InviMag Virus DNA/RNA Mini Kit/ KFmL User manual
Contents
1. danger danger H315 319 334 335 P280 305 351 338 310 405 H302 312 332 412 EUH032 P273 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 inhaled 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 call a POISON CENTER or doctor physician P405 Store locked up Emergency medical information can be obtained 24 hours a day from infotrac outside of USA 1 352 323 3500 in USA 1 800 535 5053 7 InviMag Virus DNA RNA Mini Kit KFmL 0515 Product characteristic of the InviMag Virus DNA RNA Mini Kit KFmL Starting material Time for preparation up to 200ul of fresh or frozen plasma serum sensitive recovery realizing about 20 25 min and cell free body fluids amplification with sensitive after lysis up to 200 ul cell culture supernatant detection assays from staring up to 200ul of rinse liquid from swabs material including minimum swab 100 copies2. This manual contains 4 protocols Lysis Samples are lysed under 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 100 ul Elution Buffer R The eluted nucleic acids are ready to use in different subsequent tests 9 InviMag Virus DNA RNA Mini Kit KFmL 0515 Yield and quality of viral DNA and or viral RNA Different amplification systems vary in efficiency depending on the total amount of nucleic acid present in the reaction Eluates derived by this kit will contain Carrier RNA which will greatly exceed the amount of the isolated NA Yields of viral nucleic acids isolated from biological samples are usually low concentrated and therefore almost impossible to determine photometrically Keep in mind that the Carrier RNA 5 ug per 200 ul sample will account for most of the present NA 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 The amount of purified viral NA in the InviMag Virus DNA RNA M
3. stratecee molecular User manual InviMag Mirus DNA RNA Mini Kit KFmL foruse on KingFisher mL Thermo Fisher Scientific for automated 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 swabs with magnetic beads 2441150x00 anal STRATEC Molecular GmbH D 13125 Berlin lt Instruction InviMag Virus DNA RNA Mini Kit KFmL The InviMag Virus DNA RNA Mini Kit KFmL is the ideal tool using a combination of RTP technology and InviMag technology for simultaneous isolation of high quality viral DNA and RNA from human and animal serum and plasma samples cerebrospinal fluid cell culture supernatants and other cell free body fluids like urine as well as from swabs or tissue biopsies for in vitro diagnostic purposes 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 process monitoring Due to the high purity the isolated viral DNA and RNA is ready to use for a broad panel of downstream applications or can be stored at 80 C for subsequent use The kit is neither suitable for isolation of viral DNA or RNA from whole blood or blood stains nor for isolation of RNA or DNA from bacteria fungi plants Compliance with EU Direc
4. 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 cycles before isolating the viral RNA should be avoided 8 InviMag Virus DNA RNA Mini Kit KFmL 0515 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 The PCR process is covered by US Patents 4 683 195 and 4 683 202 and foreign equivalents owned by Hoffmann La Roche AG 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 cycles of stored samples should be avoided because this leads to a low RNA yield Use of poor quality starting material influences the RNA
5. Scheme of the InviMag Virus DNA RNA Mini Kit KFmL 12 Protocol 1 Simultaneous isolation of total nucleic acids viral DNA and RNA from cell free body fluids serum plasma CSF synovial urine 13 Protocol 2 Simultaneous isolation of total nucleic acids viral DNA and RNA from cell culture supernatant 13 Protocol 3 Simultaneous isolation of total nucleic acids viral DNA and RNA from swabs or 200 ul rinse liquid 14 Protocol 4 Simultaneous isolation of total nucleic acids viral DNA and RNA from tissue biopsy 14 Starting a run on the KFmL instrument 15 The following steps will run automatically on the KingFisher mL 16 For self programming of the KingFisher mL system 17 Troubleshooting 19 Appendix 20 General notes on handling DNA 21 General notes on handling viral RNA 22 Ordering information 23 2 InviMag Virus DNA RNA Mini Kit KFmL 0515 Kit contents of InviMag Virus DNA RNA Mini Kit KFmL Store the MAP Solution A at 2 8 C Store all other kit components at room temperature 15 extractions 75 extractions 300 extractions Catalogue No 2441150100 2441150200 2441150400 Extraction Tubes 15 75 6 x 50 MAP Solution A 0 5 ml 2x1ml 7 ml Binding Solution empty bottle empty bottle empty bottle fill with 99 7 Isopropanol final volume 8 ml final volume 40 ml final volume 140 ml Wash Buffer R1 Wash Buffer R2 10 ml final volume 20 ml 12 ml final volume 60 ml 2 x 20 ml final volume 2
6. high molecular weight DNA is necessary to ensure compatibility with various downstream applications Damaged DNA could perform poorly in applications such as genomic Southern blotting long template PCR Storage of DNA A working stock of DNA can be stored at 2 8 C for several weeks For long term storage DNA should be stored at 20 C However storage at 20 C may cause shearing particularly if the DNA is exposed to repeated freeze thawing cycles Note that the solution in which the nucleic acid 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 Drying dissolving and pipetting DNA Avoid overdrying genomic DNA after ethanol precipitation It is better to air dry than to use a vacuum although vacuum drying can be used with caution Avoid vigorous pipetting Pipetting genomic DNA through small tip openings causes shearing or nicking One way to decrease shearing of genomic DNA is to use special tips that have wide openings designed for pipetting genomic DNA DNA yield The amount of purified viral DNA depends on sample source transport conditions storage and age of the sample 21 InviMag Virus DNA RNA Mini Kit KFmL 0515 General notes on handling viral RNA RNA is far less stable than DNA It is very sensitive to degradation by endogenous RNases in th
7. higher sensitivity for some viruses or some strains of viruses e g HIV or HCV After lysis transfer the lysed sample into the Tube A of the KingFisher tube strip and add the 400 ul of Binding Solution and 20 ul MAP Solution A see also below Vortex the tube MAP Solution A vigorously before use Start the program see instructions on page 17 13 InviMag Virus DNA RNA Mini Kit KFmL 0515 Protocol 3 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 Sample Lysis la Place the swab into the Extraction Tube and add 400 ul of ddH2O Close the cap and mix by vortexing for 10 s 1b Transfer 200 ul of rinse liquid or of the stabilization media into the Extraction Tube and add 200 ul of ddH20 Close the cap and mix by vortexing for 10 s 2 Place the Extraction Tube into a Thermomixer and incubate while continuously shaking for 15 min at 65 C During lysis prefill all tubes with the required buffers and appropriate volumes see page 17 3 Place the Extraction Tube into another Thermomixer and incubate under continuously shaking for 10 min at 95 C These step leads to higher sensitivity for some viruses or some strains of viruses e g HIV or HCV 4 After lysis transfer the lysed sample into the Tube A of the KingFisher tube strip and add the 400 ul of Binding Solution and 20 ul MAP Solution A see a
8. yield too The amount of purified RNA in the RTP DNA RNA Virus Mini Kits 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 separation of the cell culture supernatant Repeated freezing and thawing cycles of stored samples will 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 STRATEC Molecular will be released of its responsibilities if other sample materials than described in the Intended Use are processed or if the sample preparation protocols are changed or modified Principle and procedure The InviMag Virus DNA RNA Kit KFmL 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 high purified DNA RNA is eluted in Elution Buffer R or water
9. DNA into 1 5 ml reaction tubes store the DNA RNA at 20 or 80 TC recommended for RNA If the viral RNA DNA contains carryover of magnetic particles transfer the viral NA into a 1 5 ml reaction tube and centrifuge at maximum speed for 1 minute Transfer the viral RNA DNA containing supernatant into a new tube 15 InviMag Virus DNA RNA Mini Kit KFmL 0515 The following steps will run automatically on the KingFisher mL 1 Binding of the DNA RNA Automatically sample mixing for 5 min MAP separation Moving of the MAP with bounded nucleic acids into the Tube B 2 First Washing Automatically sample mixing for 1 min MAP separation Moving of the MAP with bounded nucleic acids into the Tube C 3 Second Washing Automatically sample mixing for 1 min MAP separation Moving of the MAP with bounded nucleic acids into the Tube D 4 Third Washing and Drying Automatically sample mixing for 1 min MAP separation Drying the MAP with bounded nucleic acids outside of the Tube for 8 min Moving of the MAP into the E wells 5 Elution of the viral DNA and or RNA Incubation of the MAP into the Tube E for 10 min by mixing MAP separation The MAP will than automatically removed into the B wells disposal The extracted viral DNA RNA can now be transferred into 1 5 ml reaction tubes Optional Carryover of magnetic particles should be removed by centrifugation at max speed for 1 min Transfer the clear supernatant into a new 1 5 ml r
10. ag Virus DNA RNA Mini Kit KFmL w o plastic 300 preparations 2441150450 Single components for InviMag Virus DNA RNA Mini Kit Extraction Tubes 60 ml 7440301500 MAP Solution A 1 ml 7440305100 Elution Buffer R 30 ml 7440304100 Wash Buffer R1 add 20 ml ethanol 60 ml 7440303500 Wash Buffer R2 add 200 ml ethanol 60 ml 7440303600 Related products RTP DNA RNA Virus Mini Kit 50 extractions 1040100200 RTP DNA RNA Virus Mini Kit 250 extractions 1040100300 InviMag Virus DNA RNA Mini Kit 50 extractions 1440100200 InviMag Virus DNA RNA Mini Kit 250 extractions 1440100300 InviMag Virus DNA RNA Mini Kit KF96 1 x 96 preparations 7441050100 InviMag Virus DNA RNA Mini Kit KF96 5 x 96 preparations 7441050200 Ordering information KingFisher mL and consumables Cat no Description 5400050 KingFisher mL Magnetic Particle Processor 100 240 V 50 60 Hz 97002111 KingFisher mL tip comb 800 pcs 97002121 KingFisher mL tube 900 pcs 20x45 pcs 97002131 KingFisher mL Combi 60 tubes and tip combs for 60 samples 97002141 KingFisher mL Combi 240 tubes and tip combs for 240 samples Possible suppliers for lsopropanol Carl Roth A 2 Propanol Applichem NEES 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 23 InviMag Virus DNA RNA Mini Kit KFmL 0515 stratecee molecular STRATEC Molecular GMbH Robert R ssle Str 10 13125 Berli
11. and add 200 ul of ddH20 Close the cap and mix by vortexing for 10 s Place the Extraction Tube into a Thermomixer and incubate while continuously shaking for 15 min at 65 C During lysis prefill all tubes with the required buffers and appropriate volumes see page 17 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 viruses or some strains of viruses e g HIV or HCV After lysis transfer the lysed sample into the Tube A of the KingFisher tube strip and add the 400 ul of Binding Solution and 20 ul MAP Solution A Vortex the tube MAP Solution A vigorously before use Start the assay file see page 17 Protocol 2 Simultaneous isolation of total nucleic acids viral DNA and RNA from cell culture supernatant Please read the instructions carefully and conduct the prepared procedure Sample Lysis 1 Note Transfer 200 ul of the cell free cell culture supernatant cell culture media into the Extraction Tube and add 200 ul of ddH20 Close the cap and mix by vortexing for 10 s Place the Extraction Tube into a Thermomixer and incubate while continuously shaking for 15 min at 65 C During lysis prefill all tubes with the required buffers and appropriate volumes see page 17 Place the Extraction Tube into another Thermomixer and incubate under continuously shaking for 10 min at 95 C These step leads to
12. d to use Thermo Microtiter Deep Well plates with KF96 KFflex96 workstations to ensure the best purification result PC requirements for KingFisher Software 3 1 PC requirements Interface Serial communication port via a RS 232 full duplex interface Supported operating systems Microsoft Windows 2000 Microsoft Windows XP Professional Serial ports available Pointing device Disk space 500 MB free disk space Processor Intel Pentium gt 700 MHz recommended Memory 220 MB RAM recommended 1 Mouse or equivalent is necessary CD ROM drive 1 Monitor color settings Service packs installed SVGA monitor with at least 1024 x 768 resolution and at least a 16 bit color environment Microsoft Windows 2000 Service Pack 4 or greater Microsoft Windows XP Professional Service Pack 2 or greater Browser Microsoft Internet Explorer 6 0 or greater installed If you do not have the correct Service Packs installed you can download them from the Microsoft web pages http www microsoft com 20 InviMag Virus DNA RNA Mini Kit KFmL 0515 General notes on handling DNA 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
13. e Virus DNA RNA A Binding Beginning of step Precollect No Release time hh mm ss 00 00 10 Release speed Medium Mixing pause parameters Pause for manual handling No Mixing time hh mm ss 00 05 00 Mixing speed Slow End of step Postmix No Collect count 4 Collect time s 3 Washing_1 Virus DNA RNA B Wash1 Beginning of step Precollect No Release time hh mm ss 00 00 10 Release speed Fast Mixing pause parameters Pause for manual handling No Mixing time hh mm ss 00 01 00 Mixing speed Medium End of step Postmix No Collect count 3 Collect time s 2 Washing_2 Virus DNA RNA C Wash2 Beginning of step Precollect No Release time hh mm ss 00 00 10 Release speed Fast Mixing pause parameters Pause for manual handling No Mixing time hh mm ss 00 01 00 Mixing speed Medium End of step Postmix No Collect count 3 Collect time s 2 Washing_3 Virus DNA RNA D Wash3 Beginning of step Precollect No Release time hh mm ss 00 00 10 Release speed Fast InviMag Virus DNA RNA Mini Kit KFmL 0515 Mixing pause parameters Pause for manual handling No Mixing time hh mm ss 00 01 00 Mixing speed Medium End of step Postmix No Collect count 3 Collect time s 2 Drying Virus DNA RNA D Wash3 Dry time hh mm ss 00 08 00 Tip position Outside well tube Elution Virus DNA RNA E Elution Beginning of
14. e 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 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 followed by ethanol and allowed to dry o Non disposable plastic ware should be treated before use to ensure that it is RNase free Plastic ware should be thoroughly rinsed with 0 1 M NaOH 1 mM EDTA followed by RNase free water Alternatively chloroform resistant plastic ware can be rinsed with chloroform to inactivate RNases o All buffers mus
15. e 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 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 Azgo Azgo 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 19 InviMag Virus DNA RNA Mini Kit KFmL 0515 Appendix KingFisher Software 3 1 The KingFisher Software 3 1 was used to create assay files for the KFmL KF96 and KFflex96 instruments The respective assay file can either be transferred onto the KingFisher workstation or be started directly from within the Bindlt software Keep in mind that directly run assay files are not stored in the workstation memory Note When creating assay files for usage with KingFisher instruments in combination with Microtiter Deep Well plates e g Thermo Electron it is essential to use the KingFisher software 3 1 for assay development as this software version includes the correct adjustments for this plate It is highly recommende
16. eaction tube Note The eluate contains viral DNA and or RNA After extraction place the elution tube on ice For a long term freeze the nucleic acids at 20 or 80 CT recommended for RNA 16 InviMag Virus DNA RNA Mini Kit KFmL 0515 For self programming of the KingFisher mL system Protocol Properties Name InviMag Virus DNA RNA KFmL Protocol template version 3 1 Instrument type KF96 KFflex96 Kit name InviMag Virus DNA RNA Kit KF96 KFflex96 Description KF96 KFflex96 protocol for Isolation of genomic DNA viral DNA or RNA from swabs tissue or suspension with the InviMag Virus DNA RNA Kit KF96 KFflex96 Plate Layouts Binding plate Plate type KingFisher tub strip 1000ul Reagents Name Binding Solution Volume ul 400 Type Reagent Name MAP A Solution Volume pl 20 Type Reagent Name Lysed sample Volume ul 400 Type Sample Washing plate_1 Plate type KingFisher tub strip 1000ul Reagents Name Wash Buffer R1 Volume ul 800 Type Reagent Washing plate_2 Plate type KingFisher tub strip 1000ul Reagents Name Wash Buffer R2 Volume ul 800 Type Reagent Washing plate_3 Plate type KingFisher tub strip 1000ul Reagents Name Wash Buffer R2 Volume yl 800 Type Reagent Elution plate Plate type KingFisher tub strip 1000ul Reagents Name Elution Buffer R Volume yl 100 Type Reagent Steps 17 Binding Plat
17. ed on a patented technology the InviMag technology for isolation of viral DNA and RNA by binding the nucleic acid onto magnetic particles without chaotropic buffer components For reproducible and high yields appropriate sample storage is essential The purified DNA RNA is of high quality and can be used for in vitro diagnostic analysis THE PRODUCT IS INDENTED FOR USE BY PROFESSIONALS 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 diagnostic results adequate controls for downstream applications should be used The kit is in compliance with EU Directive 98 79 EC on in vitro medical 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 start
18. form 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 KFmL 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 KFmL 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 5 InviMag Virus DNA RNA Mini Kit KFmL 0515 Intended use The InviMag Virus DNA RNA Mini Kit KFmL is designed for a simultaneous rapid and economical preparation of DNA and RNA from viruses from a wide range of clinical samples using RTP technology magnetic beads and the KingFisher mL workstation The whole process is bas
19. ing 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 Molecular 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 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 Con
20. ini Kit KFmL procedure from plasma etc depends on the sample type sample source transport storage and age Quantitative RT PCR is recommended for determination of viral NA yield In Gel Electrophoresis and in Capillary 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 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 7 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 contaminated gloves immediately Do not combine components of different k
21. iral RNA using magnetic particle in high throughput format STRATEC Molecular offers the InviMag Virus Kits KF96 for use on a KingFisher 96 robot For vacuum or centrifuge based isolation of viral RNA 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 21 For further information please contact 49 0 30 9489 2901 2910 in Germany and from foreign countries 49 0 30 9489 2907 or call 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 Tube or II for 1 h at room temperature overnight at 4 C and for long term 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
22. it 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 OO OO Reagents and equipment to be supplied by user ddH2O Vortexer 96 100 ethanol Isopropanol The InviMag Virus DNA RNA Mini Kit KFmL is validated with 2 Propanol Rotipuran gt 99 7 p a ACS ISO Order no 6752 from Carl Roth Measuring cylinder 250 ml Pipette and pipette tips Disposable gloves O O O o Reaction tubes 1 5 ml or 2 0 ml O 0 0 0 Possible suppliers for Isopropanol Carl Roth Applichem Sigma E 2 Propanol f r die Molekularbiologie 2 Propanol Rotipuran gt 99 7 p a ACS ISO a Order no 6752 Order no A3928 Order no 59304 1L F 10 InviMag Virus DNA RNA Mini Kit KFmL 0515 Preparing reagents and buffers 15 viral NA extractions Fill 8 ml 99 7 Isopropanol molecular biologic grade into the empty bottle Add 10 ml of 96 100 ethanol to the bottle Wash Buffer R1 mix thoroughly and keep the bottle always firmly closed Add 48 ml of 96 100 ethanol to the bottle Wash Buffer R2 mix thoroughly and always keep the bottle firmly closed 75 viral NA extractions Fill 40 ml 99 7 isopropanol molecular biologic grade into the empty bottle Add 20 ml of 96 100 etha
23. lso below Note Vortex the tube MAP Solution A vigorously before use 5 Start the program see instructions on page 17 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 Protocol 4 Simultaneous isolation of total nucleic acids viral DNA and RNA from tissue biopsy Please read the instructions carefully and conduct the prepared procedure Sample Lysis la Note Transfer 1 mg 10 mg of the tissue biopsy into the Extraction Tube Add 400 ul of ddH20 Close the cap and mix by vortexing for 10 s Place the Extraction Tube into a Thermomixer and incubate while continuously shaking for 15 min at 65 C Lysis time can be increased for up to 30 min After a prolonged lysis a reduced yield and quality of some viral RNA species may occur After lysis centrifuge the sample at max speed for 1 min to spin down unlysed material and follow exactly the next step During lysis prefill all tubes with the required buffers and appropriate volumes see page 17 Place the Extracti
24. n Germany Phone 49 30 94 89 29 01 Fax 49 30 94 89 29 09 E mail info berlin stratec com www stratec com 1G4j01 05 2015
25. nol to each bottle Wash Buffer R1 mix thoroughly and keep the bottle always firmly closed Add 120 ml of 96 100 ethanol to the bottle Wash Buffer R2 mix thoroughly and always keep the bottle firmly closed 300 viral NA extractions Fill 120 ml 99 7 isopropanol molecular biologic grade into the empty bottle Add 80 ml of 96 100 ethanol to each bottle Wash Buffer R1 mix thoroughly and keep the bottle always firmly closed Add 140 ml of 96 100 ethanol to each bottle Wash Buffer R2 mix thoroughly and always keep the bottle firmly closed 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 Special 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 freezing and thawing cycles 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 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 reduce
26. on Tube into an other thermomixer and incubate while continuously shaking for 10 min at 95 C These step leads to higher sensitivity for some viruses or some strains of viruses e g HIV or HCV After lysis transfer the lysed sample into the Tube A of the KingFisher tube strip and add the 400 ul of Binding Solution and 20 ul MAP Solution A Vortex the tube MAP Solution A vigorously before use Start the program see instructions on page 17 14 InviMag Virus DNA RNA Mini Kit KFmL 0515 Starting a run on the KFmL instrument 1 During lysis prefill the tubes of the KingFisher tube strips with the required buffers and appropriate volumes KingFisher mL Tube Strip Setup Tube A Filled with app 450 ul of the lysed sample 400 ul Binding Solution and 20 ul MAP Solution A after finishing the lysis step It is important to mix the bottle with MAP Solution A by vigorously shaking or vortexing Tube B 800 ul Wash Buffer R1 Tube C 800 ul Wash Buffer R2 Tube D 800 ul Wash Buffer R2 Tube E 100 ul Elution Buffer R 2 Insert the prefilled KingFisher tube strips into the KingFisher instrument 3 Place the KingFisher tips onto the magnetic rack After these preliminary steps start the assay file InviMag Virus DNA RNA KFmL Important Notes After finishing the extraction protocol the Tube E contains the extracted RNA DNA Store the RNA DNA under adequate conditions We recommend transferring the extracted viral RNA
27. per ml up to 10 mg biopsy samples The InviMag Virus DNA RNA Mini Kit KFmL 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 the KingFisher mL workstation 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 sample is lysed in an optimized lysis buffer The lysates are transferred to the subsequent automatic purification procedure based on magnetic beads The viral DNA and RNA bind to magnetic particles followed by washing steps and a final elution The procedure requires only minimal interaction by the user allowing safe handling of potentially infectious samples The procedure is designed to avoid sample to sample cross contamination The purified high quality viral DNA and RNA is ready to use for subsequent downstream applications see below or can be stored at 20 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 RNA from single sample from 200 ul starting material STRATEC Molecular offers the RTP DNA RNA Virus Mini Kit and the Invisorb Spin Virus RNA Mini Kit For the isolation of v
28. r R1 mix thoroughly and always keep the bottle firmly closed Add 160 ml of 96 100 ethanol to each bottle Wash Buffer R2 mix thoroughly and always keep the bottle firmly closed Plastic to be supplied by user see order information KingFisher mL Tip Combs 60 KingFisher mL Tube Strips 5x15 300 InviMag Virus DNA RNA Mini Kit KFmL 0515 Symbols Lot number Catalogue number Expiry date Temperature limitation Do not reuse Ba Consult operating instructions Storage All buffers and kit contents of the InviMag Virus DNA RNA Mini Kit KFmL except MAP Solution A are stable for at least 12 months MAP Solution A is stable for at least 6 months All buffers and kit contents of the InviMag Virus DNA RNA Mini Kit KFmL except MAP Solution A should be stored at room temperature RT MAP Solution A should be stored at 2 8 Room temperature is defined as range from 15 30 C Wash Buffer charged with ethanol should be stored at room temperature and should be appropriate sealed If there are any precipitates visible within the provided solutions dissolve them by carefully warming up to 30 C Quality control and product warranty STRATEC Molecular warrants the correct function of the InviMag Virus DNA RNA Mini Kit KFmL for applications as described in this manual Purchaser must determine the suitability of the product for its particular use Should any product fail to per
29. s at room temperature 15 extractions 75 extractions 300 extractions Catalogue No 2441150150 2441150250 2441150450 Extraction Tubes 15 75 6 x 50 MAP Solution A 0 5 ml 2ximl 7 ml Binding solution empty bottle empty bottle empty bottle fill with 98 100 Isopropanol final volume 8 ml final volume 40 ml final volume 140 ml 10 ml 2 x 20 ml 2 x 80 ml Waste Ruffer final volume 20 ml final volume 2 x 40 ml final volume 2 x 160 ml 12 ml 30 ml 3 x 40 ml Wash Buffer R2 final volume 60 ml final volume 150 ml final volume 3 x 200 ml Elution Buffer R 2 ml 15 ml 60 ml Elution Tubes 15 5x15 6 x 50 Manual 1 1 1 Initial steps Fill 8 ml 99 7 Fill 40 ml 99 7 Fill 140 ml 99 7 Isopropanol molecular biologic grade into the empty bottle Add 10 ml of 96 100 ethanol to the bottle Wash Buffer R1 mix thoroughly and always keep the bottle firmly closed Add 48 ml of 96 100 ethanol to the bottle Wash Buffer R2 mix thoroughly and always keep the bottle firmly closed Isopropanol molecular biologic grade into the empty bottle Add 20 ml of 96 100 ethanol to the bottle Wash Buffer R1 mix thoroughly and always keep the bottle firmly closed Add 120 ml of 96 100 ethanol to the bottle Wash Buffer R2 mix thoroughly and always keep the bottle firmly closed Isopropanol molecular biologic grade into the empty bottle Add 80 ml of 96 100 ethanol to each bottle Wash Buffe
30. s the chance of viral nucleic acid degradation in the rare event that RNase or DNase molecules are not denatured by the salts and detergents in the Lysis Buffer in the Extraction Tube 11 InviMag Virus DNA RNA Mini Kit KFmL 0515 Scheme of the InviMag Virus DNA RNA Kit KFmL Please read protocols prior the start of the preparation carefully Add required amount of ddH2O 200 400 ul to the sample Vortex for 10 s Place sample into a Thermomixer and incubate while continuously shaking for 15 min at 65 C followed by an incubation for 10 min at 95 C During lysis prefill all KingFisher Tube strips with required buffers and appropriate volumes Tube A 400 ul Binding Solution and 20 ul MAP Solution A Tube B 800 ul Wash Buffer R1 Tube C 800 ul Wash Buffer R2 Tube D 800 ul Wash Buffer R2 Tube E 100 ul Elution Buffer R Add approx 450 ul of the lysed sample to Tube A Viral NA binding to magnetic particle Magnetic separation Washing of the particle fixed viral NA Magnetic separation Elution of genomic DNA Magnetic Separation Pure viral NA 12 InviMag Virus DNA RNA Mini Kit KFmL 0515 Protocol 1 Simultaneous isolation of total nucleic acids viral DNA and RNA from cell free body fluids serum plasma CSF synovial urine Please read the instructions carefully and conduct the prepared procedure Sample Lysis 1 Note Transfer 200 ul of the sample into the Extraction Tube
31. step Precollect No Release time hh mm ss 00 00 10 18 Release speed Medium Mixing pause parameters Pause for manual handling No Mixing time hh mm ss 00 10 00 Mixing speed Slow End of step Postmix No Collect count 4 Collect time s 3 Bead Removal Virus DNA RNA D Washo Release time hh mm ss 00 00 30 Release speed Fast InviMag Virus DNA RNA Mini Kit KFmL 0515 Troubleshooting Problem Probable cause Comments and suggestions low amount of extracted DNA RNA insufficient lysis incomplete elution low amount of MAP Solution A 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 D be sure you pipet the Elution Buffer D with the right amount to the right position mix MAP Solution A thoroughly before pipetting to the KingFisher tube low concentration of extracted DNA RNA too much Elution Buffer incorrect storage of starting material elute the DNA with lower volume of Elution Buffer D ensure that the storage of starting material was correctly avoid thawing of the material degraded or sheared DNA RNA DNA RNA does not perform well in downstream applications e g real time PCR or PCR incorrect storage of starting material old material ethanol carryover during elution salt carryover during elution ensure that the storag
32. t 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 contaminations cavity seams shouldn t be moistened 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 Do not merge kit components from other kits unless the lot numbers are identical o Tominimize the risk of infections from potentially infectious material we recommend working under laminar air flow until the samples are lysed oO00 0 O 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 at 80 C and is stable for several years at this condition 22 InviMag Virus DNA RNA Mini Kit KFmL 0515 Ordering information Product Package Size Catalogue No InviMag Virus DNA RNA Mini Kit KFmL 15 preparations 2441150100 InviMag Virus DNA RNA Mini Kit KFmL 75 preparations 2441150200 InviMag Virus DNA RNA Mini Kit KFmL 300 preparations 2441150400 InviMag Virus DNA RNA Mini Kit KFmL w o plastic 15 preparations 2441150150 InviMag Virus DNA RNA Mini Kit KFmL w o plastic 75 preparations 2441150250 InviM
33. tive 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 RTP 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 RTP are registered trademarks of STRATEC Biomedical AG The PCR process is covered by US Patents 4 683 195 and 4 683 202 and foreign equivalents owned by Hoffmann La Roche AG 2015 STRATEC Molecular all rights reserved 1 InviMag Virus DNA RNA Mini Kit KFmL 0515 Content Kit contents of InviMag Virus DNA RNA Mini Kit KFmL 3 Kit contents of InviMag Virus DNA RNA Mini Kit KFmL w o plastic 4 Symbols 5 Storage 5 Quality control 5 Intended use 6 Product use limitation 6 Internal control IC Extraction control 6 Safety information 7 Product characteristic of the InviMag Virus DNA RNA Mini Kit KFmL 8 Sampling and storage of starting material 8 Principle and procedure 9 Yield and quality of viral DNA and or viral RNA 10 Important notes 10 Reagents and equipment to be supplied by user 10 Preparing reagents and buffers 11 Important indications 11
34. trols to the sample 6 InviMag Virus DNA RNA Mini Kit KFmL 0515 Safety information when and while working with chemicals always wear a suitable lab coat disposable gloves and protective goggles Avoid skin contact Adhere to the legal requirements for working with biological material For more information please consult the appropriate material safety data sheets MSDS These are available online in convenient and compact PDF format at www stratec com for each STRATEC Molecular product and its components If buffer bottles are damaged or leaking WEAR GLOVES AND PROTECTIVE GOGGLES when discarding the bottles in order to avoid any injuries STRATEC Molecular has not tested the liquid waste generated by the InviMag Virus DNA RNA Mini Kit KFmL 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 KFmL to which they apply are listed below as follows Wash Buffer R1 contains guanidinthiocyanat which is an irritant Wash Buffer and Elution Buffer R contain DEPC treated ddH20 DEPC is inactivated by autoclaving for 20 min at 121 C Proteinase K included in the Extraction Tube Wash Buffer R1 OD
35. x 40 ml 30 ml final volume 150 ml 2 x 80 ml final volume 2 x 160 ml 3 x 40 ml final volume 3 x 200 ml Elution Buffer R 2 ml 15 ml 60 ml KingFisher mL Tip Combs 3 15 60 KingFisher mL Tube Strips 15 5x15 300 Elution Tubes 15 5x15 6 x 50 Manual 1 1 1 Initial steps Fill 8 ml 99 7 Fill 40 ml 99 7 Fill 140 ml 99 7 Isopropanol molecular biologic grade into the empty bottle Add 10 ml of 96 100 ethanol to the bottle Wash Buffer R1 mix thoroughly and always keep the bottle firmly closed Add 48 ml of 96 100 ethanol to the bottle Wash Buffer R2 mix thoroughly and always keep the bottle firmly closed Isopropanol molecular biologic grade into the empty bottle Add 20 ml of 96 100 ethanol to the bottle Wash Buffer R1 mix thoroughly and always keep the bottle firmly closed Add 120 ml of 96 100 ethanol to the bottle Wash Buffer R2 mix thoroughly and always keep the bottle firmly closed Isopropanol molecular biologic grade into the empty bottle Add 80 ml of 96 100 ethanol to each bottle Wash Buffer R1 mix thoroughly and always keep the bottle firmly closed Add 160 ml of 96 100 ethanol to each bottle Wash Buffer R2 mix thoroughly and always keep the bottle firmly closed InviMag Virus DNA RNA Mini Kit KFmL 0515 Kit contents of InviMag Virus DNA RNA Mini Kit KFmL w o plastic Store the MAP Solution A at 2 8 C Store all other kit component
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