Invisorb Spin Virus RNA Mini Kit
Contents
1. For monolayer cells Add 600 ul Lysis Buffer RV 200 ul ddH O 20 ul Carrier RNA and 20 ul Proteinase K to monolayer cells Collect cell lysate with a rubber policeman Mix thoroughly by pipetting up and down No cell clumps should be visible before proceeding with the next step Pipet the lysed mixture into 2 0 ml reaction tube 1 Sample Lysis Place the tube with the sample into a thermomixer and incubate under continuously shaking for 10 minutes at 65 C Add the Internal Control 2 Binding of the RNA Add 400 ul Binding Solution to the tube with the lysed sample and mix the sample completely by pipetting up and down or by vortexing Transfer 650 ul of the sample into the RTA Spin Filter Set Close the tube incubate for 1 min at RT and centrifuge for 1 minute at 5 900 X g 8 000 rpm Discard the filtrate and transfer the residual sample into the RTA Spin Filter Set Close the cap incubate for 1 min at RT and centrifuge at 5 900 x g 8 000 rpm for 1 min Discard the RTA Receiver Tube with filtrate and place the RTA Spin Filter into a new RTA Receiver Tube 3 First Washing of the RTA Spin Filter Add 600 ul Wash Buffer R1 to the RTA Spin Filter and centrifuge for 1 min at 5 900 x g 8 000 rpm Discard the filtrate and place the RTA Spin Filter into a new RTA Receiver Tube 4 Second Washing of the RTA Spin Filter Add 600 ul Wash Buffer R2 to the RTA Spin Filter and centrifuge for 1 min at 5 900 x g 8 000 rpm Discard the filtrate pla2. 5415 D from Eppendorf The indicated rpm amounts are referring to this centrifuge Protocol 1 Isolation of viral RNA from serum plasma or other cell free body fluids Please read the instructions carefully and conduct the prepared procedure Important Preheat the prospective volume of the Elution Buffer R to 80 for the final elution step The protocol has been optimized for the isolation of RNA from body fluids of 200 ul For samples which have a smaller volume than 200 ul please fill up to a total volume of 200 ul with ddH2O 1 Sample Lysis Mix samples smaller than 200 ul with ddH O to a total volume of 200 ul Transfer the sample in a 2 0 ml Receiver Tube Add 600 ul Lysis Buffer RV 20 ul Carrier RNA and 20 ul Proteinase K to the sample Close the cap and vortex shortly Place the Receiver Tube into a thermomixer and incubate under continuously shaking at 65 C for 10 min Add the Internal Control 2 Binding of the RNA Add 400 ul Binding Solution to the tube with the lysed sample and mix the sample completely by pipetting up and down or by vortexing Transfer 650 ul of the sample into the RTA Spin Filter Set Close the tube incubate for 1 min at RT and centrifuge for 1 minute at 5 900 x g 8 000 rpm Discard the filtrate and transfer the residual sample into the RTA Spin Filter Set Close the cap incubate for 1 min at RT and centrifuge at 5 900 x g 8 000 rpm for 1 min Discard the RTA Receiver Tube with filtrate and place the RT
3. 556 E Mail eppendorf eppendorf com Internet www eppendorf com SIGMA Laborzentrifugen GmbH 37507 Osterode am Harz Germany Phone 49 5522 5007 0 Fax 49 5522 5007 12 E Mail info sigma zentrifugen de Internet www sigma zentrifugen de 24 Invisorb Spin Virus RNA Mini Kit 0515 stratecee molecular STRATEC Molecular GMbH Robert R ssle Str 10 13125 Berlin Germany Phone 49 30 94 89 29 01 Fax 49 30 94 89 29 09 E mail info berlin stratec com www stratec com 1A5j 05 2015
4. RNA from swab material Protocol 3 solation of viral RNA from cell culture supernatants Protocol 4 Isolation of viral RNA from 1 x 10 mammalian cells Protocol 5 solation of viral RNA from max 20 mg tissue samples Protocol 6 Isolation of virus RNA from max 50 mg stool samples Protocol 7 solation of viral RNA whole blood Troubleshooting Appendix General notes on handling RNA Storage of RNA Ordering information Invisorb Spin Virus RNA Mini Kit 0515 oO O O O DOAN GBD ODO A a a RA N N N N NNN AH ABH Ba SB SBS SS Ss 2 2 2 m id A WO WWD OO WON Oa a fF A BP WO ND NY VY O O Kit content of the Invisorb Spin Virus RNA Mini Kit Store dissolved Proteinase K at 20 C Store lyophilized Carrier RNA at 2 8 C Store dissolved Carrier RNA at 80 C Store all other kit components at room temperature RT 5 viral RNA extractions 50 viral RNA extractions 250 viral RNA extractions Catalogue No 1040300100 1040300200 1040300300 Lysis Buffer RV 2x2ml 35 ml 170 ml Carrier RNA for 240 ul for 1 2 ml for 3 x2 ml working solution working solution working solution for 250 ul for 1 1 ml for 3 x 2 ml Proteinase K working solution working solution working solution Binding Solution fill with 99 7 Isopropanol 3x1 ml ready to use empty bottle final volume 30 ml empty bottle final volume 120 ml tube with Proteinase K mix thoroug
5. RTA Spin Filter o binding of the viral RNA to the membrane of the RTA Spin Filter o washing of the membrane and elimination of contaminants and ethanol Elution of highly pure viral RNA from the membrane Repeated wash steps make sure that contaminations and enzyme inhibitors are efficiently removed and high purified RNA is eluted in Elution Buffer R or Rnase free water This manual contains 6 protocols Lysis Samples are lysed under denaturing conditions at elevated temperatures Due to the strong denaturing lyses conditions in the presence of Proteinase K and Lysis Buffer RV cells are quickly broken and RNases are inactivated simultaneously The viral RNA is secured The addition of Carrier RNA is necessary for the enhancement of viral RNA recovery so a very small number of viral RNA molecules will also be purified Carrier RNA also stabilizes nucleic acids in samples with very small nucleic acid concentrations Binding viral RNA After adding Binding Solution to optimize the binding of viral RNA to the RTA Spin Filter membrane the lysate will be applied onto the RTA Spin Filter and the viral RNA is bound to the surface of the RTA Filter membrane as the lysate is drawn through by centrifugation Removing residual contaminants Contaminants are efficiently washed away using Wash Buffer R1 and R2 while the viral RNA remains bound to the membrane of the RTA Spin Filter Elution High quality viral RNA is eluted from the membrane using
6. Rnase free water You can also take chloroform resistant plastic ware rinsed with chloroform to inactivate RNases o All buffers must be prepared from DEPC treated Rnase free ddH O 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 moisted 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 o To minimize the risk of infections from potentially infectious material we recommend working under laminar air flow until the samples are lysed oO00 0 Q O This kit should only be used by personnel trained in in vitro diagnostic laboratory practice Storage of RNA Purified RNA can be stored 80 C and is stable for months and years e g precipitated and stored in 70 ethanol 23 Invisorb Spin Virus RNA Mini Kit 0515 Ordering information Product Package size Catalogue No Invisorb Spin Virus RNA Mini Kit 5 preparations 1040300100 Invisorb Spin Virus RNA Mini Kit 50 preparations 1040300200 Invisorb Spin Virus RNA Mini Kit 250 preparations 1040
7. limitation The kit is neither validated for use with bone marrow cultured cells nor for the isolation of total RNA from serum plasma blood tissue or nor for the isolation of viral DNA 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 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 6 Invisorb Spin Virus RNA Mini Kit
8. 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 contaminations 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 gloves if they become contaminated Do not combine components of different kits 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 O O 0 0 Important indications Preparing RNA When preparing viral RNA work quickly during the manual steps of the procedure The Lysis Buffer RV of the Invisorb Spin Virus RNA Mini Kit simplifies viral RNA isolation by combining efficient lyses of the starting material and the inactivation of exogenous and endogenous RNases Extreme care should be
9. recommended for determination of viral RNA 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 11 Invisorb Spin Virus RNA Mini Kit 0515 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 Alternatively it can be mixed with the Carrier RNA Attention don t add directly these Internal Controls to the sample Preparing buffers 5 viral RNA extractions Add 250 ul ddH20 to the tube with Proteinase K mix thoroughly until completely dissolving and store at 20 C Add 240 ul RNase Free Water to the Carrier RNA Mix thoroughly until completely dissolving Binding Solution Wash Buffer R1 and R2 are ready to use 50 viral RNA extractions Add 1 1 ml ddH O to the tube with Proteinase K mix thoroughly until completely dissolving and store at 20 C Fill 30 ml 99 7 Isopropanol molecular biologic grade into the empty bottle Add 1200 ul RNase Free Water to the Carrier RNA Mix thoroughly until completely dissolving Add 20 ml 96 100 ethanol to the bottle Wash Buffer R1 Add 48 ml 96 100 ethanol to each
10. reliable and fast manual isolation and purification of high quality viral RNA from serum urine plasma cerebrospinal fluid other cell free body fluids and cell culture supernatants swab material stool cells and tissue samples For reproducible high yields an appropriate sample storage and quick operation under the rules for RNA operation is essential The purified viral RNA is ready to use for in vitro diagnostic analysis only The isolation protocol and all buffers are optimized to assure a high yield as well as a high purity of purified viral RNA All manual work is reduced to a minimum THE PRODUCT IS INTENDED 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 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
11. require a phenol chloroform extraction Only a minimum of manual work by the user is necessary The procedure is designed to avoid sample to sample cross contaminations and allow safe handling of potentially infectious samples The procedure is highly suited for simultaneous processing of multiple samples Traditional time killing procedures can be replaced using the Invisorb Spin Virus RNA Mini Kit STRATEC Molecular also offers a system for the purification of viral RNA from serum and plasma in a 96 well format Invisorb Virus RNA Mini Kit KF96 The Invisorb Virus RNA HTS 96 Kit X is designed for on X tractor Gene Corbett Robotics The InviMag Virus DNA RNA Mini Kit KFml and the RTP Virus DNA RNA Mini Kit have been developed for isolation of viral DNA or viral RNA If you are interested in using the kit on a laboratory workstation please do not hesitate to contact our technical support 49 0 30 9489 2907 For technical support or further information please contact 49 0 30 9489 2901 2903 2907 2910 or your local distributor The PCR process is covered by US Patents 4 683 195 and 4 683 202 and foreign equivalents owned by Hoffmann La Roche AG 8 Invisorb Spin Virus RNA Mini Kit 0515 Principle and procedure The Invisorb Spin Virus RNA Mini Kit procedure comprises the following steps o lyses of the virus particle o adjustment of the binding conditions followed by the transfer of the sample into the RNA binding
12. the RTA Spin Filter surface Incubate for 3 min and centrifuge at 5 900 x g 8 000 rpm for 1 min 17 Invisorb Spin Virus RNA Mini Kit 0515 Protocol 4 Isolation of viral RNA from 1 x 10 mammalian cells Please read the instructions carefully and conduct the prepared procedure Important Preheat the prospective volume of the Elution Buffer R to 80 for the final elution step Harvest cells Cells grown in suspension Spin up to 1 x 10 cells for 5 min at 1 500 rpm Discard the supernatant and remove all media completely Cells grown in a monolayer In large culture vessels dishes gt 35 mm flasks gt 12 5 cm detach cells by trypsination Transfer the cells to a centrifuge tube and sediment by centrifugation at 1 500 rpm for 5 min Remove the supernatant completely In small culture vessels 96 24 12 6 well plates 35 mm dishes 12 5 cm flasks discard the media completely and continue with the lysis immediately Important Incomplete removal of the cell culture media will inhibit the lysis and dilute the lysate which will affect the binding of RNA to the RTA Spin Filter Disrupt cells by adding Lysis Buffer RV For pelleted cells Loosen cell pellet by flicking the tube and add 600 ul Lysis Buffer RV 200 ul ddH O 20 ul Carrier RNA and 20 ul Proteinase K Close the cap and vortex shortly No cell clumps should be visible before proceeding with the next step Pipet the lysed mixture into a 2 0 ml reaction tube
13. 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 sitratec 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 Invisorb Spin Virus 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 Invisorb Spin Virus RNA Mini Kit to which they apply are listed below as follows Lysis Buffer RV Proteinase K warning danger H302 312 332 412 EUH032 P273 H315 319 334 335 P280 305 351 338 310 405 Wash Buffer R1 warning H302 312 332 412 EUH032 P273 H302 Harmful if swallowed H312 Harmful in contact with skin H332 Harmful if inhaled H412 Harmful to aquatic life with long lasting e
14. 300300 Single components for Invisorb Spin Virus RNA Mini Kit Lysis Buffer RV 35 ml 1040301000 Wash Buffer R1 20 ml 1040303500 Wash Buffer R2 2 x12 ml 1040303600 Elution Buffer R 10 ml 1040304100 Other sample sizes Invisorb Virus RNA HTS 96 Kit X 4 x 96 preparations 7143310300 Invisorb Virus RNA HTS 96 Kit X 24 x 96 preparations 7148310400 Invisorb Virus DNA HTS 96 Kit X 4 x 96 preparations 7142310300 Invisorb Virus DNA HTS 96 Kit X 24 x 96 preparations 7142310400 InviMag Virus RNA Mini Kit KF 96 2 x 96 preparations 7443300100 InviMag Virus RNA Mini Kit KF 96 5 x 96 preparations 7443300200 InviMag Virus DNA Mini Kit KF 96 2 x 96 preparations 7442300100 InviMag Virus DNA Mini Kit KF 96 5 x 96 preparations 7442300200 InviMag Virus DNA RNA Mini Kit KFmL 15 preparations 2441150100 InviMag Virus DNA RNA Mini Kit KEmL 75 preparations 2441150200 RTP Virus DNA RNA Mini Kit 50 preparations 1040100200 RTP Virus DNA RNA Mini Kit 250 preparations 1040100300 Invisorb Spin Virus DNA Mini Kit 50 preparations 1040200200 Invisorb Spin Virus DNA Mini Kit 250 preparations 1040200300 Possible suppliers for lsopropanol Carl Roth 2 Propanol Applichem Sigma Rotipuran gt 99 7 p a ACS ISO 2 Propanol f r die Molekularbiologie 2 Propanol Order no 6752 Order no A3928 Possible suppliers for Centrifuges Order no 59304 1L F Eppendorf AG 22331 Hamburg Germany Phone 49 0 40 53801 0 Fax 49 0 40 53801
15. 80 C in aliquots is recommended Repeated freezing 9 Invisorb Spin Virus RNA Mini Kit 0515 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 RNA yield Use of poor quality starting material influences the RNA yield too The amount of purified RNA in the Invisorb Spin Virus 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 Lysis Buffer RV 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 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
16. A Spin Filter into a new RTA Receiver Tube 3 First Washing of the RTA Spin Filter Add 600 ul Wash Buffer R1 to the RTA Spin Filter and centrifuge for 1 min at 5 900 x g 8 000 rpm Discard the filtrate and place the RTA Spin Filter into a new RTA Receiver Tube 4 Second Washing of the RTA Spin Filter Add 600 ul Wash Buffer R2 to the RTA Spin Filter and centrifuge for 1 min at 5 900 x g 8 000 rpm Discard the filtrate place the RTA Spin Filter again into a new RTA Receiver Tube and repeat the second washing step Remove the residual ethanol by final centrifugation for 4 min at maximum speed 5 Elution of the RNA Place the RTA Spin Filter into a 1 5 ml Elution Tube and add 100 ul of the Elution Buffer R preheated to 80 C directly onto the RTA Spin Filter surface Incubate for 3 min and centrifuge at 5 900 x g 8 000 rpm for 1 min 15 Invisorb Spin Virus RNA Mini Kit 0515 Protocol 2 isolation of viral RNA from swab material Please read the instructions carefully and conduct the prepared procedure Important Preheat the prospective volume of the Elution Buffer R to 80 for the final elution step If the swab is delivered in a stabilization media make sure that these media are suitable for RNA virus stabilization If you are not sure please contact the manufacturer Some RNA virus stabilizing media dissolve the viral particles They may be used by transferring 200 ul directly into the 2 0 ml Receiver Tubes and continue with
17. C directly onto the membrane of the RTA Spin Filter incubate for 3 min and centrifuge for 1 min at 5 900 x g 8 000 rpm discard the RTA Spin Filter and place the eluted viral RNA immediately on ice 13 Invisorb Spin Virus RNA Mini Kit 0515 Lysis procedures For easier handling we recommend to prepare a Master Mix only for the needed amount of samples consisting of Lysis Buffer RV Proteinase K and if required Carrier RNA When preparing the Master Mix it is recommended to use a volume of 5 greater than that required The Master Mix is stable for at least 2h at RT Preparation of a Master Mix Number of Amount of Amount of Amount of samples Lysis Buffer RV Carrier RNA Proteinase K 600 ul sample 20 ul sample 20 ul sample 6 3 9 ml 130 ul 130 ul 8 5 1 ml 170 ul 170 ul 10 6 3 ml 210 ul 210 ul 12 7 8 ml 260 ul 260 ul 16 10 2 ml 340 ul 340 ul 20 12 6 ml 420 ul 420 ul 24 15 0 ml 500 ul 500 ul 32 20 1 mi 670 ul 670 ul 40 25 2 ml 840 ul 840 ul 48 30 0 ml 1000 ul 1000 ul Extraction control Extraction control DNA or RNA must be combined with the provided Carrier RNA in one mixture The vials with Carrier RNA contain 240 ul 1 2 ml or 2 0 ml stock solutions depending on the package size Add the respective amount of Extraction Control Nucleic Acid to the Carrier RNA replace the according amount of RNAse free water Then you may add the respective mixture to the master mix pleas
18. Elution Buffer R or RNase Free Water The eluted RNA is ready to use in different subsequent applications 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 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 RNA contained in such deep frozen samples is stable for months RNA purification should be processed as soon as possible Samples can also be stored in Lysis Buffer RV 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 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
19. ants the correct function of the Invisorb Spin Virus 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 Invisorb Spin Virus 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 Invisorb Spin Virus 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 5 Invisorb Spin Virus RNA Mini Kit 0515 from Germany 49 0 30 9489 2901 2910 from abroad 49 0 30 9489 2903 2907 or contact your local distributor Intended use The Invisorb Spin Virus RNA Mini Kit is the ideal tool for
20. bottle Wash Buffer R2 250 viral RNA extractions Add 2 ml ddH 0O to the tube with Proteinase K mix thoroughly until completely dissolving and store at 20 C Fill 120 ml 99 7 Isopropanol molecular biologic grade into the empty bottle Add 2 ml RNase Free Water to the Carrier RNA Mix thoroughly until completely dissolving Add 80 ml 96 100 ethanol to each bottle Wash Buffer R1 Add 160 ml 96 100 ethanol to each bottle Wash Buffer R2 Equipment and reagents to be supplied by user When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information s please consult the appropriate material safety data sheets MSDS See our webpage www stratec com Microcentrifuge 11 000 rpm Thermomixer 65 C 80 C ddH20 Ethanol 96 100 Isopropanol O O O O 0 The Invisorb Spin Virus 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 Isopropanol Carl Roth 2 Propanol Applichem 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 12 Invisorb Spin Virus RNA Mini Kit 0515 Scheme of the Invisorb Spin Virus RNA Mini Kit Please work quickly and perform all extraction steps at room temperature RT fr Please read the protocols carefully prior to the start o
21. ce the RTA Spin Filter again into a new RTA Receiver Tube and repeat the second washing step Remove the residual ethanol by final centrifugation for 4 min at maximum speed 5 Elution of the RNA Place the RTA Spin Filter into a 1 5 ml Elution Tube and add 100 ul of the Elution Buffer R preheated to 80 C directly onto the RTA Spin Filter surface Incubate for 3 min and centrifuge at 5 900 x g 8 000 rpm for 1 min 18 Invisorb Spin Virus RNA Mini Kit 0515 Protocol 5 Isolation of viral RNA from max 20 mg tissue samples Please read the instructions carefully and conduct the prepared procedure Important Preheat the prospective volume of the Elution Buffer R to 80 for the final elution step Disruption and lysis of the starting material Note To maximize the final yield of viral RNA a complete disruption of tissue sample is important For the O disruption of starting material it is possible to use commercially available rotor stator homogenizer or bead mills It is also possible to disrupt the starting material using mortar and pestle in liquid nitrogen and grind the tissue sample to a fine powder using rotor stator homogenizer 1 Transfer the weighed amount of fresh or frozen starting material in a suitable reaction vesicle for the homogenizer 2 Add 600 ul Lysis Buffer RV and 200l ddH20 vigorously mixed before adding 3 Homogenize the sample 4 Transfer the sample into a 2 0 ml reaction tube and place the homogena
22. d the filtrate and place the RTA Spin Filter into a new RTA Receiver Tube Second Washing of the RTA Spin Filter Add 600 ul Wash Buffer R2 to the RTA Spin Filter and centrifuge for 1 min at 5 900 x g 8 000 rpm Discard the filtrate place the RTA Spin Filter again into a new RTA Receiver Tube and repeat the second washing step Remove the residual ethanol by final centrifugation for 4 min at maximum speed Elution of the RNA Place the RTA Spin Filter into a 1 5 ml Elution Tube and add 100 ul of the Elution Buffer R preheated to 80 C directly onto the RTA Spin Filter surface Incubate for 3 min and centrifuge at 5 900 x g 8 000 rpm for 1 min 19 Invisorb Spin Virus RNA Mini Kit 0515 Protocol 6 Isolation of viral RNA from max 50 mg stool samples Please read the instructions carefully and conduct the prepared procedure Important Preheat the prospective volume of the Elution Buffer R to 80 for the final elution step Sample Preparation 1 2 Ooh amp N Add for each stool sample 400 ul ddH20 into a 2 0 Receiver Tube Transfer with a new glass stick from each stool sample a small piece size of a lentil in the water prefilled 2 0 ml Receiver Tube Resuspend the sample from the glass stick in water and discard the stick Close the tube and vortex each sample vigorously until it is a homogeneous suspension Centrifuge the samples for 5 min at max speed e g at 15 000 rpm Hettich Universal 30 RF Pip
23. e note that this is just possible if all the samples need the same control Notes If you only have indication of amount per reaction please calculate by using eluate and template volume If the extraction control is stable in plasma serum CSF urine respiratory samples whole blood stool transport media or on dried swabs e g armored RNA it can alternatively be added to the sample shortly before beginning sample preparation If the extraction control is naked DNA or RNA it is unstable in plasma serum CSF urine respiratory samples whole blood stool transport media or on dried swabs and must not be added directly to the samples You may add it together with the carrier RNA or after finishing the Lysis Step before continuing with step 2 Refer to the manufacturer s instructions to determine the optimal amount of extraction control for specific downstream applications Using an amount other than that recommended may lead to wrong quantification results 14 Invisorb Spin Virus RNA Mini Kit 0515 Instructions The following notes are valid for all protocols Note The RNA can also be eluted with a lower but not lower than 40 ul or a higher volume of Elution Buffer R depends on the expected yield or needed concentration of RNA Important After extraction place the Elution Tube on ice For a long time storage place the nucleic acids at 20 C or 80 Note The centrifugation steps were made with the Centrifuge
24. e second washing step Remove the residual ethanol by final centrifugation for 4 min at maximum speed Elution of the RNA Place the RTA Spin Filter into a 1 5 ml Elution Tube and add 100 ul of the Elution Buffer R preheated to 80 C directly onto the RTA Spin Filter surface Incubate for 3 min and centrifuge at 5 900 x g 8 000 rpm for 1 min 20 Invisorb Spin Virus RNA Mini Kit 0515 Protocol 7 Isolation of viral RNA from whole blood Please read the instructions carefully and conduct the prepared procedure Important Preheat the prospective volume of the Elution Buffer R to 80 for the final elution step The protocol has been optimized for the isolation of RNA from 50 ul whole human blood EDTA or citrate Please fill up each blood sample to a total volume of 200 ul with ddH2O or PBS buffer Important Animal Blood The useable volume of the blood depend on animal species This is to check for every species 1 Sample Lysis Transfer the sample in a 2 0 ml Receiver Tube Add 600 ul Lysis Buffer RV 20 ul Carrier RNA and 20 ul Proteinase K to the sample Close the cap and vortex shortly Place the Receiver Tube into a thermomixer and incubate under continuously shaking at 65 C for 10 min Add the Internal Control 2 Binding of the RNA Add 400 ul Binding Solution to the tube with the lysed sample and mix the sample completely by pipetting up and down or by vortexing Transfer 650 ul of the sample into the RTA Spin Filter Se
25. endix General notes on handling 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 have to stand 12 hours at 37 C and then autoclave or heat 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 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
26. er can contain Citrate or EDTA and other samples Samples can be fresh lyophilized or frozen provided they have not been frozen and thawed more than ones The procedure can be used for isolation of viral RNA from a broad range of RNA viruses The amount of purified viral RNA in the Invisorb Spin Virus RNA Mini Kit procedure depends on the sample type the virus titer sample source transport storage and age The Invisorb Spin Virus RNA Mini Kit simplifies viral RNA isolation by combining efficient lyses of the starting material and the inactivation of exogenous and endogenous RNases The sample will be lysed in an optimized Lysis Solution and proteins will be degraded during the lyses with Proteinase K The liberated RNA is bound onto the membrane of the RTA Spin Filter Contaminants are removed by repeated wash steps and the purified viral RNA can be eluted in a small volume of Elution Buffer R The isolated viral RNA is ready to use and should be stored at 80 C Yield and quality of isolated viral 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 RNA is ready to use for a broad panel of downstream applications like RNA dot blots cDNA transcription in vitro translation RT PCR TaqMan analysis and array technologies QO O OnO O The purification procedure is rapid and does not
27. ette from each sample 200 ul supernatant and transfer the sample in a new 2 0 ml Receiver Tubes Add 600 ul Lysis Buffer RV to this supernatant and mix vigorously Sample Lysis Add 20 ul Carrier RNA and 20 ul Proteinase K to the homogenate Close the cap and vortex shortly Place the Receiver Tube into a thermomixer and incubate under continuously shaking for 10 minutes at 65 C Add the Internal Control Binding of the RNA Add 400 ul Binding Solution to the tube with the lysed sample and mix the sample completely by pipetting up and down or by vortexing Transfer 650 ul of the sample into the RTA Spin Filter Set Close the tube incubate for 1 min at RT and centrifuge for 1 minute at 5 900 X g 8 000 rpm Discard the filtrate and transfer the residual sample into the RTA Spin Filter Set Close the cap incubate for 1 min at RT and centrifuge at 5 900 x g 8 000 rpm for 1 min Discard the RTA Receiver Tube with filtrate and place the RTA Spin Filter into a new RTA Receiver Tube First Washing of the RTA Spin Filter Add 600 ul Wash Buffer R1 to the RTA Spin Filter and centrifuge for 1 min at 5 900 x g 8 000 rpm Discard the filtrate and place the RTA Spin Filter into a new RTA Receiver Tube Second Washing of the RTA Spin Filter Add 600 ul Wash Buffer R2 to the RTA Spin Filter and centrifuge for 1 min at 5 900 x g 8 000 rpm Discard the filtrate place the RTA Spin Filter again into a new RTA Receiver Tube and repeat th
28. f the preparation procedure transfer 200 ul sample into a 2 0 ml Receiver Tube add 600 ul Lysis Buffer RV 20 ul Carrier RNA and 20 ul Proteinase K or or add 640 ul Mastermix see page 14 incubate for 10 minutes at 65 C in a thermomixer Add the Internal Control to each sample or check page 14 for realization of the optimal binding conditions add 400 ul Binding Solution and mix the sample completely by pipetting up and down or by vortexing transfer 650 ul of the sample on the RTA Spin Filter incubate for 1 min and centrifuge for 1 min at 5 900 x g 8 000 rpm discard the flow through transfer the residual sample into the RTA Spin Filter incubate for 1 min and centrifuge for 1 min at 5 900 x g 8 000 rpm transfer the RTA Spin Filter into a new RTA Receiver Tube pipet 600 ul Wash Buffer R1 onto the RTA Spin Filter and centrifuge for 1 min at 5 900 x g 8 000 rpm discard the flow through and the RTA Receiver Tube transfer the RTA Spin Filter into a new RTA Receiver Tube pipet 600 ul Wash Buffer R2 onto the RTA Spin Filter and centrifuge for 1 min at 5 900 x g 8 000 rpm discard the flow through transfer the RTA Spin Filter into a new RTA Receiver Tube Repeat this washing step to eliminate any traces of ethanol centrifuge again for 1 min at maximum speed discard the RTA Receiver Tube transfer the RTA Spin Filter into a RNase free 1 5 ml Elution Tube pipet 50 100 ul of Elution Buffer R preheated to 65
29. ffects H315 Causes skin irritation H319 Causes serious eye irritation H334 May cause allergy or asthma symptoms or breathing difficulties if inhaled H335 May cause respiratory irritation EUH032 Contact with acids liberates very toxic gas P273 Avoid release to the environment P280 Wear protective gloves protective clothing eye protection face protection P305 P351 P338 IF IN EYES Rinse cautiously with water for several minutes Remove contact lenses if present and easy to do Continue rinsing P310 Immediately call a POISON CENTER or doctor physician P405 Store locked up Emergency medical information can be obtained 24 hours a day from infotrac outside of USA 1 352 323 3500 in USA 1 800 535 5053 7 Invisorb Spin Virus RNA Mini Kit 0515 Product characteristics of the Invisorb Spin Virus RNA Mini Kit up to 200 ul cell free body fluids depends on the sample storage and 20 minutes swab material rinsed liquid from swab source ss PP EE Note The added Carrier RNA will account for most of the eluted RNA max 20 mg tissue sample Quantitative RT PCR is recommended max 50 mg stool sample for determination of the viral RNA yield The Invisorb Spin Virus RNA Mini Kit provides a fast and efficient way for reliable isolation of high quality viral RNA from RNA viruses found in a diverse range of starting material The procedure is suitable for use with plasma or serum eith
30. hly until completely dissolving and store at 20 C Add 240 ul RNase Free Water to the Carrier RNA Mix thoroughly until completely dissolving Binding Solution Wash Buffer R1 and R2 are ready for use with Proteinase K mix thoroughly until completely dissolving and store at 20 C Fill 30 ml 99 7 Isopropanol molecular biologic grade into the empty bottle Add 1 2 ml RNase Free Water to the Carrier RNA Mix thoroughly until completely dissolving Add 20 ml 96 100 ethanol to the bottle Wash Buffer R1 Add 48 ml 96 100 ethanol to 15 ml 20 ml 80 ml Wash Bular Pe ready to use final volume 40 ml final volume160 ml 15 ml 2x12 ml 2x 40 ml Washbulerne ready to use final volume 2 x 60 ml final volume 2 x 200 ml Elution Buffer R 2ml 15 ml 30 ml es Free 2ml 2ml 3x2ml a Spin Filter 5 50 5x50 ao 15 3 x 50 15 x 50 a Tubes 5 50 5 x 50 Elution Tubes 5 50 5 x 50 Manual 1 1 1 Initial steps Add 250 ul ddH20 to the Add 1 1 ml ddH2O to the tube Add 2 ml ddH 0 to each tube with Proteinase K mix thoroughly until completely dissolving and store at 20 C Fill 120 ml 99 7 Isopropanol molecular biologic grade into the empty bottle Add 2 ml RNase Free Water to each tube Carrier RNA Mix thoroughly until completely dissolving Add 80 ml 96 100 ethanol to each bottle Wash Buffer R1 Add 160 ml 96 100 ethanol to each bottle Wash Buffer R2 each bottle Wash Buffer R2 4 Invisorb Spin Vi
31. in Filter Set Close the cap incubate for 1 min at RT and centrifuge at 5 900 x g 8 000 rpm for 1 min Discard the RTA Receiver Tube with filtrate and place the RTA Spin Filter into a new RTA Receiver Tube 3 First Washing of the RTA Spin Filter Add 600 ul Wash Buffer R1 to the RTA Spin Filter and centrifuge for 1 min at 5 900 x g 8 000 rpm Discard the filtrate and place the RTA Spin Filter into a new RTA Receiver Tube 4 Second Washing of the RTA Spin Filter Add 600 ul Wash Buffer R2 to the RTA Spin Filter and centrifuge for 1 min at 5 900 x g 8 000 rpm Discard the filtrate place the RTA Spin Filter again into a new RTA Receiver Tube and repeat the second washing step Remove the residual ethanol by final centrifugation for 4 min at maximum speed 5 Elution of the RNA Place the RTA Spin Filter into a 1 5 ml Elution Tube and add 100 ul of the Elution Buffer R preheated to 80 C directly onto the RTA Spin Filter surface Incubate for 3 min and centrifuge at 8 000 rpm for 1 min 16 Invisorb Spin Virus RNA Mini Kit 0515 Protocol 3 Isolation of viral RNA from cell culture supernatants Please read the instructions carefully and conduct the prepared procedure Important Preheat the prospective volume of the Elution Buffer R to 80 for the final elution step 1 Sample Lysis Transfer 200 ul of the cell culture supernatant cell culture media into a 2 0 ml Receiver Tube Add 600 ul Lysis Buffer RV 20 ul Carrier RNA and 20 ul Pr
32. ivity 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 Handling of RTA Spin Filter Due to the sensitivity of viral RNA amplification technologies the following precautions are necessary when handling RTA Spin Filter to avoid cross contamination between sample preparations carefully apply the sample or solution to the RTA Spin Filter pipet the sample into the filter without wetting the rim of the column always change pipet tips between liquid transfers we recommend the use of aerosol barrier pipet tips avoid touching the RTA Spin Filter membrane with the pipet tip oO000 0 Yield and quality of 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 RNA 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
33. ixing of the sample with Binding Solution Incomplete elution Comments and suggestions After lysis spin lysate to pellet debris and continue with the protocol using the supernatant Increase g force and or centrifugation time Reduce the amount of starting material All centrifugation steps should be conducted at room temperature Make sure that the cell culture medium is complete removed after the cell harvest Mix sample sufficient by pipetting up and down with Binding Solution prior to transfer the sample onto the RTA Spin Filter Prolong the incubation time with preheated Elution Buffer R to 5 10 min or repeat elution step once again RNA degraded Inappropriate handling of the starting material The RNA purification protocol should be performed quickly see also General notes on handling RNA page 22 Cell pellets stored at 80 C for later processing should be immediately frozen after cell harvest by liquid nitrogen treatment Viral RNA does not perform well in downstream applications e g RT PCR Ethanol carryover during elution Salt carryover during elution Increase g force or centrifugation time when drying the RTA Spin Filter Ensure that Wash Buffer R1 and R2 are at room temperature Check up Wash Buffer R1 and R2 for salt precipitates If there are any precipitates solve these precipitates by careful warming 22 Invisorb Spin Virus RNA Mini Kit 0515 App
34. oteinase K to the sample Close the cap and vortex shortly Place the tube into a thermomixer and incubate under continuously shaking for 10 minutes at 65 C Add the Internal Control Binding of the RNA Add 400 ul Binding Solution to the tube with the lysed sample and mix the sample completely by pipetting up and down or by vortexing Transfer 650 ul of the sample into the RTA Spin Filter Set Close the tube incubate for 1 min at RT and centrifuge for 1 minute at 5 900 x g 8 000 rpm Discard the filtrate and transfer the residual sample into the RTA Spin Filter Set Close the cap incubate for 1 min at RT and centrifuge at 5 900 x g 8 000 rpm for 1 min Discard the RTA Receiver Tube with filtrate and place the RTA Spin Filter into a new RTA Receiver Tube First Washing of the RTA Spin Filter Add 600 ul Wash Buffer R1 to the RTA Spin Filter and centrifuge for 1 min at 5 900 x g 8 000 rpm Discard the filtrate and place the RTA Spin Filter into a new RTA Receiver Tube Second Washing of the RTA Spin Filter Add 600 ul Wash Buffer R2 to the RTA Spin Filter and centrifuge for 1 min at 8 000 rpm Discard the filtrate place the RTA Spin Filter again into a new RTA Receiver Tube and repeat the second washing step Remove the residual ethanol by final centrifugation for 4 min at maximum speed Elution of the RNA Place the RTA Spin Filter into a 1 5 ml Elution Tube and add 100 ul of the Elution Buffer R preheated to 80 C directly onto
35. rus RNA Mini Kit 0515 Symbols Ass Manufacturer Lot Lot number Catalogue number Expiry date Consult operating instructions Temperature limitation ox BRE Do not reuse Storage All buffers and kit components except the dissolved Proteinase K and the Carrier RNA of the Invisorb Spin Virus RNA Mini Kit should be stored well sealed and dry at room temperature RT and are stable for at least 12 months under these conditions Carrier RNA is stable for at least 12 months Proteinase K Dissolved Proteinase K must be stored at 20 C Dividing Proteinase K into aliquots and storage at 20 C is recommended Carrier RNA Lyophilized Carrier RNA can be stored at 2 8 C or RT but the recommendation for long time storage is 20 C and is stable for at least 12 months Dissolved Carrier RNA must be stored at 80 C but repeated freezing and thawing will degrade the Carrier RNA and reduce the functionality of the kit Therefore dividing Carrier RNA into aliquots and storage at 80 C is recommended Dissolved Carrier RNA is stable for at least 6 months Wash Buffer R1 and R2 charged with ethanol should be appropriate sealed Before every use make sure that all components have room temperature If there are any precipitates within the provided solutions solve these precipitates by warming carefully Room temperature is defined as range from 15 30 C Quality control and product warranty STRATEC Molecular warr
36. step 1 1 Sample Lysis A If you get a swab without transport media please follow the instructions below o rinse each swab with 500 ul cooled water or cooled PBS and use a 200 ul aliquot of the liquid for viral RNA extraction o transfer each sample in a 2 0 ml Receiver Tube and follow the protocol B If you want use the swab direct place the swab in a 2 0 ml Receiver Tube and add 200 ul ddH 0 Add 600 ul Lysis Buffer RV 20 ul Carrier RNA and 20 ul Proteinase K to the sample Close the cap and vortex shortly Place the Receiver Tube into a thermomixer and incubate under continuously shaking for 10 min at 65 C Add the Internal Control 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 tube The removing of the swab from the 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 2 Binding of the RNA Add 400 ul Binding Solution to the tube with the lysed sample and mix the sample completely by pipetting up and down or by vortexing Transfer 650 ul of the sample into the RTA Spin Filter Set Close the tube incubate for 1 min at RT and centrifuge for 1 minute at 5 900 x g 8 000 rpm Discard the filtrate and transfer the residual sample into the RTA Sp
37. stratecee molecular User manual A s e e e e e Invisorb Spin Virus RNA Mini Kit for purification of viral RNA from up to 200 ul serum plasma and other cell free body fluids cell culture supernatants rinse liquidfrom swabs stool sample cells fresh or frozen tissue samples 1040300X0 taal STRATEC Molecular GmbH D 13125 Berlin Instruction for the Invisorb Spin Virus RNA Mini Kit The Invisorb Spin Virus RNA Mini Kit is the ideal tool using Invisorb technology for the isolation and purification of high quality viral RNA from RNA viruses contained in up to 200 ul serum plasma cerebrospinal fluid other cell free body fluids and cell culture supernatants swab material rinse liquid from swabs stool samples max 50 mg cells 1 x 10 mammalian cells and fresh or frozen tissue samples max 20 mg for in vitro diagnostic purposes using a spin filter format Fresh or frozen plasma or serum from blood treated with anticoagulants like EDTA or citrate but not with heparin as well as small samples whole blood can be used The kit is neither suitable for isolation of total RNA from whole blood blood stains cultured or isolated cells tissue samples bacteria fungi plants nor for purification of viral DNA 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 In
38. t Close the tube incubate for 1 min at RT and centrifuge for 1 minute at 5 900 X g 8 000 rpm Discard the filtrate and transfer the residual sample into the RTA Spin Filter Set Close the cap incubate for 1 min at RT and centrifuge at 5 900 x g 8 000 rpm for 1 min Discard the RTA Receiver Tube with filtrate and place the RTA Spin Filter into a new RTA Receiver Tube 3 First Washing of the RTA Spin Filter Add 600 ul Wash Buffer R1 to the RTA Spin Filter and centrifuge for 1 min at 5 900 x g 8 000 rpm Discard the filtrate and place the RTA Spin Filter into a new RTA Receiver Tube 4 Second Washing of the RTA Spin Filter Add 600 ul Wash Buffer R2 to the RTA Spin Filter and centrifuge for 1 min at 5 900 x g 8 000 rpm Discard the filtrate place the RTA Spin Filter again into a new RTA Receiver Tube and repeat the second washing step Remove the residual ethanol by final centrifugation for 4 min at maximum speed 5 Elution of the RNA Place the RTA Spin Filter into a 1 5 ml Elution Tube and add 100 ul of the Elution Buffer R preheated to 80 C directly onto the RTA Spin Filter surface Incubate for 3 min and centrifuge at 5 900 x g 8 000 rpm for 1 min 21 Invisorb Spin Virus RNA Mini Kit 0515 Troubleshooting Problem probable cause Clogged RTA Spin Filter Insufficient disruption or homogenization of the starting material Little or no viral RNA eluted Incomplete removal of cell culture medium Insufficient m
39. taken to avoid contaminations with RNases when handling Elution Buffer R Storing samples Frozen Serum or plasma samples must not be thawed more than ones Repeated freeze thawing leads to denaturation and precipitation of proteins resulting in reduced viral titers and therefore reduced yields of viral nucleic acids In addition cryoprecipitate formed during freeze and thawing will clog the RTA Spin Filter membrane 10 Invisorb Spin Virus RNA Mini Kit 0515 Adding carrier RNA Carrier RNA serves two purposes Firstly it enhances the binding of viral acids to the RTA Spin Filter membrane 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 RNA degradation in the rare event that RNase molecules are not denaturated by the chaotropic salt and detergents in the Lysis Buffer RV If Carrier RNA is not added to the Lysis Buffer RV this may lead to reduced viral RNA recovery The use of an internal control is recommended when using the Invisorb Spin Virus RNA Mini Kit in combination with diagnostic amplification systems Internal Control RNA and reconstituted Carrier RNA should be added to the Lysis Buffer RV and mixed thoroughly by inverting the tube 10 times To avoid foaming do not vortex Eluting viral RNA For downstream applications that require small staring volumes using viral RNA eluted in 40 ul Elution Buffer R may increase assay sensit
40. te for longer storage at 20 C or use the sample immediately for isolation of viral RNA following the protocol step 1 using a mortar and pestle and liquid nitrogen 1 Transfer the weighed amount of fresh or frozen starting material under liquid nitrogen and grind the material to a fine tissue powder 2 Transfer the powder into a 2 0 ml Receiver Tube Do not allow the sample to thaw 3 Add 600 ul Lysis Buffer RV and 200 ul ddH20 to the sample and follow protocol step 1 Sample Lysis Add 20 ul Carrier RNA and 20 ul Proteinase K to the homogenate Close the cap and vortex shortly Place the Receiver Tube into a thermomixer and incubate under continuously shaking for 10 minutes at 65 C Add the Internal Control Binding of the RNA Add 400 ul Binding Solution to the tube with the lysed sample and mix the sample completely by pipetting up and down or by vortexing Transfer 650 ul of the sample into the RTA Spin Filter Set Close the tube incubate for 1 min at RT and centrifuge for 1 minute at 5 900 X g 8 000 rpm Discard the filtrate and transfer the residual sample into the RTA Spin Filter Set Close the cap incubate for 1 min at RT and centrifuge at 5 900 x g 8 000 rpm for 1 min Discard the RTA Receiver Tube with filtrate and place the RTA Spin Filter into a new RTA Receiver Tube First Washing of the RTA Spin Filter Add 600 ul Wash Buffer R1 to the RTA Spin Filter and centrifuge for 1 min at 5 900 x g 8 000 rpm Discar
41. visorb Eppendorf 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 Invisorb is a registered trademark 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 2 Invisorb Spin Virus RNA Mini Kit 0515 Contents Kit content of the Invisorb Spin Virus RNA Mini Kit Symbols Storage Quality controland product warranty Intended use Product use limitation Safety information Product characteristics of the Invisorb Spin Virus RNA Mini Kit Principle and procedure Lysis Binding viral RNA Removing residual contaminants Elution Sampling and storage of starting material Important points before starting a protocol Important indication Yield and quality of viral RNA Internal control IC Extraction control Preparing buffers Equipment and reagents to be supplied by user Scheme of the Invisorb Spin Virus RNA Mini Kit Lysis procedures Preparation of a Master Mix Extraction control Instructions Protocol 1 Isolation of viral RNA from cell free body fluids serum plasma Protocol 2 solation of viral
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