InviMag Virus DNA/RNA Mini Kit User manual
Contents
1. stratecee molecular User manual InviMag Virus DNA RNA Mini Kit optimized for the InviMag Rack for purification of viral DNA and RNA from up to 200 ul serum plasma cell culture supernatant and other cell free body fluids biopsy samples and swab with magnetic beads 1440100x0 anal STRATEC Molecular GmbH D 13125 Berlin lt Instruction InviMag Virus DNA RNA Mini Kit The InviMag Virus DNA RNA Mini Kit is the ideal tool using a combination of InviMag and RTP technologies on a magnetic rack for simultaneous isolation of high quality viral DNA and RNA from serum plasma cerebrospinal fluid cell culture supernatants and other cell free body fluids urine as well as from swabs or tissue biopsies for in vitro diagnostic use Fresh or frozen plasma or serum from blood treated with anticoagulants like EDTA or citrate but not with heparin The customer convenient RTP technology simplifies the process handling reduces the handling steps with infectious material and allows a process monitoring Furthermore viral DNA can be isolated from plant material The kit is neither validated for the isolation of genomic DNA nor for the isolation of total RNA 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 RTP Invisorb Eppendort Registered m2. Buffer R1 mix thoroughly and always keep the bottle firmly closed Add 48 ml of 96 100 ethanol to each bottle Wash Buffer R2 mix thoroughly and always keep the bottle firmly closed isopropanol molecular biologic grade into the empty bottle Add 125 ml of 96 100 ethanol to the bottle Wash Buffer R1 mix thoroughly and always keep the bottle firmly closed Add 200 ml of 96 100 ethanol to each bottle Wash Buffer R2 mix thoroughly and always keep the bottle firmly closed 3 InviMag Virus DNA RNA Mini Kit 0113 Symbols LOT Lot number Catalogue number Date of manufacture Expiry date Consult operating instructions Temperature limitation Do not reuse Ox HKE E Storage All buffers and kit components except the dissolved Proteinase K and the MAP Solution A should be stored well sealed and dry at room temperature RT and are stable for at least 12 months under these conditions MAP Solution A is stable for at least 6 months MAP Solution A should be stored at 4 C Proteinase K Dissolved Proteinase K must be stored at 20 C Dividing Proteinase K into aliquots and storage at 20 C is recommended Wash Buffer charged with ethanol should be stored at room temperature and should be appropriately sealed If there are any precipitates within the provided solutions dissolve these precipitates by carefully warming up to room temperature up to 30 C Room temperature is de
3. The prefilled buffer and enzymes lyse the samples stabilize the viral nucleic acids After the lysis under higher temperature and shaking binding buffer and magnetic beads are transferred to the lysate to enhance the selective viral RNA and DNA adsorption to magnetic particles followed by washing steps and the final elution The procedure requires minimized interaction by the user allowing safe handling of potentially infectious samples The procedures are designed to avoid sample to sample cross contamination The advantage of the kit results from the simultaneous isolation of nucleic acids from DNA and RNA viruses That enables the user to test the sample for all kinds of RNA and DNA viruses which are of interest after one preparation Yield and quality of isolated viral DNA and RNA is suitable for any molecular diagnostic detection system The diagnostic tests should be performed according to manufacturer s specifications Due to the high purity the isolated viral DNA RNA is ready to use for a broad panel of downstream applications or can be stored at 80 C for subsequent use o RT PCR o PCR o Real time PCR for quantitative and qualitative virus diagnostic No toxic or hazardous chemicals like chaotropic components are used For the isolation of viral nucleic acids with spin filters from 200 ul starting material STRATEC Molecular offers the RTP DNA RNA Virus Mini Kit and the Invisorb Spin Virus RNA Mini Kit The PCR proc
4. RNA yield Use of poor quality starting material influences the RNA yield too The amount of purified RNA in the InviMag Virus DNA RNA Mini Kit procedure using up to 20 mg tissue sample depends on kind of starting material The thawing process could be proceed e g directly in the Extraction Tubes Cell culture supernatants Best results are obtained with fresh material or material that has been immediately frozen and stored at 20 C or 80 C after winning of the cell culture supernatant Repeated freezing and thawing of stored samples should be avoided can influence the sensitivity Swabs The protocol works with fresh prepared swabs or rinsed liquid from swabs or mouth brushes Best results are obtained with fresh material or material that has been immediately frozen and stored at 20 C or 80 C 8 InviMag Virus DNA RNA Mini Kit 0113 Principle and procedure The InviMag Virus DNA RNA Mini Kit procedure comprises following steps o Lysis of the virus particles in the prefilled Extraction Tube o Binding the viral NA to the magnetic beads o Washing of the magnetic beads and elimination of ethanol o Elution of the viral nucleic acids After lysis the viral NA bind to the magnetic beads contaminations and enzyme inhibitors are efficiently removed during the following three wash steps and highly purified DNA RNA is eluted in Elution Buffer R or water This manual contains 4 protocols Lysis Samples are lysed un
5. the sample by pipetting up and down for 4 5 times Note Vortex the tube MAP Solution A vigorously before use 5 Incubate for 5 min the mixture at RT Then insert the Magnetic Stripe and incubate for further 5 min at RT Remove supernatant by aspiration or by pipetting then remove the Magnetic Stripe 6 Add 800 ul Wash Buffer R1 to the vial Close the vial and mix it by pipetting up and down Incubate for 3 min in the Magnet Rack with inserted Magnetic Stripe Remove supernatant then remove the Magnetic Stripe 7 Add 800 ul Wash Buffer R2 to the vial Close the vial and mix it by pipetting up and down Incubate for 3 min in the Magnet Rack with inserted Magnetic Stripe Remove supernatant then remove the Magnetic Stripe Repeat once this Step 8 Insert the Magnetic Stripe and open the lid of the tube for 10 min to evaporate the ethanol completely 9 Remove the Magnetic Stripe then add 120 ul of the Elution Buffer R mix by pipetting up and down for three times Incubate at room temperature for 5 min 10 Insert the Magnetic Stripe and incubate for further 3 min Transfer the eluate to the 1 5 ml Elution Tube centrifuge the tube for 3 min at 10 000 x g and transfer the supernatant to a new 1 5 ml Elution Tube If no centrifuge is available please put the vial again in the Magnet Rack insert the Magnetic Stripe and incubate for further 5 min at RT Transfer the supernatant in a new 1 5 ml Elution Tube It contains the viral nu
6. the vial and mix it by pipetting up and down Incubate for 3 min in the Magnet Rack with inserted Magnetic Stripe Remove supernatant and then remove the Magnetic Stripe 8 Add 800 ul Wash Buffer R2 to the vial Close the vial and mix it by pipetting up and down Incubate for 3 min in the Magnet Rack with inserted Magnetic Stripe Remove supernatant and then remove the Magnetic Stripe Repeat once this Step 9 Insert the Magnetic Stripe and open the lid of the tube for 10 min to evaporate the ethanol completely 10 Remove the Magnetic Stripe then add 120 ul of the Elution Buffer R mix by pipetting up and down for three times Incubate at room temperature for 5 min 11 Insert the Magnetic Stripe and incubate for further 3 min Transfer the eluate to the 1 5 ml Elution Tube centrifuge the tube for 3 min at 10 000 x g and transfer the supernatant to a new 1 5 ml Elution Tube If no centrifuge is available please put the vial again in the Magnet Rack insert the Magnetic Stripe and incubate for further 5 min at RT Transfer the supernatant in a new 1 5 ml Elution Tube It contains the viral nucleic acids ready to use Important Notes 1 The viral nucleic acids can also be eluted with a lower volume of Elution Buffer R depends on the expected amounts of viral nucleic acids But pay attention that minimum volume for the elution is 30 ul and that this volume can reduce the maximum yield If quite large amount of viral nucleic acids a
7. 0113 Intended use The InviMag Virus DNA RNA Mini Kit is designed for a reliable and fast simultaneous isolation of high quality viral DNA and RNA from a wide range of clinical samples from fresh or frozen human or mammalian serum plasma cerebrospinal fluid cell culture supernatants and other cell free body fluids as well as from swabs or tissue biopsies using the RTP technology magnetic beads and a magnetic tool For reproducible high yields an appropriate sample storage and quick operation under the rules for RNA and DNA operation is essential The purified viral DNA and or RNA are ready to use for in vitro diagnostic analysis The isolation protocols as well as all buffers are optimized to provide high yield and purity of the extracted viral nucleic acids The procedure requires minimal interaction by the user allowing safe handling of potentially infectious samples THE PRODUCT IS INDENTED FOR USE BY PROFESSIONAL USERS ONLY 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 diagno
8. Protocol 3 Simultaneous isolation of total nucleic acids viral DNA and RNA from tissue biopsies 15 Protocol 4 Simultaneous isolation of total nucleic acids viral DNA and RNA from plant tissue 16 Troubleshooting 17 Appendix 18 General notes on handling DNA 18 General notes on handling RNA 19 Ordering information 20 2 InviMag Virus DNA RNA Mini Kit 0113 Kit contents of InviMag Virus DNA RNA Mini Kit Store the MAP Solution A at 4 C Store dissolved Proteinase K at 20 C Store all other kit components at room temperature RT 10 extractions 50 extractions 250 extractions Catalogue No 1440100900 1440100200 1440100300 Extraction Tubes 10 50 5 x 50 MAP Solution A 250 ul 1 1 ml 5 5 ml Binding Solution 5x1 ml empty bottle empty bottle fill with 99 7 Isopropanol ready to use final volume 30 ml final volume 120 ml Elution Buffer R 2 ml 15 ml 60 ml 15 ml 30 ml 125 ml wash Butter Ri ready to use final volume 60 ml final volume 250 ml 2x15 ml 2x 12 ml 2x 50 ml Wash Buffer R2 ready to use final volume 2 x 60 ml final volume 2 x 250 ml Elution Tubes 20 2x 50 10 x 50 Manual 1 1 1 Initial steps Wash Buffer R1 is ready Fill 30 ml 99 7 Fill 120 ml 99 7 to use Wash Buffer R2 is ready to use Binding Solution is ready to use isopropanol molecular biologic grade into the empty bottle Add 30 ml of 96 100 ethanol to the bottle Wash
9. Tube Internal Controls The use of an internal control is recommended when using the InviMag Virus DNA RNA Mini Kit in combination with diagnostic amplification systems STRATEC Molecular provides the needed Control DNA or RNA Detection Assays Eluting viral RNA and DNA For downstream applications that require small starting volumes elution volume may be reduced to 40 ul Elution Buffer R The volume of eluate recovered may be up to 5 ul less than the volume of elution buffer applied to the RTA Spin Filter The volume of eluate recovered depends on the nature of the sample 10 InviMag Virus DNA RNA Mini Kit 0113 Preparing buffers and reagents 10 viral DNA RNA extractions Binding Solution Wash Buffer R1 and R2 are ready to use 50 viral DNA RNA extractions Fill 30 ml 99 7 Isopropanol molecular biologic grade into the empty bottle Add 30 ml 96 100 ethanol to the bottle Wash Buffer R1 Add 48 ml 96 100 ethanol to each bottle Wash Buffer R2 250 viral DNA RNA extractions Fill 120 ml 99 7 Isopropanol molecular biologic grade into the empty bottle Add 125 ml 96 100 ethanol to each bottle Wash Buffer R1 Add 200 ml 96 100 ethanol to each bottle Wash Buffer R2 Special instruction for samples with high virus titer When using high titer samples please remember that depending on virus the system has an upper capacity limit It may be necessary to dilute the sample in advance of the preparation Samples with titer h
10. acted viral RNA DNA We recommend storage of the DNA at 20 or 80 especially for RNA 15 InviMag Virus DNA RNA Mini Kit 0113 Protocol 4 Simultaneous isolation of total nucleic acids viral DNA and RNA from plant tissue Please read the instructions carefully and conduct the prepared procedure 1 Homogenization of 10 mg plant material with mortar and pistil under liquid nitrogen or ina mixer mill Transfer the homogenized plant material into the Extraction Tube Add 400 ul of ddH2O Close the cap and mix by vortexing for 10 s 2 Place the Extraction Tube into a Thermomixer and incubate under continuously shaking for 15 minutes at 65 C Lysis time can be increased up to 30 min A longer lysis time could reduce the final yield and the quality of some viral RNA species 3 Place the Extraction Tube into another Thermomixer and incubate under continuously shaking for 10 minutes at 95 C Spin down unlysed material 1 min at 10 000 x g Transfer the Extraction Tube with the lysate in the Magnet Rack without magnet and add 400 ul Binding Solution and 20 ul MAP Solution A and mix the sample by pipetting up and down for 4 5 times Note Vortex the tube MAP Solution A vigorously before use 6 Incubate for 5 min the mixture at RT Then insert the Magnetic Stripe and incubate for further 5 min at RT Remove supernatant by aspiration or by pipetting then remove the Magnetic Stripe 7 Add 800 ul Wash Buffer R1 to the vial Close
11. applications e g real time PCR or PCR ethanol carryover during elution salt carryover during elution increase drying time for removing of ethanol check up the Wash Buffers for salt precipitates If there are any precipitates solve these precipitates by careful warming ensure that the Wash Buffers are at room temperature low A260 A2g0 ratio from UV measurement eluted DNA is brown colored small part of the magnetic particles are left in the elution centrifuge down at full speed for 1 min and transfer supernatant to a new tube 17 InviMag Virus DNA RNA Mini Kit 0113 General notes on handling viral DNA 1 Nature of DNA The length and delicate physical nature of DNA requires careful handling to avoid damage due to shearing and enzymatic degradation Other conditions that affect the integrity and stability of DNA include acidic and alkaline environments high temperature and UV irradiation Careful isolation and handling of high molecular weight DNA is necessary to ensure compatibility with various downstream applications Damaged DNA could perform poorly in applications such as long template PCR 2 Storage of DNA A working stock of DNA can be stored at 2 4 C for several days For long term storage DNA should be stored at 20 C but storing at 20 C can cause shearing particularly if the DNA is exposed to repeated freeze thaw cycles Note that the solution in which the nucleic ac
12. arks 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 RTP and Invisorb are registered trademarks of STRATEC Biomedical AG The PCR process is covered by US Patents 4 683 195 and 4 683 202 and foreign equivalents owned by Hoffmann La Roche AG 2013 STRATEC Molecular all rights reserved 1 InviMag Virus DNA RNA Mini Kit 0113 Contents Kit contents of InviMag Virus DNA RNA Mini Kit 3 Symbols 4 Storage 4 Quality control 4 Intended use 5 Product use limitation 5 Safety information 6 Product characteristic of InviMag Virus DNA RNA Mini Kit 7 Sample storage 8 Principle and procedure 9 Internal control IC Extraction control 9 Important notes 10 Important points before starting a protocol 10 Important indication 10 Preparing reagents and buffers 11 Special instruction for samples with high virus titer 11 Equipment and reagents to be supplied by user 11 Scheme 12 Protocol 1 Simultaneous isolation of total nucleic acids viral DNA and RNA from cell free body fluids serum plasma synovia urine and from cell culture supernatants 13 Protocol 2 Simultaneous isolation of total nucleic acids viral DNA and RNA from swab material 14
13. be autoclaved or heated to 100 C for 15 min to remove residual DEPC o Electrophoresis tanks should be cleaned with detergent solution e g 0 5 SDS thoroughly rinsed with RNase free water and then rinsed with ethanol and allowed to dry o Non disposable plasticware should be treated before use to ensure that it is RNase free Plasticware should be thoroughly rinsed with 0 1 M NaOH 1 mM EDTA followed by RNase free water You can also take chloroform resistant plasticware rinsed with chloroform to inactivate RNases o All buffers must be prepared from DEPC treated RNase free ddH20 When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles Change gloves frequently and keep tubes closed All centrifugation steps are carried out at room temperature To avoid cross contamination cavity seams shouldn t be moisten with fluid Reduce the preparation time as much as possible Use only sterile disposable polypropylene tubes throughout the procedure These tubes are generally RNase free Keep isolated RNA on ice o Do not use kit components from other kits with the kit you are currently using unless the lot numbers are identical oO000 0 O O This kit should only be used by personnel trained in in vitro diagnostic laboratory practice Storage of viral RNA Purified viral RNA can be stored 80 C and is stable for months and years e g precipated and stored in 70 ethanol 19 Inv
14. cleic acids ready to use Important Notes 1 The viral nucleic acids can also be eluted with a lower volume of Elution Buffer R depends on the expected amounts of viral nucleic acids But pay attention that minimum volume for the elution is 30 ul and that this volume can reduce the maximum yield If quite large amount of viral nucleic acids are expected the volume of Elution Buffer can be increased up to 200 ul 2 After finishing the extraction protocol the Elution Tube contains the extracted viral RNA DNA We recommend storage of the DNA at 20 or 80 especially for RNA 13 InviMag Virus DNA RNA Mini Kit 0113 Protocol 2 Simultaneous isolation of total nucleic acids viral DNA and RNA from swabs or 200 ul rinse liquid Please read the instructions carefully and conduct the prepared procedure See also Special instruction for samples with high virus titer page 12 1a Place the swab into the Extraction Tube and add 400 ul of ddH20 Close the cap and mix by vortexing for 10 s 1b Transfer 400 ul of rinse liquid or of the stabilization media into the Extraction Tube The stabilization medium shouldn t contain alcohol Close the cap mix by vortexing for 10 s 2 Place the Extraction Tube into a Thermomixer and incubate under continuously shaking for 15 minutes at 65 C 3 Place the Extraction Tube into another Thermomixer and incubate under continuously shaking for 10 minutes at 95 C These step leads to higher sensit
15. der denaturing conditions at elevated temperatures Binding of the viral nucleic acids After adding Binding Solution and MAP Solution A to the lysate in the Extraction Tube the nucleic acids are bound to the surface of the magnetic beads Removing residual contaminants Contaminants are efficiently washed away using Wash Buffer R1 and R2 while the nucleic acids remain bound to the magnetic beads Elution The nucleic acids are eluted from the beads using 120 ul Elution Buffer R The eluted nucleic acids are ready for use in different subsequent tests Yield and quality of viral DNA RNA Different amplification systems vary in efficiency depending on the total amount of nucleic acid present in the reaction Eluates from this kit contain viral RNA DNA and Carrier RNA and amounts of Carrier RNA will greatly exceed amounts of viral nucleic acids Yields of viral RNA DNA isolated from biological samples are normally less than 1 ug and therefore difficult to determine photometrically Keep in mind that the Carrier RNA 5 ug per 200 ul sample will account for most of the RNA present The kit is suitable for downstream analysis with NAT techniques for examples 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 RNA yield In Gel Electrophoresis and in Capillary Electrophoresis RNA extracted with the provided kit looks lik
16. ds removal of lysed material add 800 ul Wash Buffer R1 washing of the particle fixed viral NA magnetic separation of beads removal of the wash buffer eluate add 800 ul Wash Buffer R2 washing of the particle fixed viral NA magnetic separation of beads removal of the wash buffer eluate repeat this step add 120 ul Elution Buffer R magnetic separation of beads transfer of the eluate in a new 1 5 ml Receiver Tube pure viral RNA or DNA 12 InviMag Virus DNA RNA Mini Kit 0113 Protocol 1 Simultaneous isolation of total nucleic acids viral DNA and RNA from cell free body fluids serum plasma CSF synovia urine and from cell culture supernatant cell culture media Please read the instructions carefully and conduct the prepared procedure See also Special instruction for samples with high virus titer page 12 1 Transfer 200 ul of the sample serum plasma CSF synovia urine cell culture into the Extraction Tube and add 200 ul of ddH2O Close the cap and mix by vortexing for 10 s 2 Place the Extraction Tube into a Thermomixer and incubate under continuously shaking for 15 minutes at 65 C 3 Place the Extraction Tube into another Thermomixer and incubate under continuously shaking for 10 minutes at 95 C These step leads to higher sensitivity for some viral strains 4 Transfer the Extraction Tube in the Magnet Rack without magnet and add 400 ul Binding Solution and 20 ul MAP Solution A and mix
17. e 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 Internal control IC Extraction control Internal Controls IC from the PCR assay provider can be used as extraction controls if the fragments are longer than 100 bp In this case they have to be added after finalization of the lysis step Attention don t add directly these Internal Controls to the sample 9 InviMag Virus DNA RNA Mini Kit 0113 Important notes Important points before starting a protocol Immediately upon receipt of the Product inspect the Product and its components as well as the package for any apparent damages correct quantities and quality If there are any unconformities you have to 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 Always change pipet tips between liquid transfers To avoid cross contamination we recommend the use of aerosol barrier pipet tips o All centrifugation steps are carried out at room temperature When working with chemicals always wear a Suitable lab coat disposable gloves and protective goggles Discard gl
18. ecular immediately upon detection thereof The chemicals and the plastic parts are for laboratory use only they must be stored in the laboratory and must not be used for purposes other than intended The Product with its contents is unfit for consumption 5 InviMag Virus DNA RNA Mini Kit 0113 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 liquid waste generated by the InviMag Virus DNA RNA Mini Kit procedures for residual infectious materials Contamination of the liquid waste with residual infectious materials is highly unlikely but cannot be excluded completely Therefore liquid waste must be considered infectious and be handled and discarded according to local safety regulations European Community risk and safety phrases for the components of the InviMag Virus DNA RNA Mini Kit to which they apply are listed below as follows Wash Buffer R1 contains guanid
19. ess is covered by US Patents 4 683 195 and 4 683 202 and foreign equivalents owned by Hoffmann La Roche AG 7 InviMag Virus DNA RNA Mini Kit 0113 For the isolation of viral nucleic acids using magnetic particle in high throughput format STRATEC Molecular offers the InviMag Virus Kits KFmL and the InviMag Virus Kits KF96 for use on a KingFisher mL and 96 robot For vacuum or centrifuge based isolation of viral NA in 96 well format on different robotic stations STRATEC Molecular offers the Invisorb DNA Virus HTS 96 Kit and the Invisorb RNA Virus HTS 96 Kit see ordering information page 19 For further information please contact Phone 49 0 30 9489 2901 2910 in Germany and from foreign countries phone 49 0 30 9489 2907 or your local distributor Sampling and storage of starting material Best results are obtained using freshly extracted samples As long as the samples are not shock frosted with liquid nitrogen or are incubated with RNase inhibitors or denaturing reagents the viral RNA is not secured Therefore it is essential that samples are immediately flash frozen subsequent to the harvesting by using liquid nitrogen and are stored at 80 C Viral RNA contained in such deep frozen samples is stable for months Viral RNA purification should be processed as soon as possible Samples can also be stored in the dissolved Lysis Buffer in the Extraction Tubel or II for 1 h at room temperature overnight at 4 C and for long ter
20. fined as range from 15 30 C Quality control and product warranty STRATEC Molecular warrants the correct function of the InviMag Virus DNA RNA Mini Kit 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 STRATEC Molecular will replace the Product free of charge STRATEC Molecular reserves the right to change alter or modify any Product to enhance its performance and design at any time In accordance with STRATEC Molecular s ISO 9001 2000 and ISO EN 13485 certified Quality Management System the performance of all components of the InviMag Virus DNA RNA Mini Kit have been tested separately against predetermined specifications routinely on lot to lot to ensure consistent product quality If you have any questions or problems regarding any aspects of InviMag Virus DNA RNA Mini Kit 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 2907 or contact your local distributor 4 InviMag Virus DNA RNA Mini Kit
21. iMag Virus DNA RNA Mini Kit 0113 Ordering information Product Package size Catalogue No InviMag Virus DNA RNA Mini Kit 50 preparations 1440100200 InviMag Virus DNA RNA Mini Kit 250 preparations 1440100300 Related products InviMag Virus DNA RNA Mini Kit KEmL 15 preparations 2441150100 InviMag Virus DNA RNA Mini Kit KFmL 75 preparations 2441150200 InviMag Virus DNA RNA Mini Kit KEmL 300 preparations 2441150400 InviMag Virus DNA RNA Mini Kit KF96 1 x 96 preparations 7441050100 InviMag Virus DNA RNA Mini Kit KF96 5 x 96 preparations 7441050200 InviMag Virus DNA Kit KF96 1 x 96 preparations 7440300100 InviMag Virus DNA Kit KF96 5 x 96 preparations 7440300200 InviMag Virus RNA Kit KF96 1 x 96 preparations 7443300100 InviMag Virus RNA Kit KF96 5 x 96 preparations 7443300200 RTP DNA RNA Virus Mini Kit 50 extractions 1040100200 RTP DNA RNA Virus Mini Kit 250 extractions 1040100300 Invisorb Virus DNA HTS 96 Kit X 4 x 96 preparations 7142310300 Invisorb Virus DNA HTS 96 Kit X 24 x 96 preparations 7142310400 Invisorb Virus RNA HTS 96 Kit X 4 x 96 preparations 7143310300 Invisorb Virus RNA HTS 96 Kit X 24 x 96 preparations 7143310400 Single Components for InviMag Virus DNA RNA Mini Kit Extraction Tubes 10 Tubes 7440301500 MAP Solution A 1 ml 7440305100 Elution Buffer R 30 ml 7440304100 Wash Buffer R1 add 80 ml ethanol 80 ml 7440303500 Wash Buffer R2 add 160 ml ethanol 40 ml 7440303600 Possib
22. id is eluted in will affect it s stability during storage Pure water lacks buffering capacity and an acidic pH may lead to acid hydrolysis Tris or Tris EDTA buffer contains sufficient buffering capacity to prevent acid hydrolysis 3 DNA yield The amount of purified viral DNA depends on sample source transport conditions storage and age of the sample 18 InviMag Virus DNA RNA Mini Kit 0113 General notes on handling viral RNA RNA is far less stable than DNA It is very sensitive to degradation by endogenous RNases in the biological material and exogenous RNases which are permanently present everywhere in the lab To achieve satisfactory qualitative and quantitative results in RNA preparations contaminations with exogenous RNases has to be reduced as much as possible Avoid handling bacterial cultures cell cultures or other biological sources of RNases in the same lab where the RNA purification is to be carried out All glassware should be treated before use to ensure that it is RNase free Glassware should be cleaned with detergent thoroughly rinsed and oven baked at 240 C for four or more hours before use Autoclaving alone will not completely inactivate many RNases Oven baking will both inactivate RNases and ensure that no other nucleic acids such as Plasmid DNA are present on the surface of the glassware You can also clean glassware with 0 1 DEPC diethyl pyrocarbonate The glassware must stand 12 hours at 37 C and then
23. igher than 10 copies ul may be at this limit but please remember that this depends on virus strain Reagents and equipment to be supplied by user Measuring cylinder 250 ml Pipette and pipette tips Disposable gloves Reaction tubes 1 5 ml or 2 0 ml ddH2O Vortexer 96 100 Ethanol Isopropanol O70 70 TO OTO v0 O The InviMag Virus DNA RNA Mini Kit is validated with 2 Propanol Rotipuran gt 99 7 p a ACS ISO Order no 6752 from Carl Roth Possible suppliers for lsopropanol Fa Carl Roth 2 Propanol Fa Applichem Fa Sigma Rotipuran gt 99 7 p a ACS ISO 2 Propanol f r die Molekularbiologie 2 Propanol Order no 6752 Order no A3928 Order no 59304 1L F 11 InviMag Virus DNA RNA Mini Kit 0113 Scheme of the InviMag Virus DNA RNA Mini Kit Please work quickly and perform all extraction steps at room temperature RT Please read protocols prior the start of the preparation carefully transfer 200 ul sample into the provided Extraction Tubes add 200 ul dd H20 for samples which have a smaller volume than 200 ul please fill up to a total volume of 400 ul with ddH O incubate for 15 minutes at 65 C in a thermomixer incubate for 10 minutes at 95 C in a thermomixer for realization of the optimal binding conditions add 400 ul Binding Solution and 20 ul MAP Solution A mix the sample completely by pipetting up and down binding of viral NA to magnetic particle magnetic separation of bea
24. inthiocyanate which is an irritant Wash Buffer R1 Proteinase K included in the Extraction Tube danger danger H302 312 332 412 EUH032 P273 H315 319 334 335 P280 305 351 338 310 405 H319 Causes serious eye irritation H302 Harmful if swallowed H312 Harmful in contact with skin H332 Harmful if inhaled H412 Harmful to aquatic life with long lasting effects EUH032 Contact with acids liberates very toxic gas H315 Causes skin irritation H334 May cause allergy or asthma symptoms or breathing difficulties if innaled H335 May cause respiratory irritation P305 P351 P338 IF IN EYES Rinse cautiously with water for several minutes Remove contact lenses if present and easy to do Continue rinsing P273 Avoid release to the environment P280 Wear protective gloves protective clothing eye protection face protection P310 Immediately calla 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 in USA 1 800 535 5053 6 InviMag Virus DNA RNA Mini Kit 0113 Product characteristic of the InviMag Virus DNA RNA Mini Kit Starting material up to 200 ul cell free body fluids depends on the sample storage and source about 20 25 min swab material sensitive recovery realizing amplification with after lysis up to 400 ul rinse liquid from sensitive detection assays from starting
25. ivity for some viral strains Important Note To get maximum yield of viral nucleic acids it is essential to leave the swab during the complete lysis time into the reaction tube It is possible to cut the shaft of the swab so that you can close the cap of the Extraction Tube It is also possible to do the lysis step with opened cap The removing of the swab from the Extraction Tube ahead of time will be lead to a dramatically reduced final yield After lysis time carefully squeeze out the swab on the wall of the tube and discard the swab 4 Remove the swab and transfer the Extraction Tube with the lysate in the Magnet Rack without magnet Add 400 ul Binding Solution and 20 ul MAP Solution A and mix the sample by pipetting up and down for 4 5 times Note Vortex the tube MAP Solution A vigorously before use 5 Positioning of the Extraction Tube in the Magnet Rack without magnet and incubate for 5 min the mixture at RT Then insert the Magnetic Stripe and incubate for further 5 min at RT Remove supernatant by aspiration or by pipetting then remove the Magnetic Stripe 6 Add 800 ul Wash Buffer R1 to the vial Close the vial and mix it by pipetting up and down Incubate for 3 min in the Magnet Rack with inserted Magnetic Stripe Remove supernatant and then remove the Magnetic Stripe 7 Add 800 ul Wash Buffer R2 to the vial Close the vial and mix it by pipetting up and down Incubate for 3 min in the Magnet Rack with inserted Magnetic Stri
26. le suppliers for lsopropanol Fa Carl Roth f f 2 Propanol Fa Applichem Fa Sigma Rotipuran gt 99 7 p a ACS ISO 2 Propanol f r die Molekularbiologie 2 Propanol Order no 6752 Order no A3928 Order no 59304 1 L F 20 InviMag Virus DNA RNA Mini Kit 0113 Sstratecee 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 1G4j03 01 2013
27. m storage at 80 C Storage under deep frozen conditions is recommended Serum plasma urine cerebrospinal fluid or other cell free body fluids as well as cell culture supernatants swabs and 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 at 80 C Multiple thawing and freezing before isolating the viral RNA should be avoided Serum and plasma and other cell free body fluids Following centrifugation plasma or serum from blood treated with anticoagulants like EDTA or citrate but not with heparin can be stored at 2 8 C for up to 6 hours For long term storage freezing at 20 C to 80 C in aliquots is recommended Repeated freezing and thawing cycles must be avoided because denaturation and precipitation of proteins result in a decrease of the virus titer and thereby reduce the yield of the extracted viral RNA Occurring cryoprecipitates can be pelleted by briefly centrifuging 6 800 x g for 3 min The cleared supernatant should be removed without disturbing the pellet and processed immediately This step will not reduce viral titers Tissue samples biopsy material or frozen section Best results are obtained with fresh material or material that has been immediately flash frozen and stored at 20 C or 80 C Repeated freezing and thawing of stored samples should be avoided since this leads to reduced
28. nd then remove the Magnetic Stripe Add 800 ul Wash Buffer R2 to the vial Close the vial and mix it by pipetting up and down Incubate for 3 min in the Magnet Rack with inserted Magnetic Stripe Remove supernatant Remove the Magnetic Stripe Repeat once this Step 8 Insert the Magnetic Stripe and open the lid of the tube for 10 min to evaporate the ethanol completely Remove the Magnetic Stripe then add 120 ul of the Elution Buffer R mix by pipetting up and down for three times Incubate at room temperature for 5 min Insert the Magnetic Stripe and incubate for further 3 min Transfer the eluate to the 1 5 ml Elution Tube centrifuge the tube for 3 min at 10 000 x g and transfer the supernatant to a new 1 5 ml Elution Tube If no centrifuge is available please put the vial again in the Magnet Rack insert the Magnetic Stripe and incubate for further 5 min at RT Transfer the supernatant in a new 1 5 ml Elution Tube It contains the viral nucleic acids ready to use Important Notes 1 The viral nucleic acids can also be eluted with a lower volume of Elution Buffer R depends on the expected amounts of viral nucleic acids But pay attention that minimum volume for the elution is 30 ul and that this volume can reduce the maximum yield If quite large amount of viral nucleic acids are expected the volume of Elution Buffer can be increased up to 200 ul 2 After finishing the extraction protocol the Elution Tube contains the extr
29. nucleic acids viral DNA and RNA from tissue biopsy Please read the instructions carefully and conduct the prepared procedure See also Special instruction for samples with high virus titer page 12 1 Transfer 1mg up to max 10 mg of the tissue biopsy into the Extraction Tube Add 400 ul of ddH2O Close the cap and mix by vortexing for 10 s Place the Extraction Tube into a Thermomixer and incubate under continuously shaking for 15 minutes at 65 C Lysis time can be increased up to 30 min A longer lysis time could reduce the final yield and the quality of some viral RNA species Place the Extraction Tube into another Thermomixer and incubate under continuously shaking for 10 minutes at 95 C These step leads to higher sensitivity for some viral strains Spin down unlysed material 1 min at maximum speed Transfer the Extraction Tube with the lysate in the Magnet Rack without magnet and add 400 ul Binding Solution and 20 ul MAP Solution A and mix the sample by pipetting up and down for 4 5 times Note Vortex the tube MAP Solution A vigorously before use Incubate for 5 min the mixture at RT Then insert the Magnetic Stripe and incubate for further 5 min at RT Remove supernatant by aspiration or by pipetting then remove the Magnetic Stripe Add 800 ul Wash Buffer R1 to the vial Close the vial and mix it by pipetting up and down Incubate for 3 min in the Magnet Rack with inserted Magnetic Stripe Remove supernatant a
30. oves if they become contaminated Do not combine components of different kit unless the lot numbers are identical Avoid microbial contamination of the kit reagents To minimize the risk of infections from potentially infectious material we recommend working under laminar air flow until the samples are lysed o This kit should only be used by trained personnel O OTOOTO Important indications Preparing viral RNA When preparing viral RNA work quickly during the manual steps of the procedure The Lysis Buffer in the Extraction Tube simplifies viral RNA isolation by combining efficient lysis of the starting material and the inactivation of exogenous and endogenous RNases Extreme care should be taken to avoid contaminations with RNases when handling Elution Buffer R Storing samples Frozen serum or plasma samples must not be thawed more than once Repeated freeze thawing leads to denaturation and precipitation of proteins resulting in reduced viral titers and therefore reduced yields of viral nucleic acids Carrier RNA Carrier RNA serves two purposes Firstly it enhances the binding of viral acids to the MAP Solution A especially if there are very few target molecules in the sample Secondly the addition of large amounts of Carrier RNA reduces the chance of viral nucleic acid degradation in the rare event that RNase or DNase molecules are not denaturated by the salts and detergents in the Lysis Buffer in the Extraction
31. pe Remove supernatant and then remove the Magnetic Stripe Repeat once this Step 8 Insert the Magnetic Stripe and open the lid of the tube for 10 min to evaporate the ethanol completely 9 Remove the Magnetic Stripe then add 120 ul of the Elution Buffer R mix by pipetting up and down for three times Incubate at room temperature for 5 min 10 Insert the Magnetic Stripe and incubate for further 3 min Transfer the eluate to the 1 5 ml Elution Tube centrifuge the tube for 3 min at 10 000 x g and transfer the supernatant to a new 1 5 ml Elution Tube If no centrifuge is available please put the vial again in the Magnet Rack insert the Magnetic Stripe and incubate for further 5 min at RT Transfer the supernatant in a new 1 5 ml Elution Tube It contains the viral nucleic acids ready to use Important Notes 1 The viral nucleic acids can also be eluted with a lower volume of Elution Buffer R depends on the expected amounts of viral nucleic acids But pay attention that minimum volume for the elution is 30 pl and that this volume can reduce the maximum yield If quite large amount of viral nucleic acids are expected the volume of Elution Buffer can be increased up to 200 ul 2 After finishing the extraction protocol the Elution Tube contains the extracted viral RNA DNA We recommend storage of the DNA at 20 or 80 especially for RNA 14 InviMag Virus DNA RNA Mini Kit 0113 Protocol 3 Simultaneous isolation of total
32. re expected the volume of Elution Buffer can be increased up to 200 ul 2 After finishing the extraction protocol the Elution Tube contains the extracted viral RNA DNA We recommend storage of the DNA at 20 C or 80 especially for RNA 16 InviMag Virus DNA RNA Mini Kit 0113 Troubleshooting Problem Probable cause Comments and suggestions low amount of extracted DNA RNA low concentration of extracted DNA RNA insufficient lysis incomplete elution low amount of MAP Solution A too much Elution Buffer incorrect storage of starting material increase lyses time but prevent too long lyses tome because this also decrease yield reduce amount of starting material take higher volume of Elution Buffer R be sure you pipet the Elution Buffer R with the right amount mix MAP Solution A thoroughly before pipeting to the sample elute the DNA with lower volume of Elution Buffer R ensure that the storage of starting material was correctly avoid thawing of the material degraded DNA RNA incorrect storage of starting material old material ensure that the storage of starting material was correctly avoid thawing of the material ensure that the starting material is fresh or stored under appropriate condition for long time storage at 20 C avoid thawing and freezing of the material old material often contains degraded DNA RNA DNA RNA does not perform well in downstream
33. stic results adequate controls for downstream applications should 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 neither validated for the isolation of genomic DNA from stool sample dried blood stains nor from bacteria fungi parasites or the purification of total RNA The included chemicals are only useable once Differing of starting material or flow trace may lead to inoperability therefore neither a warranty nor guarantee in this case will be given neither implied nor express The user is responsible to validate the performance of the STRATEC Molecular Product for any particular use STRATEC Molecular does not provide for validation of performance characteristics of the Product with respect to specific applications STRATEC Molecular Products may be used e g in clinical diagnostic laboratory systems conditioned upon the complete diagnostic system of the laboratory the laboratory has been validated pursuant to CLIA 88 regulations in the U S or equivalents in other countries All Products sold by STRATEC Molecular are subject to extensive quality control procedures according to ISO 9001 2000 and ISO EN 13485 and are warranted to perform as described herein Any problems incidents or defects shall be reported to STRATEC Mol
34. swab material including minimum 100 copies per ml cell culture supernatants Note The added Carrier RNA will account for 1x 10 mammalian cells most of the eluted RNA Quantitative RT PCR max 10 mg tissue sample is recommended for determination of the viral RNA or DNA yield The InviMag Virus DNA RNA Mini Kit is designed for a simultaneous rapid and economical preparation of viral DNA and RNA from a wide range of clinical samples using a combination of RTP technology magnetic beads and a magnetic tool The amount of purified viral DNA and RNA in the InviMag Virus DNA RNA Mini Kit depend on the sample type the virus titer sample source transport storage and age The isolation process is based on a patented technology the InviMag technology for isolation of viral RNA and DNA by binding the nucleic acid onto magnetic particles without chaotropic buffer components The kit uses further the patented RTP technology whose special feature is an Extraction Tube containing preformulated solid lysis reagent Proteinase K Carrier Nucleic Acids and a precisely calibrated amount of an internal DNA Extraction Control or on request an internal RNA and DNA Extraction Control The technology allows the reduction of reagent preparation steps and of handling steps with infectious material Using the Extraction Tube all types of samples are transferred into the Extraction Tube together with ddH O to adjust a final sample volume of 400 ul
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