InviMag Virus RNA Mini Kit User manual
Contents
1. Important Notes After finishing the extraction protocol the Elution Tube contains the extracted viral RNA We recommend storage of the RNA at 80 C If the viral RNA contains carryover of magnetic particle transfer the viral NA into a 1 5 ml reaction tube and centrifuge at maximum speed for 1 minute and pipette the viral RNA containing supernatant into a new tube 15 InviMag Virus RNA Mini Kit 0113 Troubleshooting Problem Probable cause Comments and suggestions low amount of extracted RNA insufficient lysis incomplete elution low amount of MAP Solution A increase lysis time but prevent too long lysis time because this also decrease yield reduce amount of starting material take higher volume of Elution Buffer R be sure you pipet the Elution Buffer R with the right amount to the right position mix MAP Solution A thoroughly before pipeting to the Deep Well Plate low concentration of extracted RNA too much Elution Buffer incorrect storage of starting material incorrect Wash Buffers elute the RNA with lower volume of Elution Buffer R ensure that the storage of starting material was correctly avoid repeated thawing of the material make sure that the correct amount of ethanol is added to the Wash Buffers and that they are stored correctly degraded RNA incorrect storage of starting material old material ensure that the storage of starting material was correct2. 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 Important notes Important points before starting a protocol Immediately upon receipt of the Product inspect the Product and its components as well as the package for any apparent damages correct quantities and quality If there are any unconformities you have to notify STRATEC Molecular in writing with immediate effect upon inspection thereof If buffer bottles are damaged contact the STRATEC Molecular Technical Services or your local distributor In case of liquid spillage refer to Safety Information see page 6 Do not use damaged kit components since their use may lead to poor kit performance o Always change pipet tips between liquid transfers To avoid cross contamination we recommend the use of aerosol barrier pipet tips o All centrifugation steps are carried out at room temperature When w
3. stratecee molecular User manual InviMag Virus RNA Mini Kit optimized for the InviMag Rack for purification of viral RNA from 200 ul serum plasma cell free body fluids rinse liquid from swabs stool samples or small tissue sample with magnetic beads 1440300x00 taal STRATEC Molecular GmbH D 13125 Berlin Fe MNTAATOTAATOATOVTAVOATOOVOVOOVOOTOWNOVOQVONTOVOOVONTOVOOVOOQTOVIOTONTONTOVONTD Instruction for InviMag Virus RNA Mini Kit The InviMag Virus RNA Mini Kit combines the advantages of the innovative Invisorb technology with easy handling of magnetic particles for a very efficient and reliable isolation of nucleic acids with a high purity The RNA binding magnetic particles are characterized by a high surface area a uniform size distribution and good suspension stability and are therefore highly suitable for high throughput processing The InviMag Virus RNA Mini Kit is suitable for isolation and purification of viral RNA from fresh or frozen plasma serum cell free body fluids as well as rinsed liquid from swabs stool samples or small tissue samples The interplay of the RNA extraction and purification chemistry provided by the InviMag Virus RNA Mini Kit with the InviMag Rack was intensely tested and validated Due to the high purity the isolated RNA is ready to use for in vitro diagnostic analysis and for a broad panel of downstream applications or can be stored at 80 C for subsequent use
4. It is also possible to do the lysis step with opened cap The removing of the swab from 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 Important Notes After finishing the extraction protocol the Elution Tube contains the extracted viral RNA We recommend storage of the RNA at 80 C If the viral RNA contains carryover of magnetic particle transfer the viral NA into a 1 5 ml reaction tube and centrifuge at maximum speed for 1 minute and pipette the viral RNA containing supernatant into a new tube 13 InviMag Virus RNA Mini Kit 0113 Protocol 3 Isolation of viral RNA from tissue biopsy Please read the instructions carefully and conduct the prepared procedure 1 Transfer 1 mg up to max 10 mg of the tissue biopsy into the 2 0 ml Receiver Tube Add 200 ul of ddH20 Then add 600 ul Lysis Buffer RV 20 ul Proteinase K and 20 ul Carrier RNA 2 Place the 2 0 ml Receiver Tube into a thermomixer and incubate under continuously shaking for 10 minutes at 65 C 3 Add 400 ul Binding Solution and 20 uL MAP Solution A and mix the sample pipetting up and down for 4 5 times Incubate the mixture for 5 min at RT under shaking Note Vortex the tube MAP Solution A vigorously before use 4 Transfer the 2 0 ml Receiver Tube in the Magnetic Rack without magnet and insert the Magnetic Stripe Incubate for further 5 min at R
5. SDS thoroughly rinsed with RNase free water and then rinsed with ethanol and allowed to dry Non disposable plastic ware should be treated before use to ensure that it is RNase free Plastic ware should be thoroughly rinsed with 0 1M NaOH 1mM 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 Keep isolated RNA on ice Do not use kit components from other kits with the kit you are currently using unless the lot numbers are identical To minimize the risk of infections from potentially infectious material we recommend working under laminar air flow until the samples are lysed This kit should only be used by personnel trained in in vitro diagnostic laboratory practice 17 InviMag Virus RNA Mini Kit 0113 Ordering information Product Package size Catalogue No InviMag Virus RNA Mini Kit 10 preparations 1440300900 InviMag Virus RNA Mini Kit 50 preparations 1440300200 InviMag Virus RNA Mini Kit 250 preparations 1440300300 Related Products InviMag Virus RNA Kit KF96 1 x 96 preparations 7443300100 InviMag Virus RNA Kit KF96 5 x 96 preparations 7443300200 InviMag Virus DNA Kit KF 96 1 x 96 preparations 7442300100 InviMag Virus DNA Kit KF96 5 x 96 preparations 7442300200 I
6. The kit is neither suitable for isolation of viral DNA bacteria fungi or plants nor for purification of total RNA C Compliance with EU Directive 98 79 EC on in vitro medical devices Not for in vitro diagnostic use in countries where the EU Directive 98 79 EC on in vitro medical devices is not recognized Trademarks InviMag Invisorb Registered marks trademarks etc used in this document even when not specifically marked as such are not to be considered unprotected by law The Invisorb technology is covered by patents and patent applications US 6 110363 US 6 043 354 US 6 037 465 EP 0880535 WO 9728171 WO 9534569 EP 0765335 DE 19506887 DE 10041825 2 WO 0034463 InviMag and Invisorb are registered trademarks of STRATEC Biomedical AG The PCR process is covered by US Patents 4 683 195 and 4 683 202 and foreign equivalents owned by Hoffmann La Roche AG 2013 STRATEC Molecular all rights reserved 1 InviMag Virus RNA Mini Kit 0113 Content Kit contents of InviMag Virus RNA Mini Kit 3 Symbols 4 Storage 4 Quality control 4 Intended use 5 Product use limitation 5 Safety information 6 Product characteristic of the InviMag Virus RNA Mini Kit 7 Sampling and storage of starting material 8 Principle and procedure 8 Yield and quality of viral RNA 9 Important notes 9 Important points before starting a protocol 9 Internal control IC Extraction control 9 Reagents and equipment to be supplied by us
7. magnetic beads by saturation of non specific binding sites of the vials 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 no denaturated by chaotropic salts 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 10 InviMag Virus RNA Mini Kit 0113 Scheme of the InviMag Virus RNA Mini Kit Please read protocols prior the start of the preparation carefully Transfer the sample into the 2 0 ml Receiver Tube and add 600 ul Lysis Buffer RV 20 ul Proteinase K and 20 ul Carrier RNA to the sample Mix by pipetting 5 times Incubate under shaking for 10 minutes at 65 C Add 400 ul Binding Solution and 20 ul MAP Solution A to the lysate Mix by pipetting up and down 5 times Put the tube in the InviMag Rack Incubate for 5 min at RT Insert the Magnetic Stripe Incubate again for 5 min at RT Removal of lysed material by aspiration or pipetting Remove Magnetic Stripe Add 800 ul Wash Buffer R1 Close the vial and mix by vortexing Insert the Magnetic Stripe Incubate for 3 min at RT Removal of the Wash Buffer R1 eluate by aspiration or pipetting Remove Magnetic Stripe Add 800 ul Wash Buffer R2 A Close the vial and mix by vortexing Incubate for 3 min at RT Insert the Magnetic Stripe Incubate f
8. 50 1 5 ml Elution Tube 10 50 5 x 50 Manual 1 1 1 Initial steps Add of 250 ul of ddH20 Add of 1 1 ml of ddH2O Add of 2 ml of ddH20 to to Proteinase K mix thoroughly store at 20 C Dilute Carrier RNA by addition of 240 ul RNase Free Water Wash Buffer R1 is ready to use Wash Buffer R2 is ready to use Binding Solution is ready to use to Proteinase K mix thoroughly store at 20 C Fill 30 ml Ilsopropanol molecular biologic grade into the empty bottle Dilute Carrier RNA by addition of 1 2 ml RNase Free Water Add 30 ml of 96 100 ethanol to the bottle Wash Buffer R1 Add 48 ml of 96 100 ethanol to the bottle Wash Buffer R2 Mix thoroughly and always keep the bottles firmly closed each vial Proteinase K mix thoroughly store at 20 C Fill 120 ml lsopropanol molecular biologic grade into the empty bottle Dilute Carrier RNA by addition of 2 ml RNase Free Water to each vial Add 125 ml of 96 100 ethanol to the bottle Wash Buffer R1 Add 200 ml of 96 100 ethanol to each bottle Wash Buffer R2 Mix thoroughly and always keep the bottles firmly closed 3 InviMag Virus RNA Mini Kit 0113 Symbols Manufacturer o a4 Lot number Catalogue number Date of manufacture Expiry date Consult operating instructions Temperature limitation Do not reuse Qs BLE F Storage All buffers and kit contents of the InviMag Virus RNA M
9. avoid thawing of the material ensure that the starting material is fresh or stored under appropriate conditions for long time storage at 80 C avoid thawing and freezing of the material RNA does not perform well in downstream applications e g RT qPCR ethanol carryover during elution salt carryover during elution increase drying time for removing of ethanol check up the Wash Burffers for salt precipitates If there are any precipitates solve these precipitates by careful warming ensure that the Wash Buffers are at room temperature low A2so A2so ratio from UV measurement eluted RNA is brown colored small part of the magnetic particles are left in the elution centrifuge down at full speed for 1 min and transfer supernatant to a new tube 16 InviMag Virus RNA Mini Kit 0113 Appendix 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 contamination 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 Electrophoresis tanks should be cleaned with detergent solution e g 0 5
10. like described below Dilute Carrier RNA by addition of 1 2 ml RNase Free Water Add 30 ml of 96 100 ethanol to the bottle Wash Buffer R1 Add 48 ml of 96 100 ethanol to the bottle Wash Buffer R2 Mix thoroughly and always keep the bottles firmly closed 250 viral RNA extractions Fill 120 ml Isopropanol molecular biologic grade into the empty bottle Dilute each vial Proteinase K by addition of 2 ml of ddH20 mix thoroughly and store like described below Dilute each vial Carrier RNA by addition of 2 ml RNase Free Water Add 125 ml of 96 100 ethanol to the bottle Wash Buffer R1 Add 200 ml of 96 100 ethanol to each bottle Wash Buffer R2 Mix thoroughly and always keep the bottles firmly closed Dissolved Carrier RNA should be stored at 80 C but repeated freezing and thawing will degrade the RNA and reduce the functionality of the Kit Dividing Carrier RNA into aliquots is recommended Reagents and equipment to be supplied by user Measuring cylinder 250 ml Pipette and pipette tips Disposable gloves ddH20 Vortexer 96 100 ethanol Isopropanol Or O 0 O 0 Oo Possible suppliers for Isopropanol Carl Roth Applichem Sigma 2 Propanol 2 Propanol molecular biology grade 2 Propanol Rotipuran gt 99 7 p a ACS ISO Order no 6752 Order no A3928 Order no 59304 1L F Carrier RNA Carrier RNA serves two purposes Firstly it enhances the binding of viral acids to the
11. T Remove supernatant by aspiration or by pipetting and then remove the Magnetic Stripe 5 Add 800 ul Wash Buffer R1 to the vial Close the vial and mix it by vortexing Incubate for 3 min in the Magnet Rack with inserted Magnetic Stripe Remove supernatant and then remove the Magnetic Stripe 6 Add 800 ul Wash Buffer R2 to the vial Close the vial and mix it by vortexing Incubate for 3 min in the Magnet Rack with inserted Magnetic Stripe Remove supernatant and then remove the Magnetic Stripe Repeat once this Step 7 Insert the Magnetic Stripe and open the lid of the tube for 10 min to evaporate the ethanol completely 8 Remove the Magnetic Stripe then add 100 ul of the Elution Buffer R mix by vortexing or pipetting up and down for three times Incubate at room temperature for 5 min 9 Insert the Magnetic Stripe and incubate for further 3 min Transfer the eluate to the 1 5 ml Elution Tube It contains the viral nucleic acids ready to use Note The viral nucleic acids can also be eluted with a lower volume of Elution Buffer R depends on the expected amounts of viral nucleic acids But pay attention that minimum volume for the elution is 30 ul and that this volume can reduce the maximum yield If quite large amount of viral nucleic acids are expected the volume of Elution Buffer R can be increased Important Notes After finishing the extraction protocol the Elution Tube contains the extracted viral RNA We recommend stora
12. ation or by pipetting then remove the Magnetic Stripe Add 800 ul Wash Buffer R1 to the vial Close the vial and mix it by vortexing Incubate for 3 min in the Magnet Rack with inserted Magnetic Stripe Remove supernatant then remove the Magnetic Stripe Add 800 ul Wash Buffer R2 to the vial Close the vial and mix it by vortexing Incubate for 3 min in the Magnet Rack with inserted Magnetic Stripe Remove supernatant then remove the Magnetic Stripe Repeat once this Step Insert the Magnetic Stripe and open the lid of the tube for 10 min to evaporate the ethanol completely Remove the Magnetic Stripe then add 100 ul of the Elution Buffer R mix by vortexing or pipetting up and down for three times Incubate at room temperature for 5 min Insert the Magnetic Stripe and incubate for further 3 min Transfer the eluate to the 1 5 ml Elution Tube It contains the viral nucleic acids ready to use The viral nucleic acids can also be eluted with a lower volume of Elution Buffer R depends on the expected amounts of viral nucleic acids But pay attention that minimum volume for the elution is 30 ul and that this volume can reduce the maximum yield If quite large amount of viral nucleic acids are expected the volume of Elution Buffer R can be increased Important Notes After finishing the extraction protocol the Elution Tube contains the extracted viral RNA We recommend storage of the RNA at 80 C If the viral RNA contains carryo
13. described in the manual STRATEC Molecular will check the lot and if STRATEC Molecular investigates a problem in the lot STRATEC Molecular will replace the Product free of charge STRATEC Molecular reserves the right to change alter or modify any Product to enhance its performance and design at any time In accordance with STRATEC Molecular s ISO 9001 2000 and ISO EN 13485 certified Quality Management System the performance of all components of the InviMag Virus RNA Mini Kit have been tested separately against predetermined specifications routinely on lot to lot to ensure consistent product quality If you have any questions or problems regarding any aspects of InviMag Virus 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 4 InviMag Virus RNA Mini Kit 0113 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 Intended use The InviMag Virus RNA Mini Kit is designed for extraction and purification of viral RNA samples using magnetic beads and the InviMag Rack The nucleic acid isolation protocol is suitable for preparation of viral RNA from fresh or frozen plasma serum cell free body fluids as well as rinsed liquid from swabs stool samples or
14. e 2 0 ml Receiver Tube in the Magnetic Rack without magnet and insert the Magnetic Stripe Incubate for further 5 min at RT Remove supernatant by aspiration or by pipetting and then remove the Magnetic Stripe Add 800 ul Wash Buffer R1 to the vial Close the vial and mix it by vortexing Incubate for 3 min in the Magnet Rack with inserted Magnetic Stripe Remove supernatant and then remove the Magnetic Stripe Add 800 ul Wash Buffer R2 to the vial Close the vial and mix it by vortexing Incubate for 3 min in the Magnet Rack with inserted Magnetic Stripe Remove supernatant and then remove the Magnetic Stripe Repeat once this Step 9 10 11 Insert the Magnetic Stripe and open the lid of the tube for 10 min to evaporate the ethanol completely Remove the Magnetic Stripe then add 100 ul of the Elution Buffer R mix by vortexing or pipetting up and down for three times Incubate at room temperature for 5 min Insert the Magnetic Stripe and incubate for further 3 min Transfer the eluate to the 1 5 ml Elution Tube It contains the viral nucleic acids ready to use Note The viral nucleic acids can also be eluted with a lower volume of Elution Buffer R depends on the expected amounts of viral nucleic acids But pay attention that minimum volume for the elution is 30 ul and that this volume can reduce the maximum yield If quite large amount of viral nucleic acids are expected the volume of Elution Buffer R can be increased
15. ed The Product with its contents is unfit for consumption InviMag Virus RNA Mini Kit 0113 Safety information When and while working with chemicals always wear a suitable lab coat disposable gloves and protective goggles Avoid skin contact Adhere to the legal requirements for working with biological material For more information please consult the appropriate material safety data sheets MSDS These are available online in convenient and compact PDF format at www stratec com for each STRATEC Molecular Product and its components If buffer bottles are damaged or leaking WEAR GLOVES AND PROTECTIVE GOGGLES when discarding the bottles in order to avoid any injuries STRATEC Molecular has not tested the liquid waste generated by the InviMag Virus RNA Mini Kit procedures for residual risk materials Therefore liquid waste has to be handled and discarded according to local safety regulations European Community risk and safety phrases for the components of the InviMag 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 O danger 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 effects H315 Causes skin irritation H319 Causes serious eye irrita
16. er 10 Scheme 11 Protocol 1 Isolation of viral RNA from cell free body fluids serum plasma CSF synovia urine and from cell culture supernatant cell culture media 12 Protocol 2 Isolation of viral RNA from swabs or 200 ul rinse liquid 13 Protocol 3 solation of viral RNA from tissue biopsy 14 Protocol 4 Isolation of viral RNA from stool samples 15 Troubleshooting 16 Appendix 17 General notes on handling RNA 17 Ordering information 18 InviMag Virus RNA Mini Kit 0113 Kit contents of InviMag Virus RNA Mini Kit Store the MAP Solution A at 4 C Store lyophilized Carrier RNA 2 8 C Store dissolved Proteinase K at 207C and dissolved Carrier RNA at 80 C Store all other kit components at room temperature RT 10 extractions 50 extractions 250 extractions Catalogue No 1440300900 1440300200 1440300300 Lysis Buffer RV 7 ml 35 ml 160 ml Binding solution 5x1iml empty bottle empty bottle fill with 98 100 lsopropanol ready to use final volume 30 ml final volume 120 ml Proteinase K 250 ul 1 1 ml 3x2ml Carrier RNA 240 ul 1 2 ml 3x2ml RNase Free Water 2 ml 2 ml 3x2 ml MAP Solution A 250 ul 1 1 ml 5x1 1 ml Wash Buffer R1 ian ee final one 60 ml final Pre a ml 2x15ml 2x12 ml 2 x 50 ml Wash Buffer R2 ready to use final volume 2 x 60 ml final volume 2 x 250 ml Elution Buffer R 2 ml 15 ml 30 ml 2 0 ml Receiver Tube 10 50 5 x
17. ge of the RNA at 80 C If the viral RNA contains carryover of magnetic particle transfer the viral NA into a 1 5 ml reaction tube and centrifuge at maximum speed for 1 minute and pipette the viral RNA containing supernatant into a new tube 14 InviMag Virus RNA Mini Kit 0113 Protocol 4 Isolation of viral RNA from stool samples Please read the instructions carefully and conduct the prepared procedure 1 Pipet 400 ul ddH O in 1 5 ml reaction tube not provided Add a glass stick to the stool sample and transfer the adherent sample size of a lentil in the prefilled 1 5 ml reaction tube Resuspend the sample in the prefilled water Close the tube and vortex each sample vigorously until it becomes a homogenic suspension Centrifuge the samples for 5 min at 240 x g 15000 rpm e g Hettich Universal 30 RF Please dip carefully the pipette tip about 0 5 mm below the surface and take from there 200 ul supernatant prevent the aspiration of swimming particles and transfer the sample in a 2 0 ml Receiver Tube Add 600 ul Lysis Buffer RV 20 ul Proteinase K and 20 ul Carrier RNA Place the 2 0 ml Receiver Tube into a thermomixer and incubate under continuously shaking for 10 minutes at 65 C Add 400 ul Binding Solution and 20 uL MAP Solution A and mix the sample pipetting up and down for 4 5 times Incubate the mixture for 5 min at RT under shaking Note Vortex the tube MAP Solution A vigorously before use 6 Transfer th
18. ini Kit except MAP Solution A are stable for at least 12 months MAP Solution A is stable for at least 6 months All buffers and kit contents of the InviMag Virus RNA Mini Kit except Carrier RNA dissolved Proteinase K and MAP Solution A should be stored at room temperature RT Proteinase K Dissolved Proteinase K must be stored at 20 C Dividing the Proteinase K into aliquots and storage at 20 C is recommended MAP Solution A should be stored at 4 C Carrier RNA Lyophilized Carrier RNA can be stored at 2 8 C or at RT but the recommendation for long time storage is 20 C 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 Wash Buffer Wash Buffer charged with ethanol should be stored at room temperature and should be appropriate sealed If there are any precipitates within the provided solutions solve these precipitates by careful warming up to room temperature up to 30 C Room temperature RT is defined as range from 15 30 C Quality control and product warranty STRATEC Molecular warrants the correct function of the InviMag 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
19. is not for in vitro diagnostic use in countries where the EU Directive 98 79 EC on in vitro medical devices is not recognized Product use limitation The kit is neither validated for isolation of viral RNA from cultured or isolated cells bacteria or fungi nor for purification of RNA The included chemicals are only useable once Differing of starting material or flow trace may lead to inoperability therefore neither a warranty nor guarantee in this case will be given neither implied nor express The user is responsible to validate the performance of the STRATEC Molecular Product for any particular use STRATEC Molecular does not provide for validation of performance characteristics of the Product with respect to specific applications STRATEC Molecular Products may be used e g in clinical diagnostic laboratory systems conditioned upon the complete diagnostic system of the laboratory the laboratory has been validated pursuant to CLIA 88 regulations in the U S or equivalents in other countries All Products sold by STRATEC Molecular are subject to extensive quality control procedures according to ISO 9001 2000 and ISO EN 13485 and are warranted to perform as described herein Any problems incidents or defects shall be reported to STRATEC 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 intend
20. nviMag Virus DNA Kit KFmL 15 preparations 2442110100 InviMag Virus DNA Kit KFmL 75 preparations 2442110200 InviMag Virus RNA Kit KFmL 15 preparations 2443110100 InviMag Virus RNA Kit KFmL 75 preparations 2443110200 Invisorb Spin Virus RNA Mini Kit 50 preparations 1040300200 Invisorb Spin Virus RNA Mini Kit 250 preparations 1040300300 Invisorb Spin Virus DNA Mini Kit 50 preparations 1040200200 Invisorb Spin Virus DNA Mini Kit 250 preparations 1040200300 InviMag Rack 1 piece 5010320000 Possible suppliers for lsopropanol Carl Roth Applichem Sigma 2 Propanol 2 Pro Propanol molecular biology grade 2 Propanol Rotipuran 299 CA Pay ACS ISO Onder ine A3928 Order no 59304 1L F Order no 6752 18 InviMag Virus RNA 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 1G5j03 01 2013
21. or 3 min at RT Removal of the Wash Buffer R2 eluate by aspiration or pipetting Remove Magnetic Stripe Repeat this step from point A Insert the Magnetic Stripe open the lid Incubate for 10 min at RT to evaporate ethanol Remove Magnetic Stripe Add 100 ul Elution Buffer R Mix by vortexing Incubate for 5 min at RT Insert the Magnetic Stripe Incubate for 3 min at RT Transfer of the eluate with pure viral RNA in a 1 5 ml Elution Tube 11 InviMag Virus RNA Mini Kit 0113 Protocol 1 Isolation of viral RNA from cell free body fluids serum plasma CSF synovia urine and from cell culture supernatant cell culture media Please read the instructions carefully and conduct the prepared procedure Note 4 Transfer 200 ul of the sample serum plasma CSF synovia urine cell culture into a 2 0 ml Receiver Tube and add 600 ul Lysis Buffer RV 20 ul Proteinase K and 20 ul Carrier RNA Close the cap and mix for 10 s Place the 2 0 ml Receiver Tube into a thermomixer and incubate under continuously shaking for 10 minutes at 65 C Add 400 ul Binding Solution and 20 ul MAP Solution A and mix the sample pipetting up and down for 4 5 times Incubate the mixture for 5 min at RT under shaking Vortex the tube MAP Solution A vigorously before use Transfer the 2 0 ml Receiver Tube in the Magnetic Rack without magnet Insert the Magnetic Stripe and incubate for further 5 min at RT Remove supernatant by aspir
22. orking 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 Q O Or O 0 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 9 InviMag Virus RNA Mini Kit 0113 Preparing reagents and buffers 10 viral RNA extractions Dilute Proteinase K by addition of 250 ul ddH20 mix thoroughly and store like described below Dilute Carrier RNA by addition of 240 ul RNase Free Water Binding Solution is ready to use Wash Buffer R1 is ready to use Wash Buffer R2 is ready to use Mix thoroughly and always keep the bottles firmly closed 50 viral RNA extractions Fill 30 ml Isopropanol molecular biologic grade into the empty bottle Dilute Proteinase K by addition of 1 1 ml of ddH2O mix thoroughly and store
23. ple thawing and freezing before isolating the viral RNA should be avoided It leads to denaturation and precipitation of proteins resulting in reduced viral titers and therefore reduced yields of viral nucleic acids In addition cryoprecipitates formed during freeze thawing can give problems If cryoprecipitates are visible they should be pelleted by centrifugation at app 6800 x g for 3 minutes The cleared supernatant should be aspirated without disturbing the pellet and processed immediately This step will not reduce viral titers Best results are obtained with fresh tissue 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 thawing process could be proceed e g directly in Lysis Buffer RV Principle and procedure The InviMag Virus RNA Mini Kit procedure comprises following steps o lysis of the virus particles in the Lysis Buffer RV o binding the viral RNA to the magnetic beads o washing and elimination of ethanol o elution of viral RNA After lysis the viral RNA binds to the magnetic beads contaminations and enzyme inhibitors are efficiently removed during the following three wash steps and highly purified RNA is eluted in Elution Buffer R This manual contains 4 protocols page 13 16 Lysis Samples are ly
24. rb Virus RNA HTS 96 Kit for use on robotic stations vacuum manifold and centrifuges For further information please contact Phone 49 0 30 9489 2901 2910 in Germany and from foreign countries phone 49 0 30 9489 2907 or your local distributor Sampling and storage of starting material Best results are obtained using freshly extracted samples As long as the samples are not shock frosted with liquid nitrogen or are incubated with RNase inhibitors or denaturing reagents the 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 7 InviMag Virus RNA Mini Kit 0113 After collection and centrifugation serum plasma from blood treated with anticoagulants like EDTA or citrate but not with heparin synovial fluid samples or other cell free body fluids swabs as well as stool samples can be stored on ice for 1 2 hours for short time up to 24 h samples may be stored at 20 C For long term storage we recommend freezing samples in aliquots at 807C Frozen plasma or serum samples must not be thawed more than once Multi
25. sed under denaturing conditions at elevated temperatures in the presence of Lysis Buffer RV Carrier RNA and Proteinase K in a 2 0 ml Receiver Tube Processing a large number of samples the preparation of a master mixture of a volume 5 greater than that required for the processing of all samples is recommended Mix the master mix carefully prior to use Binding of the viral nucleic acids After adding Binding Solution and MAP Solution A to the lysate in the 2 0 ml Receiver Tube the viral RNA is bound to the surface of the magnetic beads Removing residual contaminants Contaminants are efficiently washed away using Wash Buffer R1 and R2 while the viral RNA remains bound to the magnetic beads Elution The viral RNA is eluted from the beads using 200 ul Elution Buffer R The eluted viral RNA is ready for use in different subsequent tests e g RNA dot blots cDNA transcription real time PCR quantitative RT PCR like TaqMan und LightCycler technologies or array technologies InviMag Virus RNA Mini Kit 0113 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 from this kit contain both viral RNA and Carrier RNA and amounts of Carrier RNA will greatly exceed amounts of viral nucleic acids Yields of viral RNA isolated from biological samples are normally less than 1 pg and therefore difficult to determine photometrically
26. sh Buffer R1 to the vial Close the vial and mix it by vortexing Incubate for 3 min in the Magnet Rack with inserted Magnetic Stripe Remove supernatant and then remove the Magnetic Stripe 6 Add 800 ul Wash Buffer R2 to the vial Close the vial and mix it by vortexing Incubate for 3 min in the Magnet Rack with inserted Magnetic Stripe Remove supernatant and then remove the Magnetic Stripe Repeat once this Step 7 Insert the Magnetic Stripe and open the lid of the tube for 10 min to evaporate the ethanol completely 8 Remove the Magnetic Stripe then add 100 ul of the Elution Buffer R mix by vortexing or pipetting up and down for three times Incubate at room temperature for 5 min 9 Insert the Magnetic Stripe and incubate for further 3 min Transfer the eluate to the 1 5 ml Elution Tube It contains the viral nucleic acids ready to use Note The viral nucleic acids can also be eluted with a lower volume of Elution Buffer R depends on the expected amounts of viral nucleic acids But pay attention that minimum volume for the elution is 30 ul and that this volume can reduce the maximum yield If quite large amount of viral nucleic acids are expected the volume of Elution Buffer R can be increased 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
27. small tissue samples For reproducible and high yields appropriate sample storage is essential see Sampling and storage of the starting material page 9 Fresh or frozen plasma or serum from blood treated with EDTA or citrate not with heparin from common blood collection systems can be used All utilities reagents and plastic ware necessary for preparation of viral RNA are provided by the InviMag Virus RNA Mini Kit in different package sizes The procedure of the InviMag Virus RNA Mini Kit is optimized for the isolation of viral RNA from up to 200 ul starting material For samples of a smaller volume than 200 ul please adjust a total sample volume of 200 ul with 1 x PBS prior to the start of an isolation protocol 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 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
28. tion 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 in English and German can be obtained 24 hours a day from infotrac outside of USA 1 352 323 3500 in USA 1 800 535 5053 6 InviMag Virus RNA Mini Kit 0113 Product characteristic of the InviMag Virus RNA Mini Kit The InviMag Virus RNA Mini Kit procedure is the ideal tool for efficient extraction and purification of viral RNA from fresh or frozen plasma serum cell free body fluids as well as rinsed liquid from swabs stool samples or small tissue samples using magnetic beads and the InviMag Magnetic Rack Time for Starting material preparation depends on the sample source and about 70 min up to 200 ul serum or plasma storage up to 200 ul cell free body fluids 200 ul rinse liquid from swab Note The added Carrier RNA will account for most of the eluted RNA Quantitative RT Sigol samples PCR is recommended for determination of small
29. tissue samples the viral RNA yield The RNA isolation process is based on the interaction of nucleic acids with coated magnetic particles under adapted buffer conditions The volume of buffers and other liquids necessary for RNA isolation is reduced to a minimum After a sample specific cell lysis in the optimized Lysis Buffer RV in the presence of Proteinase K and Carrier RNA the optimal binding conditions are adjusted by addition of Binding Solution The viral RNA binds to the simultaneously added magnetic particles and is separated from the solution by the InviMag Rack Subsequent to the washing steps of the particle bound nucleic acids the viral RNA is eluted in Elution Buffer R Due to the high purity the eluted viral RNA is ready to use for a broad panel of downstream applications o RT PCR o RT real time PCR o or array technologies The results from downstream applications should be interpreted with regard to other clinical or laboratory findings To minimize irregularities in diagnostic results adeguate controls for downstream applications should be used For the isolation of viral RNA from single samples STRATEC Molecular offers the Invisorb Spin Virus RNA Mini Kit for use in a centrifuge see Ordering information page 18 For automated isolation of viral RNA is offering two further bead based versions the InviMag Virus RNA Mini Kit KFmL the InviMag Virus RNA Mini Kit KF96 and a vacuum based version the Inviso
30. ver of magnetic particle transfer the viral NA into a 1 5 ml reaction tube and centrifuge at maximum speed for 1 minute and pipette the viral RNA containing supernatant into a new tube 12 InviMag Virus RNA Mini Kit 0113 Protocol 2 Isolation of viral RNA from swabs or 200 ul rinse liquid Please read the instructions carefully and conduct the prepared procedure 1a Place the swab into the 2 0 ml Receiver Tube and add 200 ul of ddH O Close the cap and mix by vortexing for 10 s Then add 600 ul Lysis Buffer RV 20 ul Proteinase K and 20 ul Carrier RNA Close the cap and mix for 10 s 1b Transfer 200 ul of rinse liquid or of the stabilization media into the 2 0 ml Receiver Tube Then add 600 ul Lysis Buffer RV 20 ul Proteinase K and 20 ul Carrier RNA Close the cap and mix by vortexing for 10 s 2 Place the 2 0 ml Receiver Tube into a thermomixer and incubate under continuously shaking for 10 minutes at 65 C We recommend breaking the swab and removing it after lysis 3 Add 400 ul Binding Solution and 20 uL MAP Solution A and mix the sample pipetting up and down for 4 5 times Incubate the mixture for 5 min at RT under shaking Note Vortex the tube MAP Solution A vigorously before use 4 Transfer the 2 0 ml Receiver Tube in the Magnetic Rack without magnet and insert the Magnetic Stripe Incubate for further 5 min at RT Remove supernatant by aspiration or by pipetting and then remove the Magnetic Stripe 5 Add 800 ul Wa
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