Viral RNA isolation - MACHEREY
Contents
1. The NucleoSpin RNA Virus kit works with 150 uL serum NucleoSpin RNA Virus F funnel columns allow the processing of 1 mL serum The funnel column of the NucleoSpin RNA Virus F kit allows a high loading capacity as well as a simultaneously small elution volume The prepared nucleic acids are suitable for applications like automated fluorescent DNA sequencing RT PCR or any kind of enzymatic reaction The detection limit for certain viruses depends on the individual procedures for example in house nested RT PCR We highly recommend using internal low copy standards as well as positive and negative controls to monitor the purification amplification and detection processes Carrier RNA poly A RNA poly A potassium salt prepared from ADP with polynucleotide phosphorylase is included for optimal performance Carrier RNA enhances binding of viral nucleic acids to the silica membrane and reduces the risk of viral RNA degradation Please note that eluates of the NucleoSpin RNA Virus kit contain both viral nucleic acids and Carrier RNA with amounts of Carrier RNA that may exceed the amount of viral nucleic acids Therefore it is not possible to quantify the nucleic acids isolated with the kit by photometric or fluorometric methods when using the carrier Thus other methods for quantification such as specific quantitative PCR or RT PCR systems are recommended Furthermore Carrier RNA may inhibit PCR reactions The amount of a2. Viral RNA isolation User manual NucleoSpin RNA Virus NucleoSpin RNA Virus F July 2014 Rev 11 MACHEREY NAGEL www mn net com Viral RNA isolation Protocol at a glance Rev 11 5 Elute highly pure RNA 0 nf 50 uL RNase free H O 70 C RT 1 2 min 11 000 x g 1 min 50 100 uL RNase free H O 70 C RT 1 2 min 3 000 x g 3 min Mini Funnel NucleoSpin RNA Virus NucleoSpin RNA Virus F 1 Lysis of 150 uL sample volume m 1 mL sample volume viruses y 600 uL RAV1 bs 4 mL RAV1 70 C 70 C 5 min 5 min 2 Adjust binding m conditions 600 uL ethanol 4 mL ethanol 3 Bind viral RNA Load sample stepwise Load sample 8 000 x g 3 000 x g 1 min g 3 5 min 4 Wash and Pe dry silica SS 1 t wash 500 uL RAW 1 wash 5 mL RAW membrane 2 wash 600 uL RAV3 2 wash 8mLRAV3 3 wash 200 uL RAV3 3 wash 2 mL RAV3 P 4 8 000 x g a i 3 000 x g D 1s and 2 1 min gt 1st and 274 3 min a 11 000 x g id 3 000 x g g gt 3 5 min 5 3 10 min MACHEREY NAGEL GmbH amp Co KG Neumann Neander Str 6 8 52355 D ren Germany Tel 49 24 21 969 270 Fax 49 24 21 969 199 tech bio mn net com www mn net com Viral RNA isolation Table of contents 1 Components 4 1 1 Kit contents 4 1 2 Reagents consumables and equipment to be supplied by user 6 1 3 About this user manual 6 2 Product description 7 2 1 The basic principle 7 2 2 Kit specificat
3. For isolation of viral RNA in general a Iysis of samples in Buffer RAV1 for 10 min at room temperature 20 25 C will be sufficient For isolation of viral RNA from viscous samples for example sputum or supernatants of tissue suspensions or stool samples a lysis at 70 C may be required For simultaneous isolation of viral RNA and DNA incubation time e g 5 15 min and temperature e g RT 56 C or 70 C should be optimized and adjusted to the sample material used MACHEREY NAGEL 07 2014 Rev 11 9 Viral RNA isolation 2 4 2 5 Remarks regarding elution Pure nucleic acids are finally eluted under low ionic strength conditions with RNase free H O pH about 7 8 or slightly alkaline Buffer RE 5 mM Tris HCl pH 8 5 Elution can be performed in a single step with water elution buffer as indicated in the protocol obtaining at least 80 of the bound nucleic acids To improve sensitivity this eluate can be used in a second elution step increasing the efficiency of elution and concentration of viral nucleic acids slightly Alternatively a second elution step can be performed with an additional volume of water elution buffer releasing practically all bound nucleic acids but resulting in a lower concentrated combined eluate RNA should be eluted with the water supplied and DNA with Elution Buffer RE Buffer RE provides better storage conditions for DNA To elute both types of nucleic acids together use the pH proof
4. 11 Viral RNA isolation 4 Safety instructions The following components of the NucleoSpin RNA Virus kits contain hazardous contents Wear gloves and goggles and follow the safety instructions given in this section GHS classification Only harmful features do not need to be labeled with H and P phrases up to 125 mL or 125 g Mindergef hrliche Eigenschaften m ssen bis 125 mL oder 125 g nicht mit H und P S tzen gekennzeichnet werden Component Hazard contents GHS symbol Hazard Precaution phrases phrases Inhalt Gefahrstoff GHS Symbol H S tze P S tze RAV1 Guanidinium thiocyanate Warning 302 412 260 273 30 60 EUHO031 301 312 330 Guanidiniumthiocyanat Achtung 30 60 RAW Guanidine hydrochloride Warning 226 302 210 233 24 36 ethanol 35 301 312 330 55 403 235 Guanidinhydrochlorid 24 36 Achtung Ethanol 35 55 Hazard phrases H 226 Flammable liquid and vapour Fl ssigkeit und Dampf entz ndbar H 302 Harmful if swallowed Gesundheitssch dlich bei Verschlucken H 412 Harmful to aquatic life with long lasting effects Sch dlich f r Wasserorganismen mit langfristiger Wirkung EUHO031 Contact with acids liberates toxic gas Entwickelt bei Ber hrung mit S ure giftige Gase Precaution phrases P 210 Keep away from heat hot surfaces sparks open flames and other ignition sources No smoking Von Hitze hei en Oberfl chen Funken offenen Flammen sowie anderen
5. Blood or a NucleoSpin Tissue kit see ordering information 5 min If the resulting solution is still turbid centrifuge the mixture for 1 min at 11 000 xg to pellet particles and to prevent clogging of the NucleoSpin RNA Virus Columns Take off the supernatant and continue with step 2 of protocol 5 1 20 MACHEREY NAGEL 07 2014 Rev 11 Viral RNA isolation 7 Appendix 7 1 Troubleshooting Problem Possible cause and suggestions Problems with Carrier RNA Carrier RNA not added See remarks concerning storage of Buffer RAV1 with Carrier RNA section 3 Small Proteinase K digestion may be necessary amounts Use and compare protocols with and without Proteinase K or no viral digestion or prolong incubation time to 10 min nucleic acids in the eluate Viral nucleic acids degraded Samples should be processed immediately If necessary add RNase inhibitor to the sample and ensure appropriate storage conditions up to the processing Check that all buffers have been prepared and stored correctly If in doubt use new aliquots of Buffer RAV1 Carrier RNA and Elution Buffer RE Reduced sensitivity Change the volume of eluate added to the PCR RT PCR Incubation time and temperature are critical for lysis as well as RNA stability For sensitive RNA preparations incubation Problems at room temperature is sufficient without significant loss of with sensitivity For parallel isolation of viral RNA and DNA incubat
6. Z ndquellenarten fernhalten Nicht rauchen P 233 Keep container tightly closed Beh lter dicht verschlossen halten P 260 Do not breathe vapours Dampf nicht einatmen P 273 Avoid release to the environment Freisetzung in die Umwelt vermeiden MACHEREY NAGEL 07 2014 Rev 11 13 Viral RNA isolation Precaution phrases P 301 312 IF SWALLOWED Call a POISON CENTER doctor if you feel unwell BEI VERSCHLUCKEN Bei Unwohlsein GIFTINFORMATIONSZENTRUM Arzt anrufen P 330 Rinse mouth Mund aussp len P 403 235 Store in a well ventilated place Keep cool K hl an einem gut bel fteten Ort aufbewahren For further information please see Material Safety Data Sheets www mn net com Weiterf hrende Informationen finden Sie in den Sicherheitsdatenbl ttern www mn net com 14 MACHEREY NAGEL 07 2014 Rev 11 NucleoSpin RNA Virus 5 NucleoSpin RNA Virus protocols 5 1 Viral RNA isolation from cell free biological fluids Before starting the preparation Check if Lysis Buffer RAV1 and Wash Buffer RAV3 were prepared according to section 3 Preheat an aliquot of Elution Buffer RE RNase free H O to 70 C 1 Lysis of viruses Add 600 pL Buffer RAV1 containing Carrier RNA to 150 uL of the sample Pipette mixture up and down and 150 sample vortex well Incubate for 5 min at 70 C 600 pL RAV1 Incubation time and temperature are critical for lysis as well as RNA stability see troubl
7. 70 C and incubate for 1 2 min at room temperature Centrifuge for 3 min at 3 000 x g 5 mL RAW 3 000 x g 3 min 8 mL RAV3 3 000 x g 3 min 2 mL RAV3 3 000 x g 10 min 50 100 pL RNase free H O 70 C RT 1 2 min 3 000 x g 3 min MACHEREY NAGEL 07 2014 Rev 11 19 NucleoSpin RNA Virus F 6 2 Isolation of viral RNA and DNA from cell free biological fluids This protocol is recommended for the purification of viral RNA and viral DNA for all types of DNA viruses like HBV and CMV for samples of up to 1 mL Before starting the preparation Check if Lysis Buffer RAV1 and Wash Buffer RAV3 were prepared according to section 3 Check if Proteinase K solution not included see ordering information was prepared Preheat an aliquot of Elution Buffer RE RNase free H O to 70 C 1 Lysis of viruses Add 4 mL Buffer RAV1 containing Carrier RNA to 1 mL of the fluid sample Add 133 pL Proteinase K 20 mg mL stock solution to the lysis mixture Pipette mixture up and down and vortex for 10 15 s Incubate for 5 min 1 mL sample a 4 mL RAVI Incubation time and temperature are critical for lysis as well as RNA stability see troubleshooting 133 uL Prot K Proteinase K is not included in this kit but can be ordered separately see ordering information For the isolation of viral DNA and genomic DNA from other matrices not cell 70 C free like blood we recommend the NucleoSpin
8. CLINICAL USE INCLUDING BUT NOT LIMITED TO DIAGNOSTIC THERAPEUTIC AND OR PROGNOSTIC USE No claim or representations is intended for its use to identify any specific organism or for clinical use included but not limited to diagnostic prognostic therapeutic or blood banking It is rather in the responsibility of the user or in any case of resale of the products in the responsibility of the reseller to inspect and assure the use of the DNA RNA protein purification products of MACHEREY NAGEL for a well defined and specific application MACHEREY NAGEL shall only be responsible for the product specifications and the performance range of MN products according to the specifications of in house quality control product documentation and marketing material This MACHEREY NAGEL product is shipped with documentation stating specifications and other technical information MACHEREY NAGEL warrants to meet the stated specifications MACHEREY NAGEL s sole obligation and the customer s sole remedy is limited to replacement of products free of charge in the event products fail to perform as warranted Supplementary reference is made to the general business terms and conditions of MACHEREY NAGEL which are printed on the price list Please contact us if you wish to get an extra copy There is no warranty for and MACHEREY NAGEL is not liable for damages or defects arising in shipping and handling transport insurance for customers excluded or out o
9. Virus F Column into a new 50 mL tube 18 MACHEREY NAGEL 07 2014 Rev 11 NucleoSpin RNA Virus F Load the residual Iysis solution onto the NucleoSpin RNA Virus F Column Centrifuge for 3 5 min at 3 000 xg Discard flow through and place the NucleoSpin RNA Virus F Column into another new 50 mL tube More than two loading steps are not recommended Wash and dry silica membrane Add 5 mL Buffer RAW to the NucleoSpin RNA Virus F Column Centrifuge for 3 min at 3 000 x g Discard flow through This washing step removes contaminants and PCR inhibitors Add 8 mL Buffer RAV3 to the NucleoSpin RNA Virus F Column Centrifuge for 3 min at 3 000 x g Discard flow through with Collection Tube Place the NucleoSpin RNA Virus F Column in a new Collection Tube 50 mL and add 2 mL Buffer RAV3 Centrifuge for 10 min at 3 000 x g to remove ethanolic Buffer RAV3 completely Optional Residual Buffer RAV3 may inhibit subsequent reactions Therefore for subsequent reactions which are extremely ethanol sensitive we recommend repeating the centrifugation with a new Collection Tube 50 mL Or alternatively incubate the NucleoSpin Virus F Columns for 1 min at 70 C to remove any remaining traces of ethanol Elute viral RNA Attach the supplied Collection Tube 0 5 mL with the adaptor to the NucleoSpin RNA Virus F Column Place the assembly in a 50 mL tube not provided Add 50 100 uL RNase free H O preheated to
10. and warranty No other statements or representations written or oral by MACHEREY NAGEL s employees agent or representatives except written statements signed by a duly authorized officer of MACHEREY NAGEL are authorized they should not be relied upon by the customer and are not a part of the contract of sale or of this warranty Product claims are subject to change Therefore please contact our Technical Service Team for the most up to date information on MACHEREY NAGEL products You may also contact your local distributor for general scientific information Applications mentioned in MACHEREY NAGEL literature are provided for informational purposes only MACHEREY NAGEL does not warrant that all applications have been tested in MACHEREY NAGEL laboratories using MACHEREY NAGEL products MACHEREY NAGEL does not warrant the correctness of any of those applications Last updated 07 2010 Rev 03 Please contact MACHEREY NAGEL GmbH amp Co KG Tel 49 24 21 969 270 tech bio mn net com 24 MACHEREY NAGEL 07 2014 Rev 11
11. in a new Collection Tube 2 mL and add 200 uL Buffer RAV3 Centrifuge for 2 5 min at 11 000 x gto remove ethanolic Buffer RAV3 completely Optional Residual Buffer RAV3 may inhibit subsequent reactions Therefore for subsequent reactions which are extremely ethanol sensitive we recommend repeating the centrifugation with a new Collection Tube 2 mL Or alternatively incubate the NucleoSpin RNA Virus Columns for 1 min at 70 C to remove any remaining traces of ethanol Elute viral RNA Place the NucleoSpin RNA Virus Column into a new sterile 1 5 mL microcentrifuge tube not provided Add 50 pL RNase free H O preheated to 70 C and incubate for 1 2 min Centrifuge for 1 min at 11 000 x g To elute viral DNA which was prepared according to the support protocol 4 2 we recommend using Buffer RE preheated to 70 C also see section 2 4 500 pL RAW 8 000 x g 1 min 600 uL RAV3 8 000 x g 1 min 5 200 pL RAV3 11 000 x g 5 min 50 uL RNase free H O 70 C RT 1 2 min 11 000 x g g 1 min MACHEREY NAGEL 07 2014 Rev 11 NucleoSpin RNA Virus 5 2 Isolation of viral RNA and DNA from cell free biological fluids This protocol is recommended for the purification of viral RNA and viral DNA for all types of DNA viruses like HBV and CMV from small samples of up to 150 uL Before starting the preparation Check if Lysis Buffer RAV1 and Wash Buffer RAV3 were prepared accor
12. 1 1 Kit contents continued NucleoSpin RNA Virus F 25 preps REF 740958 Lysis Buffer RAV1 2x 120 mL Wash Buffer RAW 150 mL Wash Buffer RAV3 Concentrate 3x25 mL RNase free H O 13 mL Elution Buffer RE 13 mL Carrier RNA lyophilized 2 x 300 ug NucleoSpin RNA Virus F Columns 25 plus Collection Tubes Collection Tubes 50 mL 25 Collection Tubes 0 5 mL 25 User manual 1 For preparation of working solutions and storage conditions see section 3 Composition of Elution Buffer RE 5 mM Tris HCl pH 8 5 MACHEREY NAGEL 07 2014 Rev 11 5 Viral RNA isolation 1 2 Reagents consumables and equipment to be supplied by user Reagents 96 100 ethanol Consumables 1 5 mL microcentrifuge tubes NucleoSpin RNA Virus or 50 mL tubes NucleoSpin RNA Virus F e Disposable tips Equipment e Manual pipettors Centrifuge for microcentrifuge tubes NucleoSpin RNA Virus or 50 mL tubes NucleoSpin RNA Virus F Vortex mixer Heating block or water bath for 70 C incubation Personal protection equipment lab coat gloves goggles 1 3 About this user manual It is strongly recommended reading the detailed protocol sections of this user manual if the NucleoSpin RNA Virus RNA Virus F kit is used for the first time Experienced users however may refer to the Protocol at a glance instead The Protocol at a glance is designed to be used only as a supplemental tool for quick referencing while
13. dded Carrier RNA may thus be carefully optimized depending on the individual PCR system used MACHEREY NAGEL 07 2014 Rev 11 7 Viral RNA isolation Table 1 Kit specifications at a glance Parameter NucleoSpin RNA Virus NucleoSpin RNA Virus F Technology Silica membrane Silica membrane technology technology Format Mini spin columns Funnel columns Sample material sg 150 uL serum plasma lt 1 mL serum plasma cell free biological fluids cell free biological fluids Fragment size 100 b approx 50 kb 100 b approx 50 kb Typical recovery rates gt 90 gt 90 Typical analysis limit 30 60 cp mL 30 60 cp mL Elution volume 50 uL 50 100 uL Preparation time 30 min 4 6 preps 45 min 2 4 preps Binding capacity 40 ug 30 ug 2 3 Remarks regarding sample quality and preparation Liquid samples Biological fluids or semi fluid samples can be processed e g serum urine or bronchoalveolar lavage For successful nucleic acid purification it is important to obtain a homogeneous clear and non viscous sample before loading onto the NucleoSpin RNA Virus Column Therefore check all samples especially old or frozen ones for presence of precipitates Precipitates remaining after lysis with Buffer RAV1 can be removed by centrifugation Avoid clearing samples before lysis because viruses of interest may be associated with particles or aggregates Incubation with Buffer RAV1 can be prolonged in order to dissolve and digest resi
14. ding to section 3 Check if Proteinase K solution not included see ordering information was prepared Preheat an aliquot of Elution Buffer RE RNase free H O to 70 C 1 Lysis of viruses Add 600 uL Buffer RAV1 containing Carrier RNA to 150 uL of the sample Add 20 uL Proteinase K 20 mg mL stock solution to the lysis mixture Pipette mixture up and down and vortex for 10 15 s Incubate for 5 min 150 pL sample t 70 C ii 600 pL RAV1 Incubation time and temperature are critical for lysis as well as RNA stability see troubleshooting y 20 uL Prot K Proteinase K is not included in this kit but can be ordered separately see ordering information For the isolation of viral DNA and genomic DNA from other matrices not cell 70 C free like blood we recommend the NucleoSpin Blood or NucleoSpin Tissue kit see ordering information 5 min If the resulting solution is still turbid centrifuge the mixture for 1 min at 11 000 xg to pellet particles and to prevent clogging of the NucleoSpin RNA Virus Columns Take off the supernatant and continue with step 2 of protocol 5 1 MACHEREY NAGEL 07 2014 Rev 11 17 NucleoSpin RNA Virus F 6 NucleoSpin RNA Virus F protocols 6 1 Viral RNA isolation from cell free biological fluids Before starting the preparation Check if Lysis Buffer RAV1 and Wash Buffer RAV3 were prepared according to section 3 Preheat an aliquot of Elution Buffer RE RNas
15. dual cell structures precipitates and virus particles RNA however is sensitive and prolonged incubation may cause decreased yields Solid samples tissue samples stool samples Prepare a 10 w v suspension of tissue in buffer e g PBS using commercial homogenization tools rotor stator or bead based homogenization tools etc Centrifuge the suspension in order to remove particles Use the clear particle free supernatant for further processing Nested PCR 8 MACHEREY NAGEL 07 2014 Rev 11 Viral RNA isolation Swab material Incubate swab in a suitable buffer e g PBS or cell culture medium for 30 min Proceed with particle free buffer or medium Blood samples Processing of blood samples is possible if using blood diluted with PBS buffer Using undiluted blood may cause clogging of the silica membrane of the NucleoSpin Virus Binding Plate The amount of PBS buffer added to blood samples has to be optimized for the individual organism As a rule of thumb we recommend to start with 50 uL blood diluted with 50 uL PBS buffer Proteinase K treatment Addition of Proteinase K solution see ordering information is necessary for the isolation of viral DNA or simultaneous viral RNA DNA isolation For isolation of viral RNA Proteinase K treatment is usually not required Proteinase K treatment is recommended for viral RNA isolation when viscous samples have to be processed e g sputum samples Sample Iysis
16. e free H O to 70 C 1 Lysis of viruses Add 4 mL Buffer RAV1 containing Carrier RNA to 1 mL 1 mL sample of the sample Pipette mixture up and down and vortex well Incubate for 5 min at 70 C 4 mL RAV1 Incubation time and temperature are critical for lysis as well as RNA stability see troubleshooting for further hints 7 o If the resulting solution is still turbid centrifuge the mixture a e for 1 min at 11 000 xg to pellet particles and to prevent min clogging of the NucleoSpin RNA Virus F Columns Take off the supernatant and proceed with step 2 2 Adjust binding conditions 4 mL EtOH Add 4 mL ethanol 96 100 to the clear lysis solution and mix by vortexing 10 15 s 3 Bind viral RNA Take the NucleoSpin RNA Virus F Column placed in a Collection Tube and load lysed sample Centrifuge for 3 5 min at 3 000 x g The use of new 50 mL tubes for every step is recommended if infectious material has to be prepared This avoids cross contamination and contamination of centrifuge units For non infectious samples we recommend discarding the flow through and reusing the 50 mL tube for loading and washing steps Additional 50 mL tubes have to be ordered separately Load sample 0 deaq The maximum loading capacity of the NucleoSpin RNA 3 000 X 9 Virus F Column is about 10 mL in order to work cross 3 5 min contamination free If more sample has to be loaded discard flow through and put the NucleoSpin RNA
17. ed pH 6 8 RNase free H O preheated to 70 C Remarks regarding quality control Buffers and NucleoSpin RNA Virus RNA Virus F Columns have been tested with rRNA and MS2 phage RNA The absence of RNases and the yield and efficiency of purification have been investigated with RT PCR 10 MACHEREY NAGEL 07 2014 Rev 11 Viral RNA isolation 3 Storage conditions and preparation of working solutions Attention Buffers RAV1 and RAW contain chaotropic salts Wear gloves and goggles CAUTION Buffer RAV1 contains guanidinium thiocyanate and Buffer RAW contains guanidine hydrochloride which can form highly reactive compounds when combined with bleach sodium hypochlorite DO NOT add bleach or acidic solutions directly to the sample preparation waste All kit components can be stored at room temperature 18 25 C and are stable up to one year Carrier RNA has a limited shelf life in Buffer RAV1 For this reason some kits contain several bottles of lyophilized Carrier RNA that should be used successively as required to avoid degradation of Carrier RNA Note Due to the production procedure and the small amount of Carrier RNA contained in the vial the carrier may hardly be visible in the vial Before use add 1 mL Lysis Buffer RAV1 to the Carrier RNA tube Dissolve the RNA and transfer it back to the RAV1 bottle Storage of Carrier RNA in Buffer RAV1 Lysis Buffer RAV1 including Carrier RNA can be stored at room temp
18. erature for 1 2 weeks Storage at room temperature prevents salt precipitation Lysis Buffer RAV1 including Carrier RNA can be stored at 4 C for up to 4 weeks or aliquoted and stored at 20 C for longer periods Storage at 4 C or below may cause salt precipitation Therefore the mixture must be preheated at 40 60 C for a maximum of 5 min in order to redissolve salts Do not warm Buffer RAV1 containing Carrier RNA more than 4 times Frequent warming temperatures gt 80 C and extended heat incubation will accelerate the degradation of Carrier RNA This leads to reduced recovery of viral RNA and eventually false negative RT PCR results in particular if low titer samples are used Before starting any NucleoSpin RNA Virus RNA Virus F protocol prepare the following Wash Buffer RAV3 Add the indicated volume of ethanol 96 100 to Wash Buffer RAV3 Concentrate Mark the label of the bottle to indicate that the ethanol is added Store Wash Buffer RAV3 at room temperature 18 25 C for up to one year MACHEREY NAGEL 07 2014 Rev 11 11 Viral RNA isolation NucleoSpin RNA Virus 10 preps 50 preps 250 preps REF 740956 10 740956 50 740956 250 Wash Buffer RAV3 6 mL 12 mL 50 mL Concentrate Add 24 mL ethanol Add 48 mL ethanol Add 200 mL ethanol NucleoSpin RNA Virus F 25 preps 740958 Wash Buffer RAV3 Concentrate 3x 25 mL Add 100 mL ethanol to each vial 12 MACHEREY NAGEL 07 2014 Rev
19. eshooting for further hints section 6 1 70 C If the resulting solution is still turbid centrifuge the mixture 5 min for 1 min at 11 000 x g to pellet particles and to prevent clogging of the NucleoSpin RNA Virus Columns Take off the supernatant and proceed with step 2 Add 600 uL ethanol 96 100 to the clear lysis solution 600 uL EtOH 2 Adjust binding conditions and mix by vortexing 10 15 s 3 Bind viral RNA lt Tubes 2 mL and load 700 uL lysed sample Centrifuge for 1 min at 8 000 x g 8 000 x g 1 min The use of new Collection Tubes 2 mL is recommended if z Load sample Place NucleoSpin RNA Virus Columns in Collection 8 stepwise infectious material has to be prepared g Load the residual lysis solution onto the NucleoSpin RNA Virus Column Centrifuge for 1 min at 8 000 x g Discard flow through and place the NucleoSpin RNA Virus Column into another new Collection Tube 2 mL More than two loading steps are not recommended MACHEREY NAGEL 07 2014 Rev 11 15 NucleoSpin RNA Virus Wash and dry silica membrane Add 500 uL Buffer RAW to the NucleoSpin RNA Virus Column Centrifuge for 1 min at 8 000 x g Discard flow through This washing step removes contaminants and PCR inhibitors Add 600 uL Buffer RAV3 to the NucleoSpin RNA Virus Column Centrifuge for 1 min at 8 000 x g Discard flow through with Collection Tube Place the NucleoSpin RNA Virus Column
20. f accident or improper or abnormal use of this product defects in products or MACHEREY NAGEL 07 2014 Rev 11 23 Viral RNA isolation components not manufactured by MACHEREY NAGEL or damages resulting from such non MACHEREY NAGEL components or products MACHEREY NAGEL makes no other warranty of any kind whatsoever and SPECIFICALLY DISCLAIMS AND EXCLUDES ALL OTHER WARRANTIES OF ANY KIND OR NATURE WHATSOEVER DIRECTLY OR INDIRECTLY EXPRESS OR IMPLIED INCLUDING WITHOUT LIMITATION AS TO THE SUITABILITY REPRODUCTIVITY DURABILITY FITNESS FOR A PARTICULAR PURPOSE OR USE MERCHANTABILITY CONDITION OR ANY OTHER MATTER WITH RESPECT TO MACHEREY NAGEL PRODUCTS In no event shall MACHEREY NAGEL be liable for claims for any other damages whether direct indirect incidental compensatory foreseeable consequential or special including but not limited to loss of use revenue or profit whether based upon warranty contract tort including negligence or strict liability arising in connection with the sale or the failure of MACHEREY NAGEL products to perform in accordance with the stated specifications This warranty is exclusive and MACHEREY NAGEL makes no other warranty expressed or implied The warranty provided herein and the data specifications and descriptions of this MACHEREY NAGEL product appearing in MACHEREY NAGEL published catalogues and product literature are MACHEREY NAGEL s sole representations concerning the product
21. ion subsequent time 5 15 min and temperature RT 56 C 72 C may be detection adapted in order to get optimal recovery rates for both species Ethanol carry over Prolong centrifugation steps in order to remove Buffer RAV3 completely Clogged membrane General Centrifuge plasma lysate before the addition of ethanol and problems subsequent loading onto the corresponding NucleoSpin RNA Virus Columns MACHEREY NAGEL 07 2014 Rev 11 21 Viral RNA isolation 7 2 Ordering information Product REF Pack of NucleoSpin RNA Virus 740956 10 50 250 10 50 250 NucleoSpin RNA Virus F 740958 25 NucleoSpin Funnel Columns 740959 30 sets Proteinase K 740506 100 mg rDNase Set Recombinant DNase and Reaction Buffer for rDNase 740963 1 set sufficient for 50 mini preps NucleoSpin Dx Virus 740895 50 250 50 250 NucleoSpin Blood 740951 10 50 250 10 50 250 NucleoSpin RNA Blood 740200 10 50 10 50 NucleoSpin Tissue 740952 10 50 250 10 50 250 Collection Tubes 2 mL 740600 1000 Visit www mn net com for more detailed product information 7 3 References M L Villahermosa M Thomson E Vazques de Parga M T Cuevas G Contreas L Perez Alvarez E Delgado N Manjon L Medrano and R Najera Improved Conditions for Extraction and Amplification of Human Immunodeficiency Virus Type 1 RNA from Plasma samples with low viral load Journal of Human Virology 3 27 34 2000 22 MACHEREY NAGEL 07 2014 Re
22. ions 7 2 3 Remarks regarding sample quality and preparation 8 2 4 Remarks regarding elution 10 2 5 Remarks regarding quality control 10 3 Storage conditions and preparation of working solutions 11 4 Safety instructions 13 5 NucleoSpin RNA Virus protocols 15 5 1 Viral RNA isolation from cell free biological fluids 15 5 2 Isolation of viral RNA and DNA from cell free biological fluids 17 6 NucleoSpin RNA Virus F protocols 18 6 1 Viral RNA isolation from cell free biological fluids 18 6 2 Isolation of viral RNA and DNA from cell free biological fluids 20 7 Appendix 21 7 1 Troubleshooting 21 7 2 Ordering information 22 7 3 References 22 7 4 Product use restriction warranty 23 MACHEREY NAGEL 07 2014 Rev 11 3 Viral RNA isolation 1 Components 1 1 Kit contents NucleoSpin RNA Virus 10 preps 50 preps 250 preps REF 740956 10 740956 50 740956 250 Lysis Buffer RAV1 10 mL 35 mL 5x35 mL Wash Buffer RAW 6 mL 30 mL 150 mL Wash Buffer RAV3 6 mL 12 mL 50 mL Concentrate RNase free H O 13 mL 13 mL 30 mL Elution Buffer RE 13 mL 13 mL 30 mL Carrier RNA 300 ug img 5x1mg lyophilized NucleoSpin RNA Virus 10 50 250 Columns dark blue rings plus Collection Tubes Collection Tubes 2 mL 30 150 750 User manual 1 1 1 For preparation of working solutions and storage conditions see section 3 Composition of Elution Buffer RE 5 mM Tris HCl pH 8 5 4 MACHEREY NAGEL 07 2014 Rev 11 Viral RNA isolation
23. performing the purification procedure All technical literature is available on the internet at www mn net com Please contact Technical Service regarding information about changes of the current user manual compared to previous revisions 6 MACHEREY NAGEL 07 2014 Rev 11 Viral RNA isolation 2 Product description 2 1 The basic principle With the NucleoSpin RNA Virus method RNA viruses are lysed quickly and efficiently by Lysis Buffer RAV1 which is a highly concentrated solution of GITC DNA viruses e g HBV are usually more difficult to lyse and require Proteinase K digestion see support protocol section 5 2 Lysis buffer and ethanol create appropriate conditions for binding of nucleic acids to the silica membrane of the NucleoSpin RNA Virus Columns Carrier RNA improves binding and recovery of low concentrated viral RNA Contaminations potential PCR inhibitors like salts metabolites and soluble macromolecular cellular components are removed in simple washing steps with ethanolic buffers RAW and RAV3 The nucleic acids can be eluted in low salt buffer or water and are ready for use in subsequent reactions 2 2 Kit specifications NucleoSpin RNA Virus Virus F kits are designed for the rapid preparation of highly pure viral nucleic acids e g HCV HIV CMV from fluid biological samples for example plasma serum urine but not blood see remarks in section 2 1 Nocross contamination due to closed systems
24. v 11 Viral RNA isolation 7 4 Product use restriction warranty NucleoSpin RNA Virus kit components are intended developed designed and sold FOR RESEARCH PURPOSES ONLY except however any other function of the product being expressly described in original MACHEREY NAGEL product leaflets MACHEREY NAGEL products are intended for GENERAL LABORATORY USE ONLY MACHEREY NAGEL products are suited for QUALIFIED PERSONNEL ONLY MACHEREY NAGEL products shall in any event only be used wearing adequate PROTECTIVE CLOTHING For detailed information please refer to the respective Material Safety Data Sheet of the product MACHEREY NAGEL products shall exclusively be used in an ADEQUATE TEST ENVIRONMENT MACHEREY NAGEL does not assume any responsibility for damages due to improper application of our products in other fields of application Application on the human body is STRICTLY FORBIDDEN The respective user is liable for any and all damages resulting from such application DNA RNA PROTEIN purification products of MACHEREY NAGEL are suitable for IN VITRO USES ONLY ONLY MACHEREY NAGEL products specially labeled as IVD are also suitable for IN VITRO diagnostic use Please pay attention to the package of the product IN VITRO diagnostic products are expressiy marked as IVD on the packaging IF THERE IS NO IVD SIGN THE PRODUCT SHALL NOT BE SUITABLE FOR IN VITRO DIAGNOSTIC USE ALL OTHER PRODUCTS NOT LABELED AS IVD ARE NOT SUITED FOR ANY
Download Pdf Manuals
Related Search