RTP DNA/ RNA Virus Mini Kit User manual
Contents
1. 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 nucleic acid degradation in the rare event that RNase or DNase molecules are not denaturated by the salts and detergents in the Lysis Buffer in the Extraction Tube Internal Control The use of an internal control is recommended when using the RTP Virus DNA RNA Mini Kit in combination with diagnostic amplification systems STRATEC Molecular provides the needed Control DNA Detection Assay Eluting viral RNA and DNA For downstream applications that require small starting volumes elution volume may be reduced to 40 ul Elution Buffer R The volume of eluate recovered may be up to 5 ul less than the volume of elution buffer applied to the RTA Spin Filter The volume of eluate recovered depends on the nature of the sample Handling of RTA Spin Filter Due to the sensitivity of viral DNA RNA amplification technologies the following precautions are necessary when handling RTA Spin Filter to avoid cross contamination between sample preparations 1 carefully apply the sample or solution to the RTA Spin Filter pipet the sample into the filter without wetting the rim of the column 2 always change pipet tips between liquid transfers we recommend the use of aerosol barrier p2. Virus RNA Kit KF96 5 x 96 preparations 7443300200 InviMag Virus DNA Kit KF96 1 x 96 preparations 7442300100 InviMag Virus DNA Kit KF96 5 x 96 preparations 7442300200 InviMag Virus DNA RNA Mini Kit 50 preparations 1440100200 InviMag Virus DNA RNA Mini Kit 250 preparations 1440100300 InviMag Virus DNA RNA Mini Kit KFmL 15 preparations 2441150100 InviMag Virus DNA RNA Mini Kit KFmL 75 preparations 2441150200 Invisorb Spin Virus DNA Mini Kit 50 preparations 1040200200 Invisorb Spin Virus DNA Mini Kit 250 preparations 1040200300 Possible suppliers for lsopropanol Carl Roth Applichem Sigma 2 Propanol 2 Propanol f r die Molekularbiologie 2 Propanol Rotipuran gt 99 7 p a ACS ISO Ordering No 6752 Ordering No A3928 Possible suppliers for Centrifuges Eppendorf AG 22331 Hamburg Germany Phone 49 0 40 53801 0 Fax 49 0 40 53801 556 E Mail eppendorf eppendorf com Phone 49 5522 5007 0 Fax 49 5522 5007 12 SIGMA Laborzentrifugen GmbH 37507 Osterode am Harz Germany Ordering No 59304 1L F E Mail info sigma zentrifugen de Internet www eppendorf com Internet www sigma zentrifugen de 21 RTP DNA RNA Virus Mini Kit 0113 stratecee molecular STRATEC Molecular GmbH Robert Rossle Str 10 13125 Berlin Germany Phone 49 30 94 89 29 01 Fax 49 30 94 89 29 09 E mail info berlin stratec com www stratec com 1A4j01 01 2013
3. 25 C Drying dissolving and pipetting DNA Avoid overdrying genomic DNA after ethanol precipitation It is better to let it air dry than to use a vacuum although vacuum drying can be used with caution Plasmid DNA and other small circular DNAs can be vacuum dried To help dissolve the DNA carefully invert the tubes several times after adding buffer and tap the tube gently on the side Alternatively let the DNA stand in buffer overnight at 2 to 8 C Minimize vortexing of genomic DNA since this can cause shearing 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 Regular pipette tips pose no problem for plasmid DNA and other small Quantification Quantification of DNA and RNA from this assay must be done by means of qPCR or Reverse Transcriptase qPCR All other methods will be disturbed by the included Carrier Nucleic Acids as well as DNA or RNA which is co purified 19 RTP DNA RNA Virus Mini Kit 0113 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
4. These tubes are generally RNase free o Keep isolated RNA on ice O 0 0 0 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 Quantification Quantification of DNA and RNA from this assay must be done by means of qPCR or Reverse Transcriptase qPCR All other methods will be disturbed by the included Carrier Nucleic Acids as well as DNA or RNA which is co purified 20 RTP DNA RNA Virus Mini Kit 0113 Ordering information Product Package size Catalogue No RTP DNA RNA Virus Mini Kit 5 preparations 1040100100 RTP DNA RNA Virus Mini Kit 50 preparations 1040100200 RTP DNA RNA Virus Mini Kit 250 preparations 1040100300 Single components for RTP DNA RNA Virus Mini Kit Extraction Tube containing solid formula 10 tubes 1040101500 of lysis reagents incl DNA control Wash Buffer R1 add 20 ml Ethanol 20 ml 1040103500 Wash Buffer R2 add 48 ml Ethanol 12 ml 1040103600 Elution Buffer R 15 ml 1040104100 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 7143310400 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 Kit KF96 1 x 96 preparations 7443300100 InviMag
5. directly onto the RTA Spin Filter surface Incubate for 3 minutes at RT and centrifuge at 11 000 x g 11 000 rpm for 1 minute 15 RTP DNA RNA Virus Mini Kit 0113 Protocol 3 Simultaneous isolation of total nucleic acids DNA and RNA from cell culture supernatants Please read the protocols carefully prior to the start of the preparation procedure Important Note Prewarm the needed amount of Elution Buffer R to 65 C for the final elution step 1 Sample Lysis Mix 200 ul of the cell culture supernatant cell culture media with 200 ul of ddH20 Transfer the sample into the provided Extraction Tube Close the cap and vortex shortly Place the Extraction Tube into a thermomixer and incubate under continuously shaking for 15 minutes at 65 C and for 10 minutes at 95 C which leads to higher sensitivity 2 Binding of the DNA and RNA Add 400 ul Binding Solution to the provided Extraction Tube and mix the sample completely by pipetting up and down or by vortexing Transfer the sample into the Spin Filter Set Close the cap and centrifuge for 2 minutes at 11 000 x g 11 000 rpm 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 500 ul Wash Buffer R1 to the RTA Spin Filter and centrifuges at 11 000 x g 11 000 rpm for 1 minute Discard the RTA Receiver Tube with filtrate and place the RTA Spin Filter into a new RTA Receiver Tube 4 Se
6. 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 2907 2910 or your local distributor Internal controls Extraction control Internal Controls IC from the PCR assay provider can be used as extraction controls if the fragments are longer than 100 bp In this case they have to be added after finalization of the lysis step Attention don t add directly these Internal Controls to the sample 8 RTP DNA RNA Virus Mini Kit 0113 Principle The RTP DNA RNA Virus Mini Kits procedure comprises following steps lysis of the virus particles adjustment of the binding conditions binding the viral nucleic acids in the lysate to the membrane of a Spin Filter RTA washing of the membrane and elimination of contaminants and ethanol elution of the viral nucleic acids SV BO Na This manual contains 4 protocols 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 pro
7. 1 min at 11 000 x g 11 000 rpm discard the flow through and the RTA Receiver Tube transfer the RTA Spin Filter into a new RTA Receiver Tube pipet 700 ul Wash Buffer R2 onto the RTA Spin Filter centrifuge for 1 min at 11 000 x g 11 000 rpm discard the flow through and the RTA Receiver Tube transfer the RTA Spin Filter into a new RTA Receiver Tube to eliminate any traces of ethanol centrifuge again for 4 min at maximum speed discard the RTA Receiver Tube transfer the RTA Spin Filter into a RNase free 1 5 ml Elution Tube pipet 60 ul of Elution Buffer R preheated to 65 C directly onto the membrane of the RTA Spin Filter incubate for 3 min centrifuge for 1 min at 11 000 x g 11 000 rpm discard the RTA Spin Filter and place the eluted viral DNA RNA immediately on ice 13 RTP DNA RNA Virus Mini Kit 0113 Instructions The following notes are valid for all protocols Note The DNA 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 the DNA RNA The eluate contains viral DNA and or viral RNA If the starting material is e g tissue the eluate contains further genomic DNA Important After extraction place the Elution Tube on ice For long time storage place the nucleic acids at 20 C or 80 C Note The centrifugation steps were made with the Centrifuge 5415 D from Eppendorf The indicated rpm amounts are
8. 95 C v Pipet wiy tips ddH20O o 1 5 ml reaction tubes o lsopropanol Ethanol 96 100 2 0 ml reaction tubes optional OOO 0 The RTP Virus DNA RNA Mini Kit is validated with 2 Propanol Rotipuran gt 99 7 p a ACS ISO Order no 6752 from Carl Roth gt Possible suppliers for lsopropanol Carl Roth Applichem Fa Sigma 2 Propanol 2 Propanol fur die Molekularbiologie 2 Propanol Rotipuran gt 99 7 p a ACS ISO Order no A3928 Order no 59304 1L F Order no 6752 12 RTP DNA RNA Virus Mini Kit 0113 Scheme of the RTP DNA RNA Virus Mini Kit Please work quickly and perform all extraction steps at room temperature RT Please read the protocols carefully prior to the start of the preparation procedure transfer 200 ul sample into the provided Extraction Tubes add 200 ul dd H20 for samples which have a smaller volume than 200 ul please fill up to a total volume of 400 ul with ddH O incubate for 15 minutes at 65 C in a thermomixer incubate for 10 minutes at 95 C in a thermomixer optional for optimal binding conditions add 400 ul Binding Solution and mix the sample completely by pipetting up and down or by vortexing transfer the sample on the RTA Spin Filter incubate for 1 min centrifuge for 2 min at 11 000 x g 11 000 rpm discard the flow through with the RTA Receiver Tube put the RTA Spin Filter in a new RTA Receiver Tube pipet 500 ul Wash Buffer R1 onto the RTA Spin Filter centrifuge for
9. A RNA Virus Mini Kit 0113 Troubleshooting Problem clogged RTA Spin Filter low amount of extracted DNA RNA low concentration of extracted DNA RNA DNA RNA does not perform well in downstream applications e g RT PCR or PCR Probable cause Comments and suggestions insufficient lysis homogenization and or too much starting material insufficient lysis incomplete elution insufficient mixing of the sample with Binding Solution incomplete removal of cell culture medium too much Elution Buffer incorrect storage of starting material ethanol carryover during elution salt carryover during elution 18 increase lysis time increase g force and or centrifugation time reduce amount of starting material all centrifugation steps should be conducted at room increase lysis time reduce amount of starting material Overloading of RTA Spin Filter reduces yield prolong the incubation time with prewarmed Elution Buffer R to 5 10 min Do the elution steps twice Take higher volume of Elution Buffer R mix sample sufficient by pipetting up and down with Binding Solution prior to transfer the sample onto the Spin Filter RTA make sure that the cell culture medium is complete removed after the cell harvest elute the DNA RNA twice with lower volume of Elution Buffer R ensure that the storage of starting material was correct avoid thawing of the material increase g force or centrifugati
10. Receiver Tube 4 Second Washing of the RTA Spin Filter Add 700 ul Wash Buffer R2 to the RTA Spin Filter and centrifuge at 11 000 x g 11 000 rpm for 1 minute Discard the RTA Receiver Tube with filtrate and place the RTA Spin Filter into a new RTA Receiver Tube Remove the residual ethanol by final centrifugation for 4 minutes at maximum speed Discard the RTA Receiver Tube with filtrate 5 Elution of the DNA RNA Place the Spin Filter into a 1 5 ml Elution Tube and add 60 ul of the Elution Buffer R prewarmed to 65 C directly onto the RTA Spin Filter surface Incubate for 3 minutes at RTand centrifuge at 11 000 x g 11 000 rpm for 1 minute 14 RTP DNA RNA Virus Mini Kit 0113 Protocol 2 Simultaneous isolation of total nucleic acids DNA and RNA from swab material Please read the protocols carefully prior to the start of the preparation procedure Important Note Prewarm the needed amount of Elution Buffer R to 65 C for the final elution step 1 Sample Lysis Place the swab into the provided Extraction Tube and add 400 ul of ddH O Place the Extraction Tube into a thermomixer and incubate under continuously shaking for 15 minutes at 65 C and for 10 minutes at 95 C which leads to higher sensitivity Important Note To get maximum yield of viral nucleic acids it is essential to leave the swab during the 2 complete lysis time into the reaction tube It is possible to cut the shaft of the swab so that you can cl
11. 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 minutes to remove residual DEPC o Electrophoresis tanks should be cleaned with detergent solution e g 0 5 SDS thoroughly rinsed with RNase free water and then rinsed with ethanol and allowed to dry o Non disposable plasticware should be treated before use to ensure that it is RNase free plastic ware should be thoroughly rinsed with 0 1 M NaOH 1 mM EDTA followed by RNase free water You can also take chloroform resistant plastic ware rinsed with chloroform to inactivate RNases All buffers must be prepared from DEPC treated RNase free ddH20 Change gloves frequently and keep tubes closed Reduce the preparation time as much as possible Use only sterile disposable polypropylene tubes throughout the procedure
12. ash Buffer R1 Add 48 ml 96 100 ethanol to each bottle Wash Buffer R2 250 viral DNA RNA extractions Fill 120 ml 99 7 Isopropanol molecular biologic grade into the empty bottle Add 80 ml 96 100 ethanol to each bottle Wash Buffer R1 Add 200 ml 96 100 ethanol to each bottle Wash Buffer R2 Harvesting 1x10 mammalian cells Cells grown in suspension Spin up to 1 x 10 cells for 5 min at 240 x g 1 500 rpm Discard the supernatant and remove all media completely Cells grown in a monolayer In large culture vessels dishes gt cells by trypsination Transfer the cells to a centrifuge tube and sediment by centrifugation at 240 x g 1 500 rpm for 5 minutes Remove the supernatant completely In small culture vessels 96 24 12 6 well plates flasks distahd th nfedia completely and continue with the lysis immediately Important Some specific cell culture media may inhibit lysis efficiency or downstream reactions For special comments on the diverse sample preparation procedures see the respective protocol on page 16 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 o Microcentrifuge gt 11 000 x g gt 11 000 o Measuring cylinder 250 ml rpm o Disposable gloves Thermomixer 65 C
13. ation procedure is rapid and does not require a phenol chloroform extraction or R Mercaptoethanol 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 RTP DNA RNA Virus Mini Kit All kit components can be stored at room temperature STRATEC Molecular also offers in combination with magnetic bead the InviMag Virus DNA RNA Mini Kit KFml Note Systems isolating simultaneously DNA and RNA from viruses using buffers adapted for the binding of DNA and RNA but the optimal binding conditions of RNA and DNA are different so that such solutions can show a little reduction in sensitivity in comparison to kits optimized to one kind of nucleic acid isolation STRATEC Molecular offers also kits for the separate isolation of viral DNA or viral RNA from serum and plasma in single tube or 96 well format Invisorb Spin Virus RNA Mini Kit the Invisorb Spin Virus DNA Mini Kit The Invisorb Virus RNA HTS 96 Kit X is designed for use on X tractor Gene Corbett Robotics the Invisorb Virus RNA HTS 96 Kit ep for use on epMotion 5075 VAC Eppendorf and the the Invisorb Virus RNA Mini Kit KF96 for use on KingFisher 96 If you are interested in using the kit on a laboratory
14. cessed as soon as possible Samples can also be stored in the dissolved Lysis Buffer in the Extraction Tubel or II for 1 h at room temperature overnight at 4 C and for long 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 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 minutes 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 materia
15. 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 RTP DNA RNA Virus Mini Kit procedure 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 RTP DNA RNA Virus Mini Kit to which they apply is listed are listed below as follows Wash Buffer R1 Extraction Tube L warning danger H302 312 332 412 EUH032 P273 H302 314 EUH208 P260 280 305 351 338 310 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 H314 Causes severe skin burns and eye damage EUH208 Contains Proteinase K May produce an allergic reaction 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 P260 Do not breathe dust fume gas mist vapours spray P280 Wear protective gloves protective clothi
16. cond Washing of the RTA Spin Filter Add 700 ul Wash Buffer R2 to the RTA Spin Filter and centrifuge at 11 000 x g 11 000 rpm for 1 minute Discard the RTA Receiver Tube with filtrate and place the RTA Spin Filter into anew RTA Receiver Tube Remove the residual ethanol by final centrifugation for 4 minutes at maximum speed Discard the RTA Receiver Tube with filtrate 5 Elution of the DNA RNA Place the RTA Spin Filter into a 1 5 ml Elution Tube Add 60 ul of the Elution Buffer R prewarmed to 65 C directly onto the RTA Spin Filter surface Incubate for 3 minutes at RT and centrifuge at 11 000 x g 11 000 rpm for 1 minute 16 RTP DNA RNA Virus Mini Kit 0113 Protocol 4 Simultaneous isolation of total nucleic acids DNA and RNA from tissue biopsies Please read the protocols carefully prior to the start of the preparation procedure Important Note Prewarm the needed amount of Elution Buffer R to 65 C for the final elution step 1 Sample Lysis Transfer 1 10 mg of the tissue sample into the provided Extraction Tube Add 400 ul of ddH O Close the cap and vortex shortly Place the provided Extraction Tube into a thermomixer and incubate under continuously shaking for 15 minutes at 65 C and for 10 minutes at 95 C which leads to higher sensitivity Lysis time at 65 C can be increased up to 30 minutes Important A longer lysis time could reduce the final yield and the quality of some viral RNA species After lysis c
17. d 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 5 RTP DNA RNA Virus Mini Kit 0113 Safety information When and while working with chemicals always wear a suitable lab coat disposable gloves and protective goggles Avoid skin contact Adhere to the legal requirements for working with biological material For more information please consult the appropriate material safety data sheets MSDS These are available online in convenient and compact PDF format at www stratec
18. entrifuge the sample at max speed for 1 minute to spin down unlysed material Transfer the cleared supernatant completely into a 1 5 ml reaction tube not provided 2 Binding of the DNA and RNA Add 400 ul Binding Solution to the 1 5 ml reaction tube and mix the sample completely by pipetting up and down or by vortexing Transfer the sample into the RTA Spin Filter Set Close the cap and centrifuge for 2 minutes at 11 000 x g 11 000 rpm 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 500 ul Wash Buffer R1 to the RTA Spin Filter and centrifuges at 11 000 x g 11 000 rpm for 1 minute Discard the RTA Receiver Tube with filtrate and place the RTA Spin Filter into a new RTA Receiver Tube 4 Second Washing of the RTA Spin Filter Add 700 ul Wash Buffer R2 to the RTA Spin Filter and centrifuge at 11 000 x g 11 000 rpm for 1 minute Discard the RTA Receiver Tube with filtrate and place the RTA Spin Filter into a new RTA Receiver Tube Remove the residual ethanol by final centrifugation for 4 minutes at maximum speed Discard the RTA Receiver Tube with filtrate 5 Elution of the DNA RNA Place the RTA Spin Filter into a 1 5 ml Elution Tube Add 60 ul of the Elution Buffer R prewarmed to 65 C directly onto the RTA Spin Filter surface Incubate for 3 minutes at RT and centrifuge at 11 000 x g 11 000 rpm for 1 minute 17 RTP DN
19. eous isolation of total nucleic acids DNA and RNA from swab material 15 Protocol 3 Simultaneous isolation of total nucleic acids DNA and RNA from cell culture supernatants 16 Protocol 4 Simultaneous isolation of total nucleic acids DNA and RNA from tissue biopsies 17 Troubleshooting 18 Appendix 19 General notes on handling DNA 19 General notes on handling RNA 20 Ordering information 21 2 RTP DNA RNA Virus Mini Kit 0113 Kit contents of RTP DNA RNA Virus Mini Kit Store all kit components at room temperature RT 5 50 250 extractions extractions extractions Catalogue No 1040100100 1040100200 1040100300 Binding Solution 3x1 ml empty bottle empty bottle Me with ray ready to use final volume 30 ml final volume 120 ml sopropano met a a Initial steps oos gt RTA Receiver 1 Wash Buffer R1 Fill 30 ml 99 7 Fill 120 ml 99 7 Wash Buffer R2 isopropanol molecular Isopropanol molecular Binding Solution are biologic grade into the biologic grade into the ready to use empty bottle empty bottle Add 20 ml of 96 Add 80 ml of 96 100 100 ethanol to the bottle ethanol to the bottle Wash Wash Buffer R1 mix Buffer R1 mix thoroughly thoroughly and always and always keep the bottle keep the bottle firmly firmly closed poten Add 200 ml of 96 100 Add 48 ml of 96 ethanol to the bottle Wash 100 ethanol to the bottle Buffer R2 mix thoroughly Wash Buffer R2 mix and alway
20. ic use in countries where the EU Directive 98 79 EC on in vitro medical devices is not recognized Trademarks Invisorb RTP InviMag 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 RTP and InviMag are registered trademarks of STRATEC Biomedical AG The PCR process is covered by US Patents 4 683 195 and 4 683 202 and foreign equivalents owned by Hoffmann La Roche AG 2013 STRATEC Molecular all rights reserved 1 RTP DNA RNA Virus Mini Kit 0113 Contents Kit contents of the RTP DNA RNA Virus Mini Kit 3 Symbols 4 Storage 4 Quality control 4 Intended use 5 Product use limitations 5 Safety information 6 Product characteristic 7 Internal controls Extraction control 8 Principle 9 Sampling and storage of starting material 9 Procedure 10 Procedure 10 Important notes 10 Important points before starting a protocol 10 Important indications 10 Preparing reagents and buffers 12 Equipment and reagents to be supplied by user 12 Scheme 13 Protocols 14 Protocol 1 Simultaneous isolation of total nucleic acids DNA and RNA from cell free body fluids serum plasma synovia urine 14 Protocol 2 Simultan
21. ipet tips 3 avoid touching the RTA Spin Filter membrane with the pipet tip Yield and quality of viral DNA RNA Different amplification systems vary in efficiency depending on the total amount of nucleic acid present in the reaction Eluates from this kit contain viral RNA DNA and Carrier RNA and amounts of Carrier RNA will greatly exceed amounts of viral nucleic acids Yields of viral RNA DNA isolated from biological samples are normally less than 1 pg and therefore difficult to determine photometrically Keep in mind that the Carrier RNA 5 ug per 200 ul sample will account for most of the RNA present The kit is suitable for downstream analysis with NAT techniques for examples qPCR RT qPCR LAMP LCR Diagnostic assays should be performed according to the manufacturer s instructions Quantitative RT PCR is recommended for determination of viral RNA yield In Gel Electrophoresis and in Capillary Electrophoresis RNA extracted with the provided kit looks 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 RTP DNA RNA Virus Mini Kit 0113 Preparing buffers 5 viral DNA RNA extractions Binding Solution Wash Buffer R1 and R2 are ready to use 50 viral DNA RNA extractions Fill 30 ml 99 7 Isopropanol molecular biologic grade into the empty bottle Add 20 ml 96 100 ethanol to the bottle W
22. l as all buffers are optimized to provide high yield and purity of the extracted viral nucleic acids The procedure requires minimal interaction by the user allowing safe handling of potentially infectious samples THE PRODUCT IS INDENTED FOR USE BY PROFESSIONAL USERS ONLY SUCH AS TECHNICIANS PHYSICIANS AND BIOLOGISTS TRAINED IN MOLECULAR BIOLOGICAL TECHNIQUES It is designed to be used with any downstream application employing enzymatic amplification or other enzymatic modifications of DNA RNA followed by signal detection or amplification Any diagnostic results generated by using the sample preparation procedure in conjunction with any downstream diagnostic assay should be interpreted with regard to other clinical or laboratory findings To minimize irregularities in 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 kits are 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 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 implie
23. l 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 RTP DNA RNA Virus Mini Kit procedure using up to 20 mg tissue sample depends on kind of starting material The thawing process could be proceed e g directly in the Extraction Tubes Cell culture supernatants Best results are obtained with fresh material or material that has been immediately frozen and stored at 20 C or 80 C after winning of the cell culture supernatant Repeated freezing and thawing of stored samples can influence the sensitivity Swabs The protocol works with fresh prepared swabs or rinsed liquid from swabs or mouth brushes Best results are obtained with fresh material or material that has been immediately frozen and stored at 20 C or 80 C 9 RTP DNA RNA Virus Mini Kit 0113 Procedure Lysis Samples are lysed under denaturing conditions at elevated temperatures Due to the strong denaturing lyses conditions in the presence of Proteinase K and the Lysis Buffer cells are quickly broken and RNases and DNases are inactivated simultaneously The viral RNA and DNA are secured The addition of Carrier RNA provided in the Extraction Tube is necessary for the enhancement of viral DNA RNA recovery so a very small
24. ng eye protection face protection P310 Immediately call a POISON CENTER or doctor physician Emergency medical information can be obtained 24 hours a day from infotrac outside of USA 1 352 323 3500 in USA 1 800 535 5053 6 RTP DNA RNA Virus Mini Kit 0113 Product characteristic of the RTP DNA RNA Virus Mini Kit up to 200 ul cell free body fluids depends on the sample storage and 20 minutes swab material cell culture supernatants source up to 400 ul rinse liquid from swab Note The added Carrier RNA will r account for most of the eluted RNA 1x 10 mammalian cells Quantitative RT PCR is recommended max 10 mg tissue sample for determination of the viral RNA or DNA yield The RTP DNA RNA Virus Mini Kit provides a fast and efficient way for reliable simultaneous isolation of high quality viral DNA and RNA from a diverse range of starting material The procedures are suitable for use with plasma or serum either can contain citrate or EDTA no heparin and other samples Samples can be fresh lyophilized or frozen provided they have not been frozen and thawed more than ones The amount of purified viral DNA and RNA in the RTP DNA RNA Virus Mini Kit procedure depend on the sample type the virus titer sample source transport storage and age The kits use the patented RTP technology whose special feature is an Extraction Tube containing preformulated solid lysis reagent Proteinase K Ca
25. number of viral DNA 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 DNA RNA to the RTA Spin Filter membrane the lysate will be applied onto the RTA Spin Filter and the viral DNA RNA are 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 DNA RNA remain bound to the membrane of the RTA Spin Filter Elution High quality viral DNA RNA is eluted from the membrane using Elution Buffer R or RNase free water The eluted DNA RNA is ready to use in different subsequent applications 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 1 Always change pipet tips bet
26. on time when drying the RTA Spin Filter ensure that the Wash Buffers are at room temperature check up the Wash Buffers for salt precipitates If there are any precipitates dissolve these precipitates by carefully warming RTP DNA RNA Virus Mini Kit 0113 Appendix General notes on handling DNA Nature of DNA The length and delicate physical nature of DNA require careful handling to avoid damage due to shearing and enzymatic degradation Other conditions that affect the integrity and stability of DNA include acidic and alkaline environments high temperature and UV irradiation Careful isolation and handling of high molecular weight DNA is necessary to ensure it will work well in various downstream applications Damaged DNA could perform poorly in applications such as genomic Southern blotting long template PCR and construction of cosmid libraries Handling fresh and stored material before the extraction of DNA For the isolation of genomic DNA from cells or tissues use either fresh samples or samples that have been quickly frozen in liquid nitrogen and stored at 70 C This procedure minimizes degradation of crude DNA by limiting the activity of endogenous nucleases Storage of DNA Store genomic DNA at 2 to 8 C Storing genomic DNA at 15 to 25 C can cause shearing of DNA particularly if the DNA is exposed to repeated freeze thaw cycles Plasmid DNA and other small circular DNAs can be stored at 2 to 8 C or at 15 to
27. 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 RTP DNA RNA Virus 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 RTP DNA RNA Virus Mini Kit or other STRATEC Molecular products please do not hesitate to contact us A copy of STRATEC Molecular s terms and conditions can be obtained upon request or are presented at the STRATEC Molecular webpage For technical support or further information please contact from Germany 49 0 30 9489 2901 2910 from abroad 49 0 30 9489 2907 or contact your local distributor 4 RTP DNA RNA Virus Mini Kit 0113 Intended use The RTP DNA RNA Virus Mini Kit is the ideal tool for reliable and fast simultaneous isolation of high quality viral DNA and RNA from fresh or frozen human or mammalian serum plasma cerebrospinal fluid cell culture supernatants and other cell free body fluids as well as from swabs or tissue biopsies For reproducible high yields an appropriate sample storage and quick operation under the rules for RNA and DNA operation is essential The purified viral DNA and or RNA is ready to use for in vitro diagnostic analysis only The isolation protocols as wel
28. ose the cap of the Extraction Tube Il It is also possible to do the lysis step with opened cap The removing of the swab from the Extraction Tube Il 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 Binding of the DNA and RNA Add 400 ul Binding Solution to the provided Extraction Tube and mix the sample completely by pipetting up and down or by vortexing Transfer the sample into the RTA Spin Filter Set Close the cap and centrifuge for 2 minutes at 11 000 x g 11 000 rpm 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 500 ul Wash Buffer R1 to the RTA Spin Filter and centrifuges at 11 000 x g 11 000 rpm for 1 minute Discard the RTA Receiver Tube with filtrate and place the RTA Spin Filter into anew RTA Receiver Tube Second Washing of the RTA Spin Filter Add 700 ul Wash Buffer R2 to the RTA Spin Filter and centrifuge at 11 000 x g 11 000 rpm for 1 minute Discard the RTA Receiver Tube with filtrate and place the RTA Spin Filter into anew RTA Receiver Tube Remove the residual ethanol by final centrifugation for 4 minutes at maximum speed Discard the RTA Receiver Tube with filtrate Elution of the DNA RNA Place the RTA Spin Filter into a 1 5 ml Elution Tube Add 60 ul of the Elution Buffer R prewarmed to 65 C
29. referring to this centrifuge Protocol 1 Simultaneous isolation of total nucleic acids DNA and RNA from cell free body fluids Please read the protocols carefully prior to the start of the preparation procedure Important Note Prewarm the needed amount of Elution Buffer R to 65 C for the final elution step The protocol has been optimized for the isolation of total nucleic acids from body fluids of 200 ul For samples which have a smaller volume than 200 ul please fill up to a total volume of 400 ul with ddH20 1 Sample Lysis Mix 200 ul of the sample with 200 ul of ddH20 Transfer the sample into the provided Extraction Tube Close the cap and vortex shortly Place the Extraction Tubes into a thermomixer and incubate under continuously shaking for 15 minutes at 65 C and for 10 minutes at 95 C this leads to higher sensitivity 2 Binding of the DNA and RNA Add 400 ul Binding Solution to the provided Extraction Tube and mix the sample completely by pipetting up and down or by vortexing Transfer the sample into the RTA Spin Filter Set Close the cap and centrifuge for 2 minutes at 11 000 x g 11 000 rpm Discard the RTA Receiver Tube with filtrate and place the RTA Spin Filter into anew RTA Receiver Tube 3 First Washing of the RTA Spin Filter Add 500 ul Wash Buffer R1 to the RTA Spin Filter and centrifuge at 11 000 x g 11 000 rpm for 1 minute Discard the RTA Receiver Tube with filtrate and place the RTA Spin Filter into anew RTA
30. rrier Nucleic Acids and a precisely calibrated amount of an internal DNA Extraction Control The technology allows the reduction of reagent preparation steps and of handling steps with infectious material Using the RTP DNA RNA Virus Mini Kit all types of samples are transferred into the Extraction Tubes together with ddH20 to adjust a final sample volume of 400 ul The prefilled buffer and enzymes lyse the samples stabilize the viral nucleic acids and enhance the selective viral RNA and DNA adsorption to the membrane in the RTA Spin Filter Contaminants are removed by repeated wash steps and the purified viral DNA and or RNA can be eluted in a small volume of Elution Buffer R The advantage of the kit results from the simultaneous isolation of nucleic acids from DNA and RNA viruses That enables the user to test the sample for all kinds of RNA and DNA viruses which are of interest after one preparation Yield and quality of isolated viral DNA and RNA is suitable for any molecular diagnostic detection system The diagnostic tests should be performed according to manufacturer s specifications Due to the high purity the isolated viral DNA RNA is ready to use for a broad panel of downstream applications or can be stored at 80 C for subsequent use RT PCR Real time PCR quantitative RT PCR like TaqMan und LightCycler technology cDNA syntheses Mircoarray application O O O O 7 RTP DNA RNA Virus Mini Kit 0113 The purific
31. s keep the bottle thoroughly and always firmly closed keep the bottle firmly closed 3 RTP DNA RNA Virus Mini Kit 0113 Symbols Manufacturer Lot number Catalogue number Date of manufacture Expiry date Consult operating instructions Temperature limitation Do not reuse HKE a ak Storage All buffers and kit components of the RTP DNA RNA Virus Mini Kit including the Extraction Tube incl Lysis Buffer Proteinase K Carrier RNA and Internal Control DNA should be stored well sealed and dry at room temperature RT and are stable for at least 12 months under these conditions Wash Buffer R1 and R2 charged with ethanol should be appropriately 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 up carefully Room temperature is defined as range from 15 30 C Quality control and product warranty STRATEC Molecular warrants the correct function of the RTP DNA RNA Virus 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
32. stratecee molecular User manual RTP DNA RNA Virus Mini Kit for simultaneous purification of viral DNA and RNA from human and animal serum and plasma samples cell culture supernatants and other cell free body fluids e g urine swabs and tissue biopsies 1040100X0 eaf STRATEC Molecular GmbH D 13125 Berlin Instruction for the RTP DNA RNA Virus Mini Kit The RTP DNA RNA Virus Mini Kit is a well established tool using RTP 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 using a spin filter format Fresh or frozen plasma or serum from blood treated with anticoagulants like EDTA or citrate but not with heparin The customer convenient RTP technology simplifies the process handling reduces the handling steps with infectious material and allows a process monitoring 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 C Compliance with EU Directive 98 79 EC on in vitro medical devices Not for in vitro diagnost
33. ween liquid transfers To avoid cross contaminations we recommend the use of aerosol barrier pipet tips 2 All centrifugation steps are carried out at room temperature 3 When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles 4 Discard gloves if they become contaminated 5 Do not combine components of different kits unless the lot numbers are identical 6 Avoid microbial contamination of the kit reagents 7 To minimize the risk of infections from potentially infectious material we recommend working under laminar air flow until the samples are lysed 8 This kit should only be used by trained personnel 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 10 RTP DNA RNA Virus Mini Kit 0113 Storing samples Frozen serum or plasma samples must not be thawed more than once Repeated freeze thawing leads to denaturation and precipitation of proteins resulting in reduced viral titers and therefore reduced yields of viral nucleic acids In addition cryoprecipitate formed during freeze thawing will clog the RTA Spin Filter membrane
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