InviMag Universal Kit/ IG User manual - Negev Bio
Contents
1. Pure total NA InviMag Universal Kit IG 0314 Preparing the samples for processing on the InviGenius system Please read the instructions carefully and conduct the prepared procedure Important Note The protocol has been optimized for the isolation of gDNA and viral nucleic acids from up to 200 ul of liquid samples To prevent possible distribution errors it is required to provide at least 400 ul of sample to ensure stable processing 1 Extraction of nucleic acids from blood serum plasma cell free body fluids urine liquor transport media These types of samples can be processed directly without any preparations Please provide at least 400 ul sample or use RNase DNase free water or PBS to adjust to this volume Please keep in mind that the first step in the equipment is the premixing of samples Therefore samples have to be at least transferable which means no presence of clots and other solid materials that will lead to blocked pipette tips and prevent a normal workflow We recommend checking samples for coagulation by mixing several times before usage on the instrument 2 Extraction of NA from rinsed liquid from swab samples a the sample will be used for cultivation Cut off the relevant part of the swab and transfer it into an RNase DNAse free 2 ml tube Add 400 yl physiological saline solutions to the swab and vortex intensely for 2 3 min Then incubate for 10 min at RT Use an aliquot for further cult2. no assay selectable eluted nucleic acids are brownish colored Probable cause pipetting of PKC failed samples transfer failed incomplete reagent buffer transfer failed incomplete sample components settled No too much ethanol added to Wash Buffer Il incorrect storage of starting material old material combination of reagents from different kits missing required reagent Comments and suggestions ensure that the lyophilized PKC is diluted with the appropriate volume of water before usage the sample tube must contain at least 500 ul sample ensure that the supplied Wash Buffer Il Binding Solution are 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 221 ml carefully premix the sample tube before inserting it into the sample rack ensure that the Wash Buffer Il 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 thawing 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 ensure that only and all reagents belonging to one kit type are used a combination of reag
3. stratecee molecular User manual InviMag Universal Kit IG foruseontheInviGenius9 STRATEC Molecular GmbH for automated purification of DNA genomic bacterial mitochondrial and viral as well asviral RNA from 200 ul clinical samples with magnetic beads 2450120100 gaal STRATEC Molecular GmbH D 13125 Berlin re Instruction for InviMag Universal Kit IG The InviMag Universal Kit IG combines the advantages of the innovative InviMag technology with easy handling of magnetic particles of high purity in combination with the InviGenius robotic platform The nucleic acid binding magnetic particles are characterized by a specific surface a uniform size distribution and good suspension stability The InviMag Universal Kit IG in combination with the InviGenius is the ideal tool for a walk away automated isolation and purification of highly pure total genomic bacterial and viral DNA and of viral RNA from 200 ul blood serum plasma cerebrospinal fluid cell culture supernatants and other cell free body fluids like urine as well as from swabs rinsed liquid sputum BAL or supernatant from stool suspension for in vitro diagnostic purposes The kit is designed for an optimal use of the InviGenius workstation The interplay of the nucleic acid extraction and purification chemistry provided by the InviMag Universal Kit IG was intensely tested and validated Due to the high purity the isolated DNA RNA is ready
4. 3 Elution volume The final elution volume is 100 ul or 200 ul depending on the selected assay file 4 Processing of bacterial samples The kit was validated with Bacillus subtilis spiked cell free medium To perform a quantitative extraction of bacterial DNA from gram positive bacteria the addition of Lysozyme is required Please add 5 ul of a 10 mg mL Lysozyme solution per 200 ul sample volume to the primary tube before starting the assay Carrier RNA Carrier RNA serves two purposes It enhances the binding of viral acids to the beads especially if there are only very few target molecules in the sample Furthermore the addition of large amounts of Carrier RNA reduces the chance of viral nucleic acid degradation InviMag Universal Kit IG 0314 Scheme of the InviMag Universal Kit IG Add the primary tubes in the sample loading rack Add the Buffers in the Buffer loading rack 200 pl sample is mixed with 200 ul Lysis Buffer HLT and 40 yl Proteinase K Carrier RNA mixture Incubation at elevated temperature is performed for 10 min 240 ul Binding Solution and 40 ul MAP Solution B are added to the lysate Nucleic acids bind to magnetic particles Magnetic separation Washing of the particle fixed viral RNA with 1 x 800 ul Wash Buffer HLT 1 x 900 ul Wash Buffer Il 1x 750 ul Wash Buffer Il Magnetic separation Elution in 100 pl or 200 ul of Elution Buffer M Magnetic separation and removal of MAP Solution B
5. 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 350 yl 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 internal 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 InviMag Universal Kit IG 0314 General overview of the InviGenius system Figure 1 Frontal view of the InviGenius system Figure 1 shows the plate positions A elution position E working position and I incubation position Disposable tips are
6. The added Carrier RNA will 200 ul cell free body fluids urine account for most of the eluted 200 yl rinsed liquid from swab nucleic acid s Quantitative 200 ul transport media Surepath Thinprep RT PCR is recommended for 200 ul supernatant from stool suspension determination of the viral RNA 200 ul from liquid sputum BAL tracheal secrete or DNA yield The DNA RNA isolation process is based on the interaction of nucleic acids with silica coated magnetic particles in optimal buffer conditions The InviGenius instrument will automatically perform all steps of sample and reagent distribution The DNA RNA purification procedure is performed without any user intervention except any sample pretreatment and the initial loading of the system This allows safe handling of potentially infectious samples Sample cross contamination and reagent cross over is effectively eliminated The usage of unique bar codes for samples and reagents avoids unwanted transpositions The InviGenius instrument uses magnetic rods to transport the DNA RNA binding magnetic particles through the various extraction phases lysis bind wash elute 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 by the addition of Binding Solution The genomic DNA RNA binds to the added magnetic particl
7. be used The kit is in compliance with EU Directive 98 79 EC on in vitro medical devices 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 STRAT
8. 40 ul Lysozyme not provided stock solution 10 mg ml to the sample Prevention of cross contamination To comply with the demanding guidelines of in vitro diagnostics we programmed the InviGenius to route the pipettor in such a way that possible contamination risks are minimized However we recommend to apply the supplied well strips and sealing foils beforehand and afterwards on the used wells on the unused wells of the Incubation Plate A and the Working Plate A Be careful not to seal any required lanes of the working plate four first free lanes InviMag Universal Kit IG 0314 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 Kit in combination with a commercially available amplification system may require introducing an internal control IC into the purification 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
9. EC Molecular immediately upon detection thereof The chemicals and the 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 InviMag Universal Kit IG 0314 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 Universal Kit IG procedures for residual infectious materials Contamination of the waste with residual infectious materials is unlikely but cannot be excluded completely Therefore the waste has to be considered infectious and should be handled and discarded accordingly to local safety regulations Subsequently European Community risk and safety phrases for the components of the InviMag Universal Kit IG to which they apply are listed Lysis Buffer HLT PKC Tube Proteinase K Carrier RNA con
10. Electrophoresis RNA extracted with the provided kit looks like degraded cause the kit contains Carrier RNA this is poly A RNA in fragments of 100 up to 1000 bases 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 unless the lot numbers are identical Avoid microbial contaminations of the kit reagents To minimize the risk of infections from potentially infectious material we recommend working under laminar air flow o This kit should only be used by trained personnel O O 0 0 Preparing reagents and buffers Before starting a run bring all reagents to room temperature Where necessary gently mix and re dissolve any precipitates b
11. ULINI E200 S200 SAMPLE 12 le leh EP management Status Ready es ser Service Figure 12 Batch definition screen 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 gt InviMag Universal Kit IG 0314 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 UYUM E200 200 709 Batch detnition Batch processing Status Ready User service Figure 13 Batch definition 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 wi
12. ample into a primary tube Add 40 ul Lysozyme not provided stock solution 10 mg ml to the tube Viscous sample Transfer 1 ml of tracheal secrete or BAL into a RNase DNase free tube and add 1 ml NAC Buffer order number 1033221100 or saturated Acetylcysteine ACC solution to the sample ratio sample to buffer must be 1 1 InviMag Universal Kit IG 0314 Incubate the mixture for 10 min at 95 C to reduce the viscosity and centrifuge at 9 300 x g 10 000 rpm for 3 min Discard the supernatant without disturbing the bacterial pellet Resuspend the bacterial pellet in 400 ul distilled water or RNase free water and transfer it into a primary tube Add 40 ul Lysozyme not provided stock solution 10 mg ml to the tube 5 Extraction of viral NA from supernatant of stool suspension Transfer 100 ul stool sample into a 2 ml tube and dilute the sample 1 10 with RNase free water Vortex the sample for 30 s followed by a 1 min centrifugation step at 12 000 x g 13 000 rpm Transfer 400 ul supernatant into a primary tube Prevent the aspiration of swimming particles 6 Extraction of bacterial NA from supernatant of stool suspension Transfer 100 ul stool sample into a 2 ml tube and add 300 yl RNase free water 300 ul Vortex the sample for 30 s followed by a 30 s centrifugation step at 3 000 rpm 1 000 x g Transfer the bacteria containing supernatant into the cavity of a primary tube prevent the aspiration of swimming particles and add
13. ations 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 single 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 22 InviMag Universal Kit IG 0314 Plate Loading Analogous to the previous loading screens the incubation working and elution plate are loaded within the plate loading screen Figure 10 Microplates Plate A Working Position management User service Figure 10 Plate loading screen In general the Incubation Plate A and Working Plate A identical are used at the incubator an
14. ces 1000 pieces 10 x 96 pieces 25 pieces Applichem 2 Propanol fur die Molekularbiologie Order no A3928 30 Catalogue No 2450120100 Catalogue No 1031100200 1031100300 1031110300 1031110500 7031300300 7031300400 1040100200 1040100300 1040300200 1040300300 1040200200 1040200300 1040500200 1040500300 5011100000 2400110100 5011100300 5011100200 5011100400 5011100100 Sigma 2 Propanol Order no 59304 1L F InviMag Universal Kit IG 0314 BALKAN KALKANA AAA AKALAN ANAK AKAN LETT TULIT LIT LOT stratecee molecular STRATEC Molecular GmbH Robert R ssle 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 1G7k01 1G 03 2014
15. creen of the InviGenius software appears Figure 3 Select Loading to start loading the system and prepare for starting a run Select Processing to define and run an assay if the system has been already loaded Main Menu Loading management 3 bic zw A InviGenius Status Ready a Ser developer Figure 3 Main menu of the InviGenius software InviMag Universal Kit IG 0314 Sample Loading After selecting Loading the sample loading screen appears Loading Disposable tips Disposable Reagents 3c sheaths Assay Selection Waste mansgemen Status Ready a ser developer Figure 4 Loading screen of the InviGenius software Select Samples to proceed with the sample loading screen Samples loading SAMPLE edited i o SAMPLE 2 edited o SAMPLE 3 edited SAMPLE 4 edited o SAMPLE 5 edited o SAMPLE 6 edited SAMPLE 7 edited SAMPLE 8 edited SAMPLE edited o SAMPLE 10 edited o SAMPLE 11 edited SAMPLE 12 edited DX C es Gn Reagent losdirg Status Ready mm Se Service Figure 5 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
16. d Please keep in mind that a small amount of Carrier RNA in the eluate will elevate the real genomic DNA content Yield and quality of pathogen DNA RNA The amount of purified pathogen DNA RNA in the InviMag Universal Kit IG procedure depends on the sample type the virus and bacteria content sample source transport storage and age Yield and quality of isolated pathogen DNA RNA is suitable for any molecular diagnostic detection system Different amplification systems vary in efficiency depending on the total amount of nucleic acid present in the reaction Eluates from this kit contain genomic or pathogen DNA RNA and Carrier RNA whereas the amount of Carrier RNA will greatly exceed the amount of viral nucleic acids Yields of viral nucleic acids isolated from biological samples are very low and very difficult to determine photometrically Keep in mind that the Carrier RNA 5 ug per 200 ul sample will account for most of the present RNA InviMag Universal Kit IG 0314 The kit is suitable for downstream analysis with NAT techniques for examples PCR qPCR RT qPCR LAMP LCR Diagnostic assays should be performed according to the manufacturer s instructions Quantitative RT PCR is recommended for determination of viral DNA or RNA yield Note f beads are visible in the eluate transfer the eluate to a new reaction tube and centrifuge for 1 min at maximum speed e g 13000 rpm In Gel Electrophoresis and in Capillary
17. d 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 the 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 23 InviMag Universal Kit IG 0314 Waste management Please make sure that the waste tray is capacity is sufficient for your planned assay If the capacity is not sufficient empty the solid waste Note The waste is potential infectious i Waste management Waste capacity 180 Dropshal satus ini place Fill teal 0 disposables a Aires of wasted disposable sheaths O Humber oi washed disposable tips i Mirraglanes Hack aad Sour mitius Hed Figure 11 Waste management screen If you have cleaned the waste tray please use the Empty solid waste button A Batch definition Batch definition Assay descriplions suitable to loaded teagents UUNI 100 S200
18. ed 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 Name Exp date Unknown Unknown PJ124045130704391506 Lysis B HLT PJ 2015 06 30 Unknown Unknown PJ410068130604241500 Elution B M PJ 2015 06 30 PJ618025302539951506 MAP Sol B PJ 2015 06 30 PJ218068100005201500 Binding Sol PJ 2015 06 30 PJ707700230228201506 PKC PI 2015 05 30 PJ306108140110851506 Wash B II P I 2015 05 30 ve 0909990000909099 Unknown Unknown Soy seeclion Figure 6 Reagent loading screen of the InviGenius software Insert all provided reagents into the provided reagent rack of the InviGenius system Verify 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 try again slowly Assay Selection Assay selection Assay description UUNI E100 S200 suitable to loaded UUNI E200 S200 reagents No Assay Selected Version Rea
19. ents belonging to different kit types is not supported Residual magnetic particles are left centrifuge the eluate plate at full in eluate 29 speed for 1 min and transfer supernatant to a new plate tube InviMag Universal Kit G 0314 Ordering information Product InviMag Universal Kit IG Related products Invisorb Spin Blood Mini Kit Invisorb Spin Blood Mini Kit Invisorb Spin Blood Midi Kit Invisorb Spin Blood Midi Kit Invisorb Blood Mini 96 HTS C Invisorb Blood Mini 96 HTS C RTP DNA RNA Virus Mini Kit RTP DNA RNA Virus Mini Kit Invisorb Spin Virus RNA Mini Kit Invisorb Spin Virus RNA Mini Kit Invisorb Spin Virus DNA Mini Kit Invisorb Spin Virus DNA Mini Kit RTP Pathogen Kit RTP Pathogen Kit InviGenius and consumables InviGenius Starting Box I IG Sheath Box Conductive filter tips 1 ml 2 x 2 rack pack 384 pieces 5 Waste Trays 120 sample tubes Sheath Bundle Sheaths Conductive filter tips 1 ml Waste tray IG Possible suppliers for Isopropanol Carl Roth 2 Propanol Rotipuran gt 99 7 p a ACS ISO Order no 6752 Package size 8 x 12 preps Package size 50 preparations 250 preparations 50 preparations 250 preparations 4 x 96 preparations 24 x 96 preparations 50 preparations 250 preparations 50 preparations 250 preparations 50 preparations 250 preparations 50 preparations 250 preparations 1 unit 1 box 10 x 48 pie
20. es MAP Solution B and is separated from the solution by magnetic rods controlled by the InviGenius system Subsequent to three washing steps of the particle bound nucleic acids the DNA RNA is finally eluted in Elution Buffer M Due to the high purity the eluted total DNA RNA is ready to use in a broad panel of downstream applications o PCR real time PCR o Restriction Enzyme Digestion o HLA Typing For the isolation of DNA from a single blood sample STRATEC Molecular offers the Invisorb Spin Blood Mini Kit or for 8 96 samples the Invisorb Blood Mini 96 HTS Kits for use on a centrifuge vacuum manifold or other robotic station For the isolation of viral RNA from single samples or viral DNA or DNA RNA together STRATEC Molecular offers a variety of kits see page 29 For further information please contact phone 49 0 30 9489 2901 2910 in Germany and from foreign countries phone 49 0 30 9489 2907 2903 or 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 Universal Kit IG 0314 Sampling and storage of starting material For reproducible and high yields the appropriate sample storage is essential Yields may vary from sample to sample depending on factors such as health of the donor sample age kind of sample transport and storage conditions Blood Best results are obtained using fresh blood samples Blood samples
21. gent loading qe Status Ready es oo Service disa tip lesding Figure 7 Assay Selection screen of the InviGenius software 20 InviMag Universal Kit IG 0314 Select the appropriate protocol and proceed with disposable tip loading After selection of the protocol the system checks the availability of the buffers the shelf live and the buffer volume If something is wrong the system will give a respective remark Disposable Tip Loading Disposable tips loading t Tip Load Position 1 ASSay selection Status Ready User developer Figure 8 Disposable tip loading screen There are three tip rack positions on the InviGenius system Fig 8 A1 A3 Remaining tip numbers are shown in field 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 Each position can be filled either with 50 ul or 1100 ul filter tips However for the Universal assays only 1100 yl filtered tips can be used and will be shown 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
22. her switched off or used for sample processing We recommend decontaminating the instrument daily 27 InviMag Universal Kit IG 0314 Appendix General notes on handling DNA RNA Nature of DNA RNA The length and delicate physical nature of DNA RNA requires careful handling to avoid damage due to shearing and enzymatic degradation Other conditions that affect the integrity and stability of DNA RNA include acidic and alkaline environments high temperature and UV irradiation Careful isolation and handling of high molecular weight gDNA is necessary to ensure its functionality in various downstream applications Damaged DNA RNA could perform poorly in applications such as Southern blotting long template PCR and construction of cosmid libraries Handling fresh and stored material before the extraction of DNA RNA For the isolation of DNA RNA use either fresh samples or samples that have been quickly frozen in liquid nitrogen and stored at 70 C This procedure minimizes degradation of crude DNA RNA by limiting the activity of endogenous nucleases Storage of DNA RNA Store DNA RNA at 2 8 C Storing DNA RNA at 20 C can cause shearing of DNA RNA particularly if the DNA RNA is exposed to repeated freeze thaw cycles Virus RNA should be stored for long term storage at 80 C 28 InviMag Universal Kit IG 0314 Troubleshooting Problem pipetting distribution errors low concentration of extracted RNA DNA degraded RNA DNA
23. ications General overview of the InviGenius system Scheme of the InviMag Universal Kit IG Preparing the samples for processing on the InviGenius system Preparing of the internal control for the InviGenius system Preparing and loading of the InviGenius system Daily maintenance Appendix General notes on handling DNA RNA Troubleshooting Ordering information oOo O O DAN Oa a 5 BP PE N _ BB AN BR 6 Na run 206 6 InviMag Universal Kit IG 0314 Kit contents of InviMag Universal 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 Component 8 x 12 preps Catalogue No 2450120100 96 samples PKC tube Proteinase K 8 x 12 mg 40 yg 12 samples Carrier RNA final volume 800 ul in max 2 runs MAP Solution B IG 2x26ml dS amp in max 8 runs Binding Solution empty bottle 96 samples fill with 99 7 Isopropanol final volume 80 ml in max 16 runs 2 x 36 mi 48 samples Wash bimer hi final volume 2 x 120 ml 96 samples 8 runs per plate Incubator Stripe Foils Initial steps Resuspend lyophilized material in PKC Tube by addition of 800 ul RNase free Water included and mix thoroughly Add 80 ml of 99 7 Isopropanol molecular biologic grade into the empty bottle Add 84 ml of 96 100 ethanol to each bot
24. ions and enzyme inhibitors are efficiently removed during the following three washing steps O O 0 o0 Lysis Samples are lysed at elevated temperatures in the presence of Lysis Buffer HLT and Proteinase K Carrier RNA PKC tube Binding of the genomic DNA RNA After addition of Binding Solution and MAP Solution B magnetic beads to the lysate the DNA RNA is bound to the surface of the beads Removing residual contaminants Contaminants are efficiently removed while the DNA RNA remains bound to the magnetic beads Elution The DNA RNA is finally eluted in Elution Buffer M The eluted DNA RNA is ready to use in different subsequent downstream applications e g for PCR amplification digestion with restriction enzymes Southern hybridizations HLA typing etc Yield and quality of genomic DNA from Blood The amount of purified genomic DNA in the InviMag Universal Kit IG procedure from whole blood depends on the leucocytes content the sample source transport storage and age Typically a volume of a 200 ul whole blood sample from a healthy individual with a white blood cell content ranging from 3 x10 to 1x10 cells ml will yield in at least 3 ug of genomic DNA The typical yield usually expected from the InviMag Universal Kit IG is about 3 6 ug DNA If a whole blood sample is mixed with anticoagulant containing buffer solutions the overall leukocyte concentration decreases and the yield of the DNA extraction procedure is reduce
25. ivation Transfer 350 ul of the rinsed liquid into a primary tube If bacterial DNA is processed 40 ul Lysozyme not provided stock solution 10 mg ml has to be added Note This does not include any warranty for efficiency of the used cultivation method b the sample will not be used for cultivation Cut off the relevant part of the swab and transfer this part into an RNase and DNAse free 2 ml tube Add 400 ul RNase free water to the swab and vortex intensely for 3 min Optional incubate for 3 min at 95 C Transfer the rinsed liquid into a primary tube If bacterial DNA is processed 40 ul Lysozyme not provided stock solution 10 mg ml has to be added 3 Extraction of NA from sputum Transfer 200 ul from the sputum sample into an RNase DNAse free tube and add 200 ul NAC Buffer order number 1033221100 or saturated Acetylcysteine ACC solution to the sample ratio sample to buffer must be 1 1 Incubate the mixture for 10 min at 95 C to reduce the viscosity and transfer the mixture into the primary tube If bacterial DNA is processed 40 ul Lysozyme not provided stock solution 10 mg ml has to be added 4 Extraction of NA from slimy tracheal secretes or BAL Non viscous samples Transfer 1 ml of tracheal secret or BAL into a RNase DNAse free tube and centrifuge at 9 300 x g 10 000 rpm for 3 min Discard the supernatant without disturbing the bacterial pellet Resuspend the bacterial pellet in 400 ul RNase free water and transfer the s
26. ll only be unlocked after a run has been successfully finished or if an error occurs 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 BBO2E6 User interacton Assay descripton Basic 01 01 00003 request Actual step lt Remaining me 00 0000 Process state ie Baich checking i Status Ready a L developer Figure 14 Batch processing screen At the end of the process the nucleic acid containing elates are located in the appropriate eluate position and can be used for any further downstream application Note The complete process will take approximately 70 minutes 58 InviMag Universal Kit IG 0314 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 deconta
27. mination go to the main menu of the InviGenius software and select Maintenance Main Menu InviGenius Logout rocessm gt Shusdovn Administration Status Ready Se developer Figure 15 Main screen of the InviGenius software When the sub item Maintenance is opened select UV decontamination Maintenance GRD decontamination Periodical maintenance Diaqgnoslics Li Cleanup procedures Cleanup drop catcher Back Status Ready m ce service Figure 16 Maintenance screen of the InviGenius software 38 InviMag Universal Kit IG 0314 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 A Duration 00 15 00 9 HHMM ss Remaining me 00 00 00 UV decontaminaton status There was no UV decontamination B completed x GB Status Ready mm 0 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 eit
28. no Venosafe 5 5ml Ref VF 076SDK Terumo Vacuette 2ml Ref A1105001 Greiner bio one Vacuette 9ml Ref 455036 Greiner bio one BD Vacutainer 2 7 ml Ref 363048 BD Vacutainer 6 ml Ref 367864 BD Vacutainer 10 ml Ref 367525 BD Vacutainer 5 0 ml Re Sarstedt Monovette 8 5m PS Tube Sarstedt 5ml Ref 55 476 Sarstedt Monovette 4 5 m Sarstedt Monovette 7 5 m Sarstedt Monovette 9 0 ml Or Q OQ Q9 9 FOse OO OO Possible supplier for lysozyme Fa Applichem Lysozyme freeze dried Order No A4972 0010 InviMag Universal Kit IG 0314 Important indications 1 Minimum volume of samples in primary tubes Please provide at least 400 ul sample in the primary tubes The procedure of the InviMag Universal Kit IG is optimized for the isolation of DNA RNA from up to 200 pl UUNI E100 S200 with E100 100 ul elution volume S200 200 ul sample volume UUNI E200 S200 with E200 200 yl elution volume S200 200 ul sample volume The dead volume is dependent on sample tube If the InviGenius detects a low sample volume 400 ul it will aspirate 200 ul sample from the tube bottom and informs the user with the warning Too low volume detected in sample container XX This feature is a remark to check the sample tube after the run for a correct sample transfer 2 Sample volume smaller than 400 ul Please adjust the sample volume to at least 400 ul Samples lower than 400 ul are flagged in the result report see point 1
29. on of DNA and RNA from fresh or frozen samples like human blood serum plasma cerebrospinal fluid cell culture supernatants and other cell free body fluids rinsed liquid from swabs urine supernatant from stool suspension BAL sputum etc Targets are nucleic acids from viruses genomic DNA from blood and bacterial DNA For efficient extraction appropriate sample storage is essential see Sampling and storage of the starting material page 8 Fresh or frozen blood plasma or serum treated with EDTA or citrate not with heparin The procedure of the InviMag Universal Kit IG is optimized for the isolation of nucleic acids from up to 200 ul starting material However we advise to provide at least 400 ul sample per a 12 mm diameter tube to prevent pipetting distribution errors The final processed sample volume is 200 yl 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
30. placed on position B1 B3 and disposable sheaths on F The waste tray C is located on the lower side of the InviGenius system behind the red flap The waste shaft D is completely stainless steel and easy to remove for autoclaving The loading bay is divided into sample loading bay J1 and reagent loading bay J2 The magnetic separator head MSH H is located on top of the incubator I and can reach all necessary positions The single head pipettor G starting positions are in the right front of the machine All movable parts only work when the InviGenius machine is closed and locked InviMag Universal Kit IG 0314 Preparing and loading of the InviGenius system Preparing the reagents Before you start a new kit add ethanol to Wash Buffer II Before starting a new run dissolve or unfreeze one vial of PKC with 800 ul RNase free water Preparing the system Switch on the InviGenius system using the power switch located on the right side of the back part of the instrument The InviGenius software will be automatically loaded after the system has booted up Please keep the door of the InviGenius system closed during system initialization After successful initialization of the InviGenius system a login screen appears Figure 2 Login use the provided user name and password Login User identifier Password Status Ready a m Figure 2 Login screen of the InviGenius software After logging in the main s
31. 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 sample 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 sample identification remove the rack check the bar code orientation and reinsert the rack slowly It is InviMag Universal Kit IG 0314 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 proce
32. ree 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 Universal 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 Universal 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 o 49 0 30 9489 2907 or contact your local distributor InviMag Universal Kit IG 0314 Intended use The InviMag Universal Kit IG is designed for fully automated extraction and purification of DNA and or RNA from 12 samples per run by using magnetic beads in combination with the InviGenius system Common collection tubes can be used to assemble a set of samples All utilities reagents and plastics except filter tips necessary for preparation of nucleic acids are provided by the InviMag Universal Kit IG The nucleic acid isolation protocol is suitable for routinely walk away automated preparati
33. stabilized with EDTA or citrate but not heparin can be stored at room temperature 18 25 C for 2 3 hours For short time storage up to 24 h samples should be stored at 4 8 C For long term storage we recommend to freeze the samples at 20 C or 80 C Avoid multiple thawing and freezing cycles of the sample s before isolating the DNA RNA Various different primary tubes blood collection system e g Sarstedt Greiner and anticoagulants except heparin can be used to collect blood samples for the InviMag Universal Kit IG procedure Serum and plasma After collection and centrifugation serum plasma 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 on ice for 1 2 hours For short time up to 24 h samples may be stored at 20 C For long term storage we recommend freezing samples in aliquots at 80 C Frozen plasma or serum samples must not be thawed more than once Multiple thawing and freezing before isolating any viral DNA RNA should be avoided It can lead to denaturation and precipitation of proteins resulting in reduced viral titers and yields of viral nucleic acids In addition cryoprecipitates formed during freeze thawing could cause problems If cryoprecipitate is visible they should be pelleted by centrifugation at app 6 800 x g for 3 minutes The cleared supernatant should be aspirated without dis
34. tains guanidine hydrochloride H302 315 319 P280 505 351 338 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 clothing eye protection face protection P305 P351 P338 IF IN EYES Rinse cautiously with water for several minutes Remove contact lenses if present and continue rinsing P310 Immediately call a POISON CENTER or doctor physician P405 Store locked up P210 Keep away from heat sparks open flames hot surfaces No smoking 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 InviMag Universal Kit IG 0314 Product characteristics of the InviMag Universal Kit IG The InviMag Universal Kit IG is the ideal tool for an efficient and fully automated DNA extraction and purification from fresh or frozen whole blood samples or DNA RNA from fresh or frozen plasma serum using magnetic beads in combination with the InviGenius robotic platform Starting Material Time for preparation depending on sample storage about 70 min per run 200 ul blood EDTA citrate stabilized but not and source 12 samples heparin Note 200 ul fresh or frozen blood 200 ul fresh or frozen plasma serum
35. 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 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 24 InviMag Universal Kit IG 0314 Disposable Sheaths Loading The sheaths are used as protection devices for the magnetic rods The sheaths are picked up automatically during the run Disposable sheaths loading Sheath Load Position pe Msesoplates Disp amp p loading loading Status Ready amm mm 0 developer Figure 9 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 contamin
36. tle Wash Buffer Il Mix thoroughly and always keep the bottle firmly closed InviMag Universal Kit IG 0314 Symbols bill Manufacturer LOT Lot number m m Tm Catalogue number Date of manufacture Expiry date Consult operating instructions Temperature limitation E E Do not reuse Storage All buffers and kit contents of the InviMag Universal Kit IG except the PKC and MAP Solution B Tube should be stored at room temperature and are stable for at least 12 months PKC Tube Lyophilized Proteinase K Carrier RNA should be stored at 2 8 C It must be stored at 20 C when dissolved in RNase free water MAP Solution B The magnetic micro particles should be stored at 2 8 C Wash Buffer Il Wash Buffer Il 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 Universal 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 STRATEC Molecular investigates a problem in the lot the product will be replaced f
37. to use for a broad panel downstream applications or can be stored at 20 C for subsequent use C Compliance with EU Directive 98 79 EC on in vitro medical devices Not for in vitro diagnostic use in countries where the EU Directive 98 79 EC on in vitro medical devices is not recognized 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 and 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 2014 STRATEC Molecular all rights reserved InviMag Universal Kit G 0314 Contents Kit contents of InviMag Universal Kit IG Symbols Storage Quality control and product warranty Intended use Product use limitation Safety information Product characteristics of the InviMag Universal Kit IG Sampling and storage of starting material Principle and procedure Yield and quality of genomic DNA RNA Yield and quality of pathogen DNA RNA Important notes Preparing reagents and buffers Reagents and equipment to be supplied by user Important ind
38. turbing the pellet and processed immediately This step will not reduce viral titers Swabs The protocol works with rinsed liquids from fresh prepared swabs as well as with dried swabs The protocol has not been validated for isolation of RNA from swabs which are stored in storage buffers of other providers Cultivated bacteria or bacterial suspension s Bacteria have to be centrifuged after cultivation and resuspended in defined conditions Best results are obtained with fresh material Urine The bacteria should be pelleted while the supernatant is discarded urea contaminations can inhibit PCR reactions For some application fresh urine can be used directly Best results are obtained with freshly pelleted material Stool samples Best results are obtained with fresh material Stool samples contain DNases and RNases which quickly lead to digestion and degradation of nucleic acids These samples should be stored at 80 C STRATEC Molecular will not take responsibility if other sample types than described above are used or if the prepared sample preparation advices are modified InviMag Universal Kit G 0314 Principle and procedure The InviMag Universal Kit IG procedure comprises following steps lysis and protein digestion binding of the DNA RNA to the magnetic beads washing the bead bound DNA RNA and elimination of alcohol elution of DNA RNA After lysis the nucleic acids bind to the magnetic beads whereas contaminat
39. y incubation at 30 C Swirl gently to avoid foaming Lysis Buffer HLT MAP Solution B and Elution Buffer M are ready to use 8 x 12 DNA RNA extractions Resuspend the lyophilized mixture of the PKC Tubes by adding of 800 ul of the provided RNase free H O and mix thoroughly Store diluted Proteinase K Carrier RNA PKC Tube at 20 C Add 80 ml of 99 7 Isopropanol molecular biologic grade into the empty bottle labeled with Binding Solution Add 84 ml of 96 100 ethanol to each bottle Wash Buffer Il Mix thoroughly and always keep the bottles firmly closed InviMag Universal Kit IG 0314 Reagents and equipment to be supplied by user When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information please consult the appropriate material safety data sheets MSDS See our webpage www stratec com Measuring cylinder 250 ml Pipette tips Disposable gloves ddH O Vortex 96 100 ethanol 99 7 96 Isopropanol molecular biological grade Q O OO 0 80 The InviMag Universal Kit IG is validated with 2 Propanol Rotipuran 799 796 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 LA Order No 6752 Order No 59304 1L F Possible primary tubes manufacturer cat
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