Viral RNA and DNA isolation - MACHEREY
Contents
1. with Cap Strips or Round well Block acids to the the risk of cross contami membrane nation used for vacuum processing only 7 Wash silica MN Square well Block Can be used for waste membrane collection if required 8 Elute DNA Rack of Tubes Strips Round well Blocks and Tube Strips can be closed with Cap Strips Use of MN Square well Block is optional For waste collection the waste tray of the NucleoVac Vacuum Manifold can be used MACHEREY NAGEL 04 2014 Rev 05 9 Viral RNA and DNA isolation 2 5 Automated processing on robotic platforms For automated use we recommend using the NucleoSpin 96 Virus Core Kit which can be automated on many common laboratory workstations For a protocol which can be used as a guideline to create robotic script see section 5 2 For the availability of scripts and general considerations about adapting NucleoSpin 96 Virus Core Kit on a certain workstation please contact MACHEREY NAGEL For vacuum processing the use of the disposable MN Wash Plate inside the vacuum manifold is recommended Use of the MN Wash Plate reduces the risk of cross contamination caused by spraying of solutions during vacuum filtration steps Visit MN at www mn net com or contact your local MACHEREY NAGEL distributor for technical support regarding hardware software setup instructions and selection of the protocol 2 6 Sample material Liquid samples Biological fluids or semi f2. P 273 P 280 P 301 312 P 302 352 P 304 340 P 305 351 338 P 312 P 330 P 332 313 P 337 313 P 342 311 P 403 233 P 403 235 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 Z ndquellenarten fernhalten Nicht rauchen Keep container tightly closed Beh lter dicht verschlossen halten Do not breathe vapours Dampf nicht einatmen Avoid breathing dust Einatmen von Staub vermeiden Avoid release to the environment Freisetzung in die Umwelt vermeiden Wear protective gloves eye protection Schutzhandschuhe Augenschutz tragen IF SWALLOWED Call a POISON CENTER doctor if you feel unwell BEI VERSCHLUCKEN Bei Unwohlsein GIFTINFORMATIONSZENTRUM Arzt anrufen IF ON SKIN Wash with plenty of water BEI KONTAKT MIT DER HAUT Mit viel Wasser waschen IF INHALED Remove victim to fresh air and keep at rest in a position comfort able for breathing BEI EINATMEN An die frische Luft bringen und in einer Position ruhigstellen die das Atmen erleichtert IF IN EYES Rinse cautiously with water for several minutes Remove contact lenses if present and easy to do Continue rinsing Bei Kontakt mit den Augen Einige Minuten lang behutsam mit Wasser sp len Vorhandene Kontaktlinsen nach M glichkeit entfernen Weiter sp len Call a POISON CENTER doctor if you feel
3. additional MN Square well Blocks are not included in the kit see ordering information Wash silica membrane Remove Self adhering PE Foil and add 500 pL Buffer RAW to each well of the NucleoSpin Virus Binding Plate Seal the NucleoSpin Virus Binding Plates with new Self adhering PE Foil Centrifuge at 5 600 6 000 x g for 1 2 min Remove Self adhering PE Foil and place NucleoSpin Virus Binding Plate onto anew MN Square well Block Add 700 uL Buffer RAV3 to each well of the NucleoSpin Virus Binding Plate Seal with new Self adhering PE Foil Centrifuge at 5 600 6 000 x g for 1 2 min 20 MACHEREY NAGEL 04 2014 Rev 05 NucleoSpin 96 Virus centrifuge processing Repeat second wash step once Prolong centrifugation to 15 min in order to remove ethanol from residual Wash Buffer RAV3 Alternatively remove the adhesive foil and place the NucleoSpin Virus Binding Plate into an incubator for 20 min at 37 C to evaporate residual ethanol Removal of ethanol by evaporation at 37 C is more effective than additional prolonged centrifugation 15 min 6 000 x g Elute viral RNA and DNA Place the NucleoSpin Virus Binding Plate onto the Rack of Tube Strips Dispense 75 100 uL RNase free water or Buffer RE preheated to 70 C to each well of the NucleoSpin Virus Binding Plate Pipette the buffer directly onto the membrane Incubate at room temperature for 1 min Seal with a new Self adhesive PE Foil
4. 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 2 7 Carrier RNA The NucleoSpin 96 Virus kits include Carrier RNA that 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 96 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 added Carrier RNA may thus be carefully optimized depending on the individual PCR system used Lysis must be done in MN Square well Blocks if sample size is 150 uL Additional MN Square well Blocks may be necessary see ordering information MACHEREY NAGEL 04 2014 Rev 05 11 Viral RNA and DNA isolation 2 8 Elution procedures Recovery of viral RNA or DNA from the membrane depends on the elution volume Elution volumes of 75 200 uL are possible with an optimum of 100 125 uL dispensed volume The dead volume of the membrane is approx 45 uL and the recovered elution
5. and 3000 Qiagen MultiPROBE II PerkinElmer Biomek 2000 and FX Beckman Coulter Starter Set A 740682 1 for use of 8 well strips on the NucleoVac 96 and automation platforms MACHEREY NAGEL 04 2014 Rev 05 29 Viral RNA and DNA isolation Product REF Pack of Starter Set C 740684 1 for use of 8 well strips under centrifugation NucleoVac 96 Vacuum Manifold 740681 1 NucleoVac Vacuum Regulator 740641 1 Support Frame for Column Holder A 740480 1 6 3 Product use restriction warranty NucleoSpin 96 Virus Core 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 PROT
6. g to collect any sample from the cover of the wells if required before opening the Iysis vessels Adjust binding conditions Remove the cover of the wells and add 400 uL ethanol 96 100 to each sample Take care not to moisten the rims of the individual wells while dispensing Close the wells with a new cover invert 10 x and mix by shaking for 15 s Spin briefly 30 s 1 500 x g to collect any sample from the cover of the wells If 150 uL sample has been prepared add 600 uL ethanol 96 100 to each lysate Transfer samples to binding plate Place waste tray into vacuum manifold base Other plates for waste collection can also be used Insert spacers labeled MTP MULTI 96 PLATE notched side up and rest the MN Wash Plate on them Close manifold and place NucleoSpin Virus Binding Plate on top of the manifold Transfer samples to the wells of the binding plate and be careful no to moisten the rims of the wells Bind viral nucleic acids to silica membrane Apply vacuum of 0 2 to 0 4 bar reduction of atmospheric pressure to allow samples to pass through the membrane 2 5 min Flow through rate should be about 1 2 drops per second Adjust vacuum strength accordingly Wash and dry silica membrane Add 500 uL Buffer RAW to each well of the NucleoSpin Virus Binding Plate Apply vacuum 0 2 to 0 4 bar until all buffer has passed through the wells of the NucleoSpin Virus Binding Plate 2 5 min Release t
7. if low titer samples are used Do not heat up Buffer RAV1 containing Carrier RNA more than 4 times MACHEREY NAGEL 04 2014 Rev 05 13 Viral RNA and DNA isolation NucleoSpin 96 Virus 2x 96 preps 4x 96 preps REF 740691 2 740691 4 Wash Buffer RAV3 100 mL 2x 100 mL Concentrate Add 400 mL ethanol Add 400 mL ethanol to each bottle Proteinase K 2x50 mg 3x75mg Iyophilized Add 2 5 mL Proteinase Buffer Add 3 5 mL Proteinase Buffer to each vial to each vial Carrier RNA 3x 1mg 6x 1mg lyophilized Transfer each vial to one Transfer each vial to one bottle of 40 mL Buffer RAV1 bottle of 40 mL Buffer RAV1 NucleoSpin 96 Virus Core Kit 4x 96 preps REF 740452 4 Wash Buffer RAV3 2x 100 mL Concentrate Add 400 mL ethanol to each bottle Carrier RNA 6x 1mg lyophilized Transfer each vial to one bottle of 40 mL Buffer RAV1 14 MACHEREY NAGEL 04 2014 Rev 05 Viral RNA and DNA isolation 4 Safety instructions The following components of the NucleoSpin 96 Virus and NucleoSpin 96 Virus Core kits contain hazardous contents Wear gloves and goggles and follow the safety instructions given in this section GHS classification Only harmful features need not 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 phrase
8. kit determines most of the consumables to be used Tube Strips MN Square well Blocks etc the vacuum use of the kit allows for more variation and higher flexibility Especially when processing a large number of samples under vacuum cross contamination is a major concern due to spraying of liquids or aerosol formation The use of the MN Wash Plate prevents the contamination by droplets at the outlets of the individual wells of the NucleoSpin Binding Plate This very assistant tool is thus recommended for vacuum processing When using the NucleoSpin 96 Virus and the Core Kit under vacuum the NucleoVac 96 Vacuum Manifold is required see ordering information Place NucleoSpin 96 Virus Binding Plate on NucleoVac Vacuum Manifold If processing less than 96 samples seal unused wells with a Self adhering PE Foil in order to ensure proper vacuum during the filtration steps This standard protocol is recommended for purification of viral RNA from for example HCV or HIV DNA viruses such as CMV can also be isolated but lysis including Proteinase K digestion is recommended not included in the core kit For hardware requirements refer to section 2 3 For detailed information regarding the vacuum manifold setup see page 26 For use of the NucleoSpin 96 Virus Core Kit REF 740452 4 refer to section 2 4 regarding recommended accessories Before starting the preparation Check if Buffer RAV1 Buffer RAV3 and Proteinase K were prepa
9. swab material or diluted blood samples may also be processed For detailed information on sample pre treatment please refer to section 2 6 e The purified nucleic acids are suitable for applications like real time PCR RT PCR PCR or any kind of enzymatic manipulation The detection limit for certain viruses depends on individual procedures for example in house nested RT PCR Use of internal extraction control samples as well as positive and negative amplification controls in order to monitor the purification amplification and detection processes is highly recommended NucleoSpin 96 Virus Core Kit is primarily designed for vacuum use for manual use or automated use on robotic platforms Processing under vacuum allows easy automation on common liquid handling instruments For more information about the automation process and the availability of ready to run scripts for certain platforms please refer to section 2 5 and contact your local distributor or MN directly Table 2 Kit specifications at a glance Parameter NucleoSpin 96 Virus Core Kit Technology Silica membrane technology Format 96 well plates Processing Manual or automated vacuum or entrifugation Sample volume 100 150 uL Typical recovery gt 90 Analysis limit 30 60 cp mL Elution volume 70 100 uL Preparation time 60 min plate Binding capacity 40 ug Lysis must be done in MN Square well Blocks if sample size is 150 uL Additional MN Squa
10. takes this into account by introducing the NucleoSpin 96 Virus Core Kit which is primarily recommended for manual or automated vacuum use Core kits contain the core items like binding plates and buffers but no accessories like plastics or enzymes The core kits together with a large variety of suitable disposables ensure the highest degree of flexibility for the user For lower or medium throughput the NucleoSpin 96 Virus kit is also available in 8 well strip format see ordering information Table 1 Kit selection guide Application Kit recommendation Manual use centrifuge Low medium throughput NucleoSpin 8 Virus High throughput NucleoSpin 96 Virus Manual use vacuum Low medium throughput NucleoSpin 8 Virus NucleoSpin 8 Virus Core Kit High throughput NucleoSpin 96 Virus NucleoSpin 96 Virus Core Kit Automated use Low medium throughput NucleoSpin 8 Virus Core Kit vacuum or centrifuge High throughput NucleoSpin 96 Virus Core Kit Please refer to the NucleoSpin 8 Virus user manual See section 6 2 for ordering information 6 MACHEREY NAGEL 04 2014 Rev 05 Viral RNA and DNA isolation 2 2 Kit specifications NucleoSpin 96 Virus allows the parallel purification of viral DNA and RNA from 100 150 uL plasma serum or other cell free biological fluids Samples can either be fresh or frozen Furthermore particle free supernatants of tissue suspensions supernatants of stool samples
11. unwell Bei Unwohlsein GIFTINFORMATIONSZENTRUM Arzt anrufen Rinse mouth Mund aussp len IF skin irritation occurs Get medical advice attention Bei Hautreizung rztlichen Rat einholen rztliche Hilfe hinzuziehen If eye irritation persists Get medical advice attention Bei anhaltender Augenreizung Arztlichen Rat einholen rztliche Hilfe hinzuziehen If experiencing respiratory symptoms Call a POISON CENTERF doctor Bei Symptomen der Atemwege GIFTINFORMATIONSZENTRUM Arzt anrufen Store in a well ventilated place Keep container tightly closed Beh lter dicht verschlossen an einem gut bel fteten Ort aufbewahren 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 16 MACHEREY NAGEL 04 2014 Rev 05 NucleoSpin 96 Virus centrifuge processing 5 1 Protocols NucleoSpin 96 Virus centrifuge processing For hardware requirements refer to section 2 3 For detailed information on each step see page 21 For use of the NucleoSpin 96 Virus Core Kit REF 740452 4 refer to section 2 4 regarding recommended accessories Before starting the preparation Check if Buffer RAV1 Buffer RAV3 and Proteinase K were prepared according to section 3 Set incubator or ove
12. Centrifuge at 5 600 6 000 x g for 2 3 min Tube Strips containing eluted RNA DNA can be conveniently closed with Cap Strips for storage Yields will be 10 15 higher when eluting in 100 200 uL water The concentration of nucleic acids in the complete eluate however will be lower For RT PCR PCR a more concentrated eluate is favorable If only viral DNA is processed elution should be done with Elution Buffer RE optimized for elution and storage of DNA MACHEREY NAGEL 04 2014 Rev 05 21 NucleoSpin 96 Virus vacuum processing 5 2 NucleoSpin 96 Virus Core Kit vacuum processing For hardware requirements refer to section 2 3 For detailed information on each step see page 27 For detailed information regarding the vacuum manifold setup see page 26 For use of the NucleoSpin 96 Virus Core Kit REF 740452 4 refer to section 2 4 regarding recommended accessories Before starting the preparation Check if Buffer RAV1 Buffer RAV3 and Proteinase K were prepared according to section 3 Set incubator or oven to 25 70 C Preheat Elution Buffer RE or water to 70 C Protocol at a glance 1 Lyse samples 100 pL sample 400 uL Buffer RAV1 20 uL Proteinase K Mix 25 70 C 10 min 2 Adjust binding conditions 400 uL ethanol 96 100 Mix 3 Transfer samples to NucleoSpin Virus Binding Plate 4 Bind nucleic acid to 0 2 bar NucleoSpin Virus Binding Plate 5 min Reduct
13. DNA RNA added to the PCR RT PCR Use diluted eluates in order to exclude inhibition Reduce Carrier RNA concentration in Buffer RAV1 Optimal Problems concentration may require some preliminary experiments with subsequent Ethanol carry over detection Extend centrifugation times in order to remove Buffer RAV3 completely PCR inhibition Add an additional wash step with 96 ethanol following the last wash with Buffer RAV3 Clogged membrane General 99 ar problems Centrifuge sample lysate before the addition of ethanol and subsequent loading onto NucleoSpin Virus Binding Plate 28 MACHEREY NAGEL 04 2014 Rev 05 Viral RNA and DNA isolation 6 2 Ordering information Product REF Pack of NucleoSpin 96 Virus 740691 2 2 x 96 preps 740691 4 4 x 96 preps NucleoSpin 96 Virus Core Kit 740452 4 4 x 96 preps NucleoSpin 8 Virus 740643 12 x 8 preps 740643 5 60 x 8 preps NucleoSpin 8 Virus Core Kit 740451 4 48 x 8 preps Proteinase K 740506 100 mg MN Square well Block 740476 4 Square well Block 740481 4 Round well Block with Cap Strips 740475 4 set consists of 1 Round well Block and12 Cap Strips Rack of Tube Strips with Cap Strips 740477 4 set consists of 1 Rack 12 Tube Strips with 8 tubes each and 12 Cap Strips Cap Strips 740478 48 MN Wash Plates 740479 4 Self adhering PE Foil 740676 50 MN Frame 740680 1 for optimized handling of 96 well plates with vacuum manifold on BioRobot 9600 9604
14. EIN purification products of MACHEREY NAGEL are suitable for N 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 expressly 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 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 30 MACHEREY NAGEL 04 2014 Rev 05 Viral RNA and DNA isolation 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 s
15. EL 04 2014 Rev 05 19 NucleoSpin 96 Virus centrifuge processing Adjust viral nucleic acid binding conditions Remove Cap Strips and add 400 uL ethanol 96 100 to each lysate Take care not to moisten the rims of the individual wells while dispensing Close the individual wells with new Cap Strips supplied Invert 10 times and mix by shaking for 15 s Spin down droplets 30 s 1 500 x g from the Cap Strips If 150 uL sample has been prepared add 600 uL ethanol 96 100 to each lysate Transfer samples to binding plates Remove the first Cap Strip and transfer all of each sample into the wells of a NucleoSpin Virus Binding Plate positioned on top of the MN Square well Block Do not moisten the rims of the individual wells while dispensing samples moistened rims may cause cross contamination during centrifugation Seal NucleoSpin Virus Binding Plates with Self adhering PE Foil Bind viral nucleic acids to silica membrane Place the MN Square well Blocks with Binding Plate onto the centrifuge carrier and insert it into the rotor buckets Centrifuge at 5 600 6 000 x g for 2 min Typically samples will pass through the columns within s 1 min Optional If 150 uL sample has been prepared load it in successive steps onto the NucleoSpin Virus Binding Plate as described in step 3 In this case use a new MN Square well Block for the washing steps as the maximum volume of the MN Square well Block may be exceeded
16. Sa A Viral RNA and DNA isolation User manual NucleoSpin 96 Virus NucleoSpin 96 Virus Core Kit April 2014 Rev 05 MACHEREY NAGEL MN www mn net com Viral RNA and DNA isolation Table of contents 1 Components 1 1 Kit contents 1 2 Reagents and to be supplied by user 5 2 Product description 6 2 1 The basic principle 6 2 2 Kit specifications 7 2 3 Required hardware 8 2 4 Recommended accessories for use ofthe NucleoSpin 96 Virus CoreKit 8 2 5 Automated processing on robotic platforms 10 2 6 Sample material 10 2 7 Carrier RNA 11 2 8 Elution procedures 12 3 Storage conditions and preparation of working solutions 13 4 Safety instructions 15 5 Protocols 17 5 1 NucleoSpin 96 Virus centrifuge processing 17 5 2 NucleoSpin 96 Virus Core Kit vacuum processing 22 6 Appendix 28 6 1 Troubleshooting 28 6 2 Ordering information 29 6 3 Product use restriction warranty 30 MACHEREY NAGEL 04 2014 Rev 05 3 Viral RNA and DNA isolation 1 Components 1 1 Kit contents NucleoSpin 96 Virus 2 x 96 preps 4 x 96 preps REF 740691 2 740691 4 Lysis Buffer RAV 1 3 x 40 mL 6 x 40 mL Wash Buffer RAW 2x75 mL 300 mL Wash Buffer RAV3 Concentrate 100 mL 2x 100 mL RNase free H O 125 mL 125 mL Elution Buffer RE 125 mL 125 mL Carrier RNA lyophilized 3x 1mg 6x 1mg Proteinase K lyophilized 2x 50mg 3x75 mg Proteinase Buffer PB 8 mL 15 mL NucleoSpin Virus Binding Plates 2 4 blue
17. Virus Binding Plate The amount of PBS buffer added to blood samples has to be optimized 10 MACHEREY NAGEL 04 2014 Rev 05 Viral RNA and DNA isolation for the individual organism As a rule of thumb we recommend to start with 50 uL blood diluted with 50 uL PBS buffer Sample volume The NucleoSpin 96 Virus and NucleoSpin 96 Virus Core Kits are specified for a sample volume of 100 uL If necessary the sample volume can be increased to 150 uL For sample volumes of 150 uL the volumes of Lysis Buffer RAV1 and ethanol have to be increased to 600 uL each Depending on the size of pipetting tips the total lysate volume of 1300 uL may be loaded in two steps onto the NucleoSpin Virus Binding Plate The buffers supplied with the kit are sufficient for processing a sample volume of 150 uL Proteinase K treatment Addition of Proteinase K solution 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 lysis For isolation of viral RNA in general a lysis of samples in Buffer RAV1 for 10 min at room temperature 18 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
18. act MACHEREY NAGEL GmbH amp Co KG Tel 49 0 24 21 969 270 e mail tech bio mn net com Trademarks Biomek is a registered trademark of Beckman Coulter Inc BioRobot is a registered trademark of the Qiagen Group MultiPROBE is a registered trademark of Perlin Elmer Inc NucleoSpin is a registered trademark of MACHEREY NAGEL GmbH amp Co KG All used names and denotations can be brands trademarks or registered labels of their respective owner also if they are not special denotation To mention products and brands is only a kind of information i e it does not offend against trademarks and brands and can not be seen as a kind of recommendation or assessment Regarding these products or services we can not grant any guarantees regarding selection efficiency or operation 32 MACHEREY NAGEL 04 2014 Rev 05
19. aration Check if Buffer RAV1 Buffer RAV3 and Proteinase K were prepared according to section 3 Set incubator or oven to 25 70 C Preheat Elution Buffer RE or water to 70 C 1 Lyse samples Pipette 400 uL Buffer RAV1 into the wells of a Rack of Tube Strips or Round well Block according to the number of samples Dispense solution to the bottom of the wells If 150 uL sample are to be prepared pipette 600 uL Buffer RAV1 into the wells We recommend using an electronic 8 channel pipetting device with extra long tips capable of holding more than 650 uL Add 100 uL sample to each Buffer RAV 1 filled well Take care to dispense the sample directly into Buffer RAV1 Pipette mixture up and down several times Do not moisten the rims Close Tube Strips or Round well Block with Cap Strips Incubate mixture for 10 min at room temperature 18 25 C Optional Add 20 pL Proteinase K to each sample pre mixed with Buffer RAV1 Close the lysis vessels with Cap Strips and incubate for 5 10 min at 56 70 C Addition of Proteinase K is required for viral DNA extraction and may be useful for viral RNA extraction from some sample types For details on incubation time and temperature see section 2 6 Spin down droplets 30 s 1 500 x g before opening the Cap Strips Lysis must be done in MN Square well Blocks if sample size is 150 uL Additional MN Square well Blocks may be necessary see ordering information MACHEREY NAG
20. ase K solution is stable at 20 C for 6 months Dividing the solution into aliquots is recommended Before use add 1 mL Lysis Buffer RAV1 to the Carrier RNA tube Dissolve the RNA and transfer it back to the Buffer RAV1 bottle Mark the label of the bottle to indicate that Carrier RNA was added Due to the production procedure and the small amount of Carrier RNA contained in the vial the Carrier RNA may hardly be visible in the vial Carrier RNA has a limited shelf life in Buffer RAV1 For this reason the NucleoSpin 96 Virus Core Kit kit contains several vials of lyophilized Carrier RNA which should be used successively as required Storage of Carrier RNA in Buffer RAV1 Buffer RAV1 with Carrier RNA can be stored at room temperature for 1 2 weeks Storage at room temperature prevents salt precipitation and avoids pre heating of the buffer solution For storage for up to 4 weeks storage of Buffer RAV1 with added Carrier RNA at 4 C is recommended For long time storage Buffer RAV1 with added Carrier RNA can be stored in aliquots at 20 C 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 dissolve precipitated salts Attention Frequent heating temperatures gt 80 C and extended heat incubation will lead to the degradation of the Carrier RNA and to reduced recovery of viral RNA and eventually false negative RT PCR results in particular
21. buffer can thus easily be estimated Highly concentrated eluates When using a minimal elution volume 75 100 uL about 70 80 of bound nucleic acids can be eluted resulting in highly concentrated RNA DNA Alternatively elution can be done in two steps with for example 75 uL each resulting in a higher elution efficiency but with a lower concentrated eluate Preheated elution buffer 70 C Use preheated elution buffer to increase overall yield Optionally following addition of preheated elution buffer incubate the NucleoSpin Virus Binding Plate for 3 min at 60 70 C before elution 12 MACHEREY NAGEL 04 2014 Rev 05 Viral RNA and DNA isolation 3 Storage conditions and preparation of working solutions Attention Buffers RAV1 and RAW contain guanidinium salts Wear gloves and goggles CAUTION Buffer RAV1 contains guanidinium thiocyanate and Buffer RW 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 Before starting any NucleoSpin 96 Virus Core Kit protocol prepare the following Wash Buffer RAV3 Add indicated volume of 96 100 ethanol to the Wash Buffer RAV3 Concentrate Mark the label of the bottle to indicate that ethanol was added Before first use of the kit add the indicated volume of Proteinase Buffer PB to dissolve lyophilized Proteinase K Protein
22. es from a variety of suitable accessory plates according to his requirements for highest flexibility For use of the NucleoSpin 96 Virus Core Kit follow the standard protocol see section 5 Recommended accessories for use of the NucleoSpin 96 Virus Core Kits are available from MACHEREY NAGEL see ordering information Protocol step Suitable consumables Remarks not supplied with the core kits Round well Blocks and Tube Strips can be closed with Cap Strips 1 Lyse Round well Block with _ samples 12 Cap Strips or Rack of Tube Strips with 12 Cap Strips 8 MACHEREY NAGEL 04 2014 Rev 05 Viral RNA and DNA isolation Protocol step Suitable consumables not supplied with the core kits Remarks or MN Square well Block Square well Block Proteinase K Square well Blocks can not be closed with Cap Strips Closing with a Self adhering PE Foil is not recommended no tight sealing when mixing Repeated pipetting up and down is recommended for mixing samples with Buffer RAV1 For certain samples and for viral DNA isolation use of Proteinase K is required 2 Adjust Cap Strips When using Round well binding Block or Tube Strips for conditions lysis new Cap Strips are required for closure of wells 3 Transfer MN Square well Block Can be used for waste samples collection if required 4 Bind nucleic MN Wash Plate MN Wash Plate minimizes
23. f the samples have passed If only viral DNA is processed elution should be done with Elution Buffer RE optimized for elution and storage of DNA Optional Repeat elution step once incubation not required Note Elution by vacuum may cause cross contamination due to aerosol formation and spraying of droplets If possible it is thus recommended to use centrifugation for the elution step Reduction of atmospheric pressure MACHEREY NAGEL 04 2014 Rev 05 27 Viral RNA and DNA isolation 6 Appendix 6 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 2 7 Small Proteinase K digestion amounts e For certain sample types and for viral DNA isolation use of or no viral Proteinase K is required for the sample lysis step Compare Hueleic protocols with and without Proteinase K digestion acids in the eluate Viral nucleic acids degraded Samples should be processed immediately If necessary add RNase inhibitor to the sample Create a nuclease free environment and ensure that no nucleases are present Use suitable tips and buffer reservoirs Check that all buffers have been prepared and stored correctly If in doubt use new aliquots of Buffer RAV1 and elution buffer Reduced sensitivity Carrier RNA may interfere with the PCR RT PCR system used Change the volume of eluted viral
24. he vacuum Reduction of atmospheric pressure 26 MACHEREY NAGEL 04 2014 Rev 05 NucleoSpin 96 Virus vacuum processing Add 700 pL Buffer RAV3 to each well of the NucleoSpin Virus Binding Plate Apply vacuum 0 2 to 0 4 bar until all buffer has passed through the wells of the NucleoSpin Virus Binding Plate 2 5 min Release the vacuum Add 700 pL Buffer RAV3 to each well of the NucleoSpin Virus Binding Plate Apply vacuum 0 2 to 0 4 bar until all buffer has passed through the wells of the NucleoSpin Virus Binding Plate 2 5 min Release the vacuum Remove MN Wash Plate After the final washing step close the valve release the vacuum and remove the NucleoSpin Virus Binding Plate Put it on a clean paper towel to remove residual EtOH containing wash buffer Remove manifold lid MN Wash Plate and waste container from the vacuum manifold Reassemble the vacuum manifold and dry the membrane by applying maximum vacuum e g 0 6 bar for 15 minutes Elute viral RNA and DNA Place a suitable vessel used for elution on appropriate spacers e g MICROTUBE RACK into the manifold base Close manifold and insert NucleoSpin Virus Binding Plate onto manifold top Dispense 100 pL RNase free water or Buffer RE preheated to 70 C to each well of the plate Pipette water directly onto the membrane Incubate at room temperature for 2 3 min and apply vacuum of 0 4 bar until all o
25. ion of atmospheric pressure 22 MACHEREY NAGEL 04 2014 Rev 05 NucleoSpin 96 Virus vacuum processing Wash and dry silica membrane 500 uL RAW 0 2 bar 5 min 700 pL RAV3 0 2 bar 2 min 700 uL RAV3 0 2 bar 5 min Remove MN Wash Plate 0 6 bar 15 min Elute viral RNA and DNA 100 pL RE 70 C 0 4 bar 2 min Optional Repeat elution step once Note Elution under centrifugation is recommended Reduction of atmospheric pressure MACHEREY NAGEL 04 2014 Rev 05 23 NucleoSpin 96 Virus vacuum processing Setup of vacuum manifold Binding Washing steps Step 4 Place the NucleoSpin Binding Plate on top of the manifold lid Step 3 Place the manifold lid on top of the manifold base Step 2 Place the MN Wash Plate in the manifold Step 1 Insert spacers MTP MULTI 96 PLATE and waste container in the manifold base Final setup Elution step Step 4 Place the NucleoSpin Binding Plate on top of the manifold lid Step 3 Place the manifold lid on top of the manifold base Step 2 Place the Rack of Tube Strips in the manifold Step 1 Insert spacers MICROTUBE RACK in the manifold base Final setup 24 MACHEREY NAGEL 04 2014 Rev 05 NucleoSpin 96 Virus vacuum processing Detailed protocol Whereas the use of a centrifuge for the processing of the NucleoSpin 96 Virus
26. luid 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 Virus Binding Plate Therefore check all samples especially old or frozen ones for presence of precipitates Precipitates can be removed after addition of Lysis Buffer RAV1 and lysis incubation by centrifugation Avoid clearing samples by centrifugation filtration before the Buffer RAV1 lysis step because viruses of interest may be associated with particles or aggregates Incubation with Buffer RAV1 can be prolonged in order to dissolve and digest residual 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 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
27. n to 25 70 C Preheat Elution Buffer RE or water to 70 C Protocol at a glance 1 Lyse samples 100 pL sample 400 uL Buffer RAV1 20 uL Proteinase K Mix 25 70 C 10 min 2 Adjust binding conditions 400 uL ethanol 96 100 Mix 3 Transfer samples to NucleoSpin Virus Binding Plate 4 Bind viral RNA and DNA 5 600 6 000 x g to silica membrane of the 2 min NucleoSpin Virus Binding Plate MACHEREY NAGEL 04 2014 Rev 05 17 NucleoSpin 96 Virus centrifuge processing Wash silica membrane 500 uL RAW 5 600 6 000 x g 2 min 700 pL RAV3 5 600 6 000 x g 2 min 700 pL RAV3 5 600 x g 15 min Elute viral RNA and DNA 100 pL RE 70 C 5 600 6 000 x g 2 min Optional Repeat elution step once 18 MACHEREY NAGEL 04 2014 Rev 05 NucleoSpin 96 Virus centrifuge processing Detailed protocol This standard protocol is recommended for purification of viral RNA from for example HCV or HIV DNA viruses such as CMV can also be isolated but Iysis including Proteinase K digestion is recommended Place the NucleoSpin Virus Binding Plate on an MN Square well Block The use of a second plate placed on an MN Square well Block avoids the need to balance the centrifuge For hardware requirements refer to section 2 3 For use of the NucleoSpin 96 Virus Core Kit REF 740452 4 refer to section 2 4 regarding recommended accessories Before starting the prep
28. or the simultaneous purification of viral RNA and DNA The kit combines the selectivity of well established silica membrane binding of nucleic acids with high throughput 96 well format With the NucleoSpin 96 Virus method RNA viruses are quickly and efficiently lysed by Lysis Buffer RAV1 which is a highly concentrated GITC solution Compared to RNA viruses DNA viruses e g HBV are usually more difficult to isolate and require a digestion of samples with Proteinase K which is provided in the kit Lysis buffer and ethanol create appropriate conditions for binding of nucleic acids to the silica membrane of the NucleoSpin Virus Binding Plate Carrier RNA included in Lysis Buffer RAV1 improves binding and recovery of low concentrated viral RNA DNA Contaminations potential PCR inhibitors like salts metabolites and soluble macromolecular cellular components are removed in washing steps with ethanolic Wash Buffer RAW and Buffer RAV3 The purified viral nucleic acids can be eluted in low salt buffer or water and are ready to use in subsequent downstream applications like RT PCR or PCR Choice of NucleoSpin Virus kits The NucleoSpin 96 Virus kit allows the purification of up to 96 samples The kit is primarily designed for centrifugation use vacuum use is also possible Use of the kit on liquid handling instruments mainly vacuum allows more variation and higher flexibility in the consumables used for lysis washing and elution MACHEREY NAGEL
29. pecifications 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 of accident or improper or abnormal use of this product defects in products or 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 a
30. re well Blocks may be necessary see ordering information MACHEREY NAGEL 04 2014 Rev 05 7 Viral RNA and DNA isolation 2 3 Required hardware Centrifugation For centrifugation a microtiterplate centrifuge which is able to accommodate the NucleoSpin Virus Binding Plate stacked on a Round or Square well Block and reaches accelerations of 5 600 6 000 x g is required bucket height 85 mm Vacuum processing Although the NucleoSpin 96 Virus kit is designed primarily for processing under centrifugation processing under vacuum is also possible The dead volume for the elution step is higher in comparison to centrifuge based elution In order to achieve highly concentrated eluates and to avoid contamination it is recommended performing the elution step by centrifugation Consumables for vacuum processing differ from the consumables required for centrifugation Therefore for vacuum processing we recommend using the NucleoSpin 96 Virus Core Kit For manual processing under vacuum a NucleoVac 96 Vacuum Manifold see ordering information is required for NucleoSpin 96 Virus Core Kit 2 4 Recommended accessories for use of the NucleoSpin 96 Virus Core Kit The NucleoSpin 96 Virus Core Kit provides the buffers Carrier RNA and NucleoSpin Virus Binding Plates Accessory plates e g lysis plates elution plates and Proteinase K are not provided with the core kits The user can individually select additional consumabl
31. red according to section 3 Set incubator or oven to 25 70 C Preheat Elution Buffer RE or water to 70 C 1 Lyse samples Pipette 400 pL Buffer RAV1 into the wells of a suitable vessel used for lysis Dispense solution to the bottom of the wells If 150 uL sample are to be prepared pipette 600 uL Buffer RAV1 into the wells We recommend using an electronic 8 channel pipetting device with extra long tips capable of holding more than 650 uL Add 100 uL sample to each Buffer RAV 1 filled well Take care to dispense the samples directly into Buffer RAV1 The MN Wash Plate is not part of the kits Please order separately see ordering information Lysis must be done in MN Square well Blocks if sample size is 150 uL Additional MN Square well Blocks may be necessary see ordering information MACHEREY NAGEL 04 2014 Rev 05 25 NucleoSpin 96 Virus vacuum processing Pipette mixture up and down several times Do not moisten the rims Close the wells and incubate the mixture for 10 min at room temperature 18 25 C Optional Add 20 uL Proteinase K 20 mg mL to each sample pre mixed with Buffer RAV1 Close the Iysis vessels and incubate for 5 10 min at 56 70 C Addition of Proteinase K is required for viral DNA extraction and may be useful for viral RNA extraction from some sample types For details on incubation time and temperature please also refer to section 2 6 Spin briefly 30 s 1 500 x
32. rings Round well Block with Cap Strips 2 4 Cap Strips 24 48 MN Square well Blocks 6 12 Rack of Tube Strips 2 4 Self adhering PE Foil 10 20 User manual 1 1 1 For preparation of working solutions and storage conditions see section 3 2 Elution Buffer RE 5 mM Tris HCl pH 8 5 Set of 1 rack 12 strips with 8 tubes each Cap Strips included 4 MACHEREY NAGEL 04 2014 Rev 05 Viral RNA and DNA isolation 1 1 Kit contents continued NucleoSpin 96 Virus Core Kit 4 x 96 preps REF 740452 4 Lysis Buffer RAV1 6 x 40 mL Wash Buffer RAW 300 mL Wash Buffer RAV3 Concentrate 2x 100 mL RNase free H O 125 mL Elution Buffer RE 125 mL Carrier RNA Iyophilized 6x 1mg NucleoSpin Virus Binding Plates 4 blue rings User manual 1 1 2 Reagents and to be supplied by user Reagents 96 100 ethanol for preparation of working solutions see section 3 For more detailed information regarding special hardware required for centrifuge or vacuum processing please see section 2 3 For recommended accessories for use of the flexible NucleoSpin 96 Virus Core Kit reduced kit composition REF 740452 4 please see section 2 4 1 For preparation of working solutions and storage conditions see section 3 2 Elution Buffer RE 5 mM Tris HCl pH 8 5 MACHEREY NAGEL 04 2014 Rev 05 5 Viral RNA and DNA isolation 2 Product description 2 1 The basic principle The NucleoSpin 96 Virus kit is designed f
33. rising 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 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 MACHEREY NAGEL 04 2014 Rev 05 31 Viral RNA and DNA isolation Please cont
34. s Inhalt Gefahrstoff GHS Symbol H S tze P S tze RAV1 Guanidinium thiocyanate Warning 302 412 260 273 30 60 EUH031 301 312 330 Guanidiniumthiocyanat Achtung 30 60 RAW Guanidinium hydrochlo Warning 226 302 210 233 ride 24 36 isopropa 301 312 330 nol 35 55 403 235 Guanidiniumhydrochlorid Achtung 24 36 Isopropanol 35 55 Proteinase K Proteinase K lyophilized Danger 315 319 261 280 Proteinase K lyophilisiert D Gefahr 334 335 302 352 304 340 305 351 338 312 332 313 337 313 342 311 403 233 Hazard phrases H 226 H 302 H 315 H 319 H 334 H 335 Flammable liquid and vapour Fl ssigjkeit und Dampf entz ndbar Harmful if swallowed Gesundheitssch dich bei Verschlucken Causes skin irritation Verursacht Hautreizungen Causes serious eye irritation Verursacht schwere Augenreizung May cause allergy or asthma symptoms or breathing difficulties if inhaled Kann bei Einatmen Allergie asthmaartige Symptome oder Atembeschwerden verursachen May cause respiratory irritation Kann die Atemwege reizen MACHEREY NAGEL 04 2014 Rev 05 15 Viral RNA and DNA isolation H 412 EUH031 Harmful to aquatic life with long lasting effects Sch dlich f r Wasserorganismen mit langfristiger Wirkung Contact with acids liberates toxic gas Entwickelt bei Ber hrung mit S ure giftige Gase Precaution phrases P 210 P 233 P 260 P 261
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