MAP Solution B magnetic beads to the lysate the gDNA is bound to the beads Removing residual contaminants Contaminants are efficiently removed using Wash Buffer HLT and Wash Buffer while the gDNA remains bound to the beads Elution The gDNA is finally eluted in Elution Buffer M and is ready to use for different subsequent downstream applications like PCR amplification digestion with restriction enzymes Southern hybridizations HLA typing etc 8 InviMag Blood DNA Maxi Kit IG 0515 Yield and quality of genomic DNA The amount of purified gDNA using the InviMag Blood DNA Maxi Kit IG procedure from whole blood depends on the leucocytes content sample source transport condition storage and age Typically a 2 ml whole blood sample from a healthy individual with an elevated white blood cell content ranging from 3x10 to 1x10 cells per ml will yield app 20 ug of gDNA The overall purified yield is in the range of 15 50 ug gDNA derived from 2 ml whole blood How
21. nol added to Wash Buffer Binding Buffer Incorrect storage of starting material Old material Combination of reagents from different kits or blocked barcode during reagent loading procedure Small part of the magnetic particles are left in the elution co Comments and suggestions Ensure that lyophilized Proteinase K is resuspended with the appropriate volume of water The sample tube must contain at least 2 5 ml sample Ensure that the Wash Buffer and Binding Buffer is filled up properly with ethanol isopropanol Do not reuse bottles more often than described in Tab 1 In case of large sample volumes gt gt 2 ml carefully premix the sample tube before inserting it into the sample rack Assure that the Wash Buffer Binding Buffer is filled up with ethanol isopropanol properly as indicated in Tab 1 Ensure that the storage condition of the starting material was correct Avoid multiple freezing and thawing cycles of the sample Ensure that the starting material is fresh or stored at appropriate conditions for long term storage at 20 C Old material often contains degraded DNA Assure that only reagents belonging to one kit type are used a combination of reagents belonging to different kit types is not supported by the system Ensure that the reagent barcode label is visible within the reagent rack window Centrifuge the eluates at full speed for 1 min and transfer supernatant to a new plate tube Invi
22. on blood bank etc In case that no primary tubes are available common collection tubes can be ordered at Sarstedt for example order no 55 476 5 ml tubes 75x12 mm polystyrene For each reaction a total sample volume of 2 ml is processed However we advise that the total provided sample volume should be at least 2 5 ml to ensure stable processing If primary tubes are used that are completely filled with blood 2 5 ml please premix by inverting the tube several time before usage to ensure a homogenous solution Always decap inserted sample tubes Keep in mind that only 12 positions of the sample rack can be processed per run due to the limited number of wells per row of the plastic ware For correct identification of the sample tubes bar codes if available must face to the bar code scanner window located at the right side of the loading bay 13 InviMag Blood DNA Maxi Kit IG 0515 After inserting the sample rack into the very left lane of the loading bay an updated screen will show the identifiers read out from the sample bar codes Figure 5 In case of unsuccessful sample identification remove the rack check the bar code orientation and reinsert the rack slowly If no sample barcodes is present the corresponding sample will be flagged as unknown sample In that case the sample name has to be entered changed manually using the Edit button Keep in mind that samples marked as unknown sample will not be processed It is
23. ow the instructions printed on the instrument screen If no error is displayed proceed by pressing the button Batch processing Batch checking Selected assay DBLD E400 2000 i x Batch processing User service 009o9o9ooo 99999 d d A s b ae E A Ee Batch definition Status Ready Figure 13 Batch definition screen Batch processing After the instrument door has been closed the run can be started by pressing the Start Button A The door will be locked during the complete run and will be unlocked only after a run has been successfully finished or if an error occurs which demands user interaction Do not try to force open the door during a run or the run will be aborted If there is an error present the Start button is blocked The user will not be able to start the instrument until the error has been solved To do so go back to Batch definition and solve the error that is displayed by red flashing color of the affected position see chapter Batch definition for detailed information Batch processing Batch identifier Assay description Actual step s Remaining time 00 00 00 Process state Idle A Events Status Ready m User service Figure 14 Batch processing screen 19 InviMag Blood DNA Maxi Kit IG 0515 At the end of the process the nucleic acid containing eluates are located in the appropriate eluate position and can be u
24. requires careful handling to avoid damage due to shearing and or enzymatic degradation Other conditions that affect the integrity and stability of DNA include acidic and alkaline environments high temperature or UV irradiation Careful isolation and handling of high molecular weight DNA is necessary to ensure its functionality in various downstream applications Damaged DNA could perform poorly in applications such as genomic Southern blotting long template PCR and construction of cosmid libraries Handling fresh and stored material before the extraction of DNA For the isolation of genomic DNA use either fresh or frozen samples 20 C This procedure minimizes degradation of crude DNA by limiting the activity of endogenous nucleases Storage of DNA Store the genomic DNA at 2 8 C or 20 C Keep in mind that the storage of genomic DNA at 20 C may cause shearing particularly if the DNA is exposed to repeated freezing and thawing cycles Plasmid DNA and other small circular DNAs can be stored at 2 8 C or at 20 C without risk 22 InviMag Blood DNA Maxi 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 Transfer of Proteinase K failed Samples transfer failed Reagent buffer transfer failed incomplete Blood components settled No too much ethanol isopropa
25. rification process The use of unique bar codes for samples and reagents prevents unwanted transpositions The InviGenius instrument uses magnetic rods to transport the DNA binding magnetic particles through the various extraction phases such as binding washing and elution The volume of reagents required 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 HLT and Proteinase K optimal binding conditions are adjusted upon addition of Binding Solution The genomic DNA binds to the simultaneously added magnetic particles and is separated from solution using magnetic rods which are controlled by the InviGenius system Subsequent to seven washing steps of the particle bound nucleic acids the pure DNA is finally eluted Due to the high purity the eluted total DNA genomic and mitochondrial is ready to use in a broad panel of downstream applications PCR real time PCR Restriction enzyme digestion HLA typing o Southern Blotting O O O For the isolation of DNA from single blood samples STRATEC Molecular offers the Invisorb Spin Blood Mini Kit or for 8 96 samples the Invisorb DNA Blood Mini HTS 96 Kits for use on a centrifuge or robotic stations For further information please contact 49 0 30 9489 2901 2910 in Germany 49 0 30 9489 2903 2907 from foreign countrie
26. ry right side of the instrument The sample rack is loaded into the far left lane whereas the reagent rack is located at the right position of the loading bay occupies 3 lanes The moveable Magnetic Separation Head MSH G is located on top of the incubator parking position The automatic pipettor head H is located above the loading bay parking position The disposable waste tray I is located behind the lower cover door of the InviGenius system User interaction with the InviGenius instrument is performed by use of the touch LCD J located at the top front right side of the instrument 11 InviMag Blood DNA Maxi Kit IG 0515 Preparing and loading of the InviGenius system Preparing the reagents Before starting a run prepare one vial of Proteinase K and add ethanol to the Wash Buffer and isopropanol to the Binding Solution bottle as indicated in Tab 1 see page 3 or page 9 Preparing the system Switch on the InviGenius system using the power switch located at the back right side of the instrument The InviGenius software will automatically be loaded after the system has booted up The door of the InviGenius system must be closed during system initialization and during a run After booting and successful initialization of the InviGenius system a log in screen appears Figure 2 Log in with the provided user name and password Login User identifier e a O Password Status Ready mm
27. s or ask your local distributor TE InviMag Blood DNA Maxi Kit IG 0515 Sampling and storage of starting material For reproducible and high yields the appropriate sample storage is essential Yields may be varying from sample to sample depending on factors such as health of the donor sample age sample type transport and storage conditions Blood Best results are obtained if fresh whole blood samples are used Mammalian blood samples stabilized with EDTA or citrate but not heparin are stable at room temperature 15 C 30 C for 2 3 hours after collection For short term storage up to 24 h samples can be stored at 4 8 C For long term storage freezing the samples at 20 C or 80 C is recommended Avoid multiple thawing and freezing cycles of samples before isolating the DNA because this may lead to degradation of the DNA Various different primary tubes blood collection systems e g Sarstedt Greiner and anticoagulants except heparin can be used to collect blood samples for the InviMag Blood DNA Maxi Kit IG procedure STRATEC Molecular will be released of its responsibilities if other sample materials than described above are used or if the sample preparation protocols are changed or modified Principle and procedure The InviMag Blood DNA Maxi Kit IG procedure comprises the following steps Lysis of blood cells and protein digestion Binding of released gDNA by magnetic beads Washing of the bead bound gDNA and el
28. se 8 x 12 DNA extractions Resuspend each lyophilized Proteinase K tube by addition of 2 4 ml dd H2O mix thoroughly and store diluted and unused Proteinase K at 20 C Add 84 ml of 96 100 ethanol to each bottle Wash Buffer II Mix thoroughly and always keep the bottle firmly closed Add 110 ml of 99 7 isopropanol to the bottle Binding Solution and always keep the bottle firmly closed Reagents and equipment to be supplied by user Measuring cylinder 250 ml Pipette tips Disposable gloves ddH2O Vortexer 96 100 ethanol 99 7 96 Isopropanol O O OF 0000 9 InviMag Blood DNA Maxi Kit IG 0515 The InviMag Blood DNA Maxi 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 Important indications 1 Minimum volume of samples in primary tubes The procedure of the InviMag Blood DNA Maxi Kit IG has been optimized for the isolation of genomic DNA from up to 2 ml human whole blood We advise to provide at least 2 5 ml blood per sample tube 12 mm diameter more if a wider diameter is used to prevent pipetting distribution errors during processing and to avoid unwanted flagged sample results due to used low sample volumes 2 Sample volume small
29. sed for any further downstream application Note The run duration of the Blood Maxi DNA assay using 12 samples is approximately 200 minutes After a run After the run is completed and no additional run should be started remove all plates and reagents from the instrument and store them accordingly to GLP guidelines Please keep in mind that the plates could contain possibly infectious material Important Never erase not completely filled plates from within the software if the plates shall be used for a later run It is not possible to register partially used plates within the software An erased plate will lead to loss of all stored information and the plate has to be discarded As with all medical clinical and diagnostically equipment all waste products 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 switch to the main menu of the InviGenius software and select Maintenance Main Menu Loading EUM management Maintenance D Administration InviGenius Status Ready mm User developer Processing Shutdown Figure 15 Main screen
30. sing on the offered plate s in C Attention Be aware that it is not possible to manually enter filled rows of a previously used plate within the system software If a partially filled plate info is erased from the system software all used lanes of that plate will be deleted from the system memory Therefore it is not possible to reenter the filling status of an erased partially filled plate One run with the DNA Blood Maxi Kit on the InviGenius using 12 samples will require one new Incubation Plate D at the incubator position one new Working Plate A at the working position and one free row within the Elution Plate E located at the eluate position Please ensure that the depicted lanes displayed on the monitor are consistent with the real lanes in the corresponding positions To avoid contaminations never wash or reuse consumed plates Continue with Waste management A7 InviMag Blood DNA Maxi Kit IG 0515 Waste management Ensure that the waste tray capacity is sufficient for the planned assay If not empty or exchange the solid waste container Handle the generated waste accordingly as required by local disposal laws Waste management Waste capacity 190 Dropshaftstatus In place Fill level 0 disposables 0 Number of wasted disposable sheaths 0 Number of wasted disposable tips 0 Microplates Back Batch definition loading h Status Ready i User service Figure 11 Waste management scre
31. utomated extraction and purification of genomic DNA from up to 2 ml whole blood in the range of 1 12 blood samples using the InviGenius robotic platform The nucleic acid isolation protocol is suitable for routinely walk away automated preparation of DNA from fresh or frozen blood samples For reproducible and high yields the appropriate sample storage is essential see Sampling and storage of the starting material page 8 Common blood collection tubes not provided and anticoagulants EDTA and citrate but not heparin can be used All utilities reagents and plastics besides filter tips required for preparation of DNA from blood are provided by the InviMag Blood DNA Maxi 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 DNA 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 Product use limitation The kit is only validated for the extraction of human whole blood samples The isolation of DNA from oth
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