Viral RNA / DNA isolation
Contents
1. Viral RNA DNA isolation User manual NucleoMag 96 Virus November 2012 Rev 03 MACHEREY NAGEL MN i www mn net com Viral RNA DNA isolation Table of contents 1 Components 1 1 Kit contents 1 2 Material to be supplied by user 5 2 Product description 6 2 1 The basic principle 6 2 2 Kit specifications 6 2 3 Magnetic separation systems 7 2 4 Adjusting the shaker settings 8 2 5 Handling of beads 8 2 6 Elution procedures 9 3 Storage conditions and preparation of working solutions 10 4 Safety instructions 11 4 1 Risk and safety phrases 11 4 2 GHS classification 13 5 Protocol for the isolation of viral RNA DNA from cell free body fluids 15 6 Appendix 19 6 1 Troubleshooting 19 6 2 Ordering information 20 6 3 Product use restriction warranty 21 MACHEREY NAGEL 11 2012 Rev 03 3 Viral RNA DNA isolation 1 Components 1 1 Kit contents NucleoMag 96 Virus 1x 96 preps 4 x 96 preps REF 744800 1 744800 4 NucleoMag V Beads 3 mL 4 x3 mL Lysis Buffer MVL 20 mL 80 mL Binding Buffer MV2 60 mL 2x 120 mL Wash Buffer MV3 50 mL 2x 100 mL Wash Buffer MV4 50 mL 2x 100 mL Wash Buffer MV5 55 mL 2x 110 mL Elution Buffer MV6 10 mL 40 mL Carrier RNA 400 ug 4 x 400 ug Carrier RNA Buffer 500 uL 4 x 500 uL Proteinase K lyophilized 22 mg 4x 22mg Proteinase Buffer PB 3 6 mL 8 mL User manual 1 1 For preparation of working solutions and storage conditions see section 3 4 MACHEREY NAGEL 11 2012 Rev2. 403 235 Natriumperchlorat 20 40 Gefahr Ethanol 35 55 MV3 Sodium perchlorate Warning 226 210 233 403 235 5 20 ethanol 20 35 Natriumperchlorat 5 20 Achtung Ethanol 20 35 MV4 Ethanol 55 75 Danger 225 210 233 403 235 Ethanol 55 75 Gefahr Carrier RNA Guanidinium thiocyanate Warning 302 412 260 273 301 312 Buffer 30 60 EU031 330 Guanidiniumthiocyanat Achtung 30 60 Proteinase K Proteinase K lyophilized Danger 315 317 261 280 302 352 Proteinase K lyophilisiert lt gt Gefahr 319 334 304 340 335 305 351 338 312 333 313 337 313 342 311 363 403 233 Hazard phrases H 225 Highly flammable liquid and vapour Fl ssigkeit und Dampf leicht entz ndbar H 226 Flammable liquid and vapour Fl ssigkeit und Dampf entz ndbar H 302 Harmful if swallowed Gesundheitssch dlich bei Verschlucken H 315 Causes skin irritation Verursacht Hautreizungen H 317 May cause an allergic skin reaction Kann allergische Hautreaktionen verursachen H 319 Causes serious eye irritation Verursacht schwere Augenreizung MACHEREY NAGEL 11 2012 Rev 03 13 Viral RNA DNA isolation Hazard phrases H 334 H 335 H 412 EUH 031 May cause allergy or asthma symptoms or breathing difficulties if inhaled Kann bei Einatmen Allergie asthmaartige Symptome oder Atembeschwerden verursa chen May cause respiratory irritation Kann die Atemwege reizen Harmful
3. Von Z ndquellen fernhalten Nicht rauchen Do not breathe dust Staub nicht einatmen Avoid contact with the skin Ber hrung mit der Haut vermeiden In case of contact with the eyes rinse with plenty of water and seek medical advice Bei Ber hrung mit den Augen gr ndlich mit Wasser absp len und Arzt konsultieren Wear suitable protective clothing and gloves Bei der Arbeit geeignete Schutzhandschuhe und Schutzkleidung tragen Wear suitable gloves and eye face protection Bei der Arbeit geeignete Schutzhandschuhe und Schutzbrille Gesichtsschutz tragen Avoid release to the environment Refer to special instructions safety data sheet Freisetzung in die Umwelt vermeiden Besondere Anweisungen einholen Sicherheitsdaten blatt zu Rate ziehen 12 MACHEREY NAGEL 11 2012 Rev 03 Viral RNA DNA isolation 4 2 GHS classification Only harmful features do not need to be labeled with H and P phrases until 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 Hazard Precaution symbol phrases phrases Inhalt Gefahrstoff GHS Symbol H S tze P S tze MVL Guanidine hydrochloride Warning 302 315 280 301 312 50 66 319 302 352 Guanidinhydrochlorid 50 66 Achtung 305 351 338 330 332 313 337 313 MV2 Sodium perchlorate 20 Danger 226 302 210 233 301 312 40 ethanol 35 55 330
4. 4 Safety instructions The following components of the NucleoMag 96 Virus kits contain hazardous contents Wear gloves and goggles and follow the safety instructions given in this section 4 1 Risk and safety phrases Component Hazard contents Hazard Risk Safety symbol phrases phrases Inhalt Gefahrstoff Gefahrstoff R S tze S S tze symbol MVL Guanidine hydrochloride 50 66 x Xn R 22 36 38 S 26 Guanidinhydrochlorid 50 66 37 39 MV2 Sodium perchlorate 20 40 Xn R 10 22 S 13 16 ethanol 35 55 Natriumperchlorat 20 40 Ethanol 35 55 MV3 Sodium perchlorate 5 20 R 10 S 16 ethanol 20 35 Natriumperchlorat 5 20 Ethanol 20 35 MV4 Ethanol 55 75 F R 11 S 7 16 Ethanol 55 75 Carrier RNA Guanidinium thiocyanate 30 60 x Xn R 20 21 22 S 13 61 Buffer Guanidiniumthiocyanat 30 60 y N 32 52 53 Proteinase K Proteinase K lyophilized x Xn R 36 37 38 S 22 24 Proteinase K lyophilisiert 42 26 36 37 Hazard labeling not necessary if quantity per bottle below 125g or mL certificate of exemption according to 67 548 EEC Art 25 1999 45 EC Art 12 and German GefStoffV 20 3 and TRGS 200 7 1 For further information see Material Safety Data Sheet Hazard labeling not necessary if quantity per bottle below 25g or mL certificate of exemption according to 67 548 EEC Art 25 1999 45 EC Art 12 and German GefStoffV 20 3 and TRGS 200 7 1 For further informati
5. 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 expressly marked as IVD on the packaging IF THERE IS NO IVD SIGN THE PRODUCT SHALL NOT BE SUITABLE FOR N 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 MACHEREY NAGEL shall only be responsible for the product specifications and the performance range of MN products according to the specifications of in
6. 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 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 tha
7. uL Buffer MV5 to each well and incubate for 45 60 s while the beads are still attracted to magnets Then aspirate and discard the supernatant Do not resuspend the beads in Buffer MV5 This step is to remove traces of ethanol and eliminates a drying step Do not exceed incubation time of max 1 min Elution Add desired volume of Buffer MV6 50 100 uL to each well of the Square well Block and resuspend the beads by shaking 5 min at 56 C Alternatively resuspend beads completely by repeated pipetting up and down and incubate for 5 min at 56 C Separate the magnetic beads by placing the Square well Block on the NucleoMag SEP magnetic separator Wait at least 2 min until all the beads have been attracted to the magnets Transfer the supernatant containing the purified viral RNA DNA to either microtubes or Tube Strips see ordering information 18 MACHEREY NAGEL 11 2012 Rev 03 Viral RNA DNA isolation 6 Appendix 6 1 Troubleshooting Problem Possible cause and suggestions Insufficient elution buffer volume Beads pellet must be covered completely with elution buffer Insufficient performance of elution buffer during elution step Remove residual buffers during the separation steps completely Remaining buffers decrease the efficiency of following wash and elution steps Beads dried out Poor yield Do not let the beads dry as this might result in lower elution low sensitivity efficiencies Aspirat
8. versa Beads follow this movement and are thus pulled through the buffer during the wash and elution steps Separation takes place when the system stops Automated separators Separators with moving magnets Magnetic beads are transferred into suitable plates or tubes Beads are resuspended from the rod covered magnets Following binding washing or elution beads are collected again with the rod covered magnets and transferred to the next plate or tube MACHEREY NAGEL 11 2012 Rev 03 T Viral RNA DNA isolation 2 4 Adjusting the shaker settings When using a plate shaker for the washing and elution steps the speed settings have to be adjusted carefully for each specific separation plate and shaker to prevent cross contamination from well to well Proceed as follows Adjusting shaker speed for binding and wash steps Load 1000 uL for checking the settings for the binding step or 600 uL for checking the settings for the washing steps dyed water to the wells of the separation plate Place the plate on the shaker and start shaking with a moderate speed setting for 30 seconds Turn off the shaker and check the plate surface for small droplets of dyed water Increase speed setting shake for an additional 30 seconds and check the plate surface for droplets again Continue increasing the speed setting until you observe droplets on top of the separation plate Reduce speed setting check again and use this setting for the wash
9. 00 uL sample 10 uL Proteinase K 4 uL Carrier RNA 200 pL MVL Mix 56 C 10 min 2 _ Bind viral RNA DNA to 600 pL MV2 NucleoMag V Beads 30 uL V Beads Mix by shaking for 5 min at RT Optional Mix by pipetting up and down Remove supernatant after 2 min separation 3 Wash with MV3 Remove Square well Block from NucleoMag SEP 500 pL MV3 Resuspend Shake 1 min at RT MACHEREY NAGEL 11 2012 Rev 03 15 NucleoMag 96 Virus Remove supernatant after 2 min separation 4 Wash with MV4 Remove Square well Block from NucleoMag SEP 500 pL MV4 Resuspend Shake 1 min at RT Remove supernatant after 2 min separation 5 Wash with MV5 550 uL MV5 Incubate for 45 60 s Note Do not resuspend the beads in Buffer MV5 Remove supernatant 6 Elute RNA DNA Remove Square well Block from NucleoMag SEP 50 100 pL MV6 Shake 5 min at 56 C Optional Mix by pipetting up and down Separate 2 min and transfer viral RNA DNA into elution plate tubes 16 MACHEREY NAGEL 11 2012 Rev 03 NucleoMag 96 Virus Detailed protocol This protocol is designed for magnetic separators with static pins e g NucleoMag SEP and suitable plate shakers It is recommended using a Square well Block for separation see ordering information Alternatively isolation of viral RNA DNA can be performed in reaction tubes with suitable magnetic sepa
10. 03 Viral RNA DNA isolation 1 2 Material to be supplied by user Product REF Pack of Separation plate for magnetic beads separation e g Square well Block 96 well block with 2 1 mL square wells Lysis tubes for incubation of samples and lysis e g Rack of Tubes Strips 1 set consists of 1 Rack 12 Strips with 8 tubes 1 2 mL wells each and 12 Cap Strips Elution plate for collecting purified nucleic acids e g Elution Plate U bottom 96 well 0 3 mL microtiterplate with 300 uL u bottom wells e g Elution Plate Flat bottom 96 well 0 3 mL microtiterplate with 300 uL flat bottom wells For use of kit on KingFisher 96 instrument e g KingFisher 96 Accessory Kit A Square well Blocks Deep well tip combs Elution Plates for 4 x 96 NucleoMag 96 Virus preps using KingFisher 96 platform 740481 740481 24 740477 740477 24 740486 24 740673 744950 4 sets 24 sets 24 20 1 set MACHEREY NAGEL 11 2012 Rev 03 Viral RNA DNA isolation 2 Product description 2 1 The basic principle The NucleoMag 96 Virus kit is designed for the isolation of viral DNA or RNA from cell free body fluids such as serum or plasma This kit provides reagents and magnetic beads for isolation of 96 samples from 200 uL serum or plasma The procedure is based on the reversible adsorption of nucleic acids to paramagnetic beads under appropriate buffer conditions Sample lysis is achieved by
11. VL may form salt precipitates upon storage To re dissolve the salt precipitate incubate the buffer bottle at 40 C until all of the precipitate is re dissolved Lysis Buffer MVL with Carrier RNA Lysis Buffer MVL with added Carrier RNA can be stored at room temperature for 1 2 weeks Frequent warming temperatures gt 80 C and extended heat incubation will cause degradation of the 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 Do not warm Buffer MVL containing Carrier RNA more than 6 times Before starting any NucleoMag 96 Virus protocol prepare the following Proteinase K Before first use of the kit add 1 1 mL Proteinase Buffer PB to each vial of the lyophilized Proteinase K Dissolved Proteinase K solution should be stored in aliquots at 20 C Carrier RNA Before first use of the kit add 500 uL Carrier RNA Buffer to each vial lyophilized Carrier RNA Store dissolved Carrier RNA solution in aliquots at 20 C NucleoMag 96 Virus 1 x 96 preps 4 x 96 preps REF 744800 1 744800 4 Proteinase K lyophilized 1 vial 22 mg 4 vials 22 mg vial Add 1 1 mL Add 1 1 mL Proteinase Proteinase Buffer Buffer to each vial Carrier RNA lyophilized 1 vial 400 ug 4 vials 400 ug vial Add 500 uL Add 500 uL Carrier RNA Carrier RNA Buffer Buffer to each vial 10 MACHEREY NAGEL 11 2012 Rev 03 Viral RNA DNA isolation
12. 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 of accident or improper or abnormal use of this product defects in products or MACHEREY NAGEL 11 2012 Rev 03 21 Viral RNA DNA 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
13. igh elution step High aspiration speed during the elution step may cause bead carry over Reduce aspiration speed for elution step 6 2 Ordering information Product REF Pack of NucleoMag 96 Virus 744800 1 1 x 96 preps 744800 4 4 x 96 preps NucleoMag SEP 744900 1 Square well Blocks 740481 4 740481 24 24 Self adhering PE Foil 740676 50 sheets Rack of Tube Strips 740477 4 sets set consists of 1 Rack 12 Tube Strips 740477 24 24 sets with 8 tubes each and 12 Cap Strips KingFisher 96 Accessory Kit A 744950 1 set Square well Blocks Deep well tip combs Elution Plates for 4 x 96 NucleoMag 96 Virus preps using King Fisher 96 platform Visit www mn net com for more detailed product information 20 MACHEREY NAGEL 11 2012 Rev 03 Viral RNA DNA isolation 6 3 Product use restriction warranty NucleoMag 96 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
14. incubation with a new and improved Lysis Buffer MVL containing chaotropic ions supported by Proteinase K digestion For binding of nucleic acids to the paramagnetic beads Binding Buffer MV2 and the NucleoMag V Beads are added to the lysate After magnetic separation the paramagnetic beads are washed to remove contaminants and salts using Wash Buffers MV3 and MV4 Residual ethanol from previous wash steps is removed by a short incubation of the beads in Wash Buffer MV5 Finally highly pure viral RNA DNA is eluted with low salt Elution Buffer MV6 or water Purified viral RNA DNA can directly be used for downstream applications The NucleoMag 96 Virus kit can be used either manually or automated on standard liquid handling instruments or automated magnetic separators 2 2 Kit specifications NucleoMag 96 Virus is designed for rapid manual and automated small scale preparation of viral RNA DNA from cell free body fluids such as serum or plasma samples The kit is designed for use with NucleoMag SEP magnetic separator plate see ordering information or other magnetic separation systems see section 2 3 Manual time for the preparation of 96 samples is about 120 minutes The purified RNA DNA can be used directly as template for RT PCR PCR or any kind of enzymatic reactions NucleoMag 96 Virus allows easy automation on common liquid handling instruments or automated magnetic separators The actual processing time depends on the configuratio
15. ing step Adjusting shaker speed for the elution step Load 100 uL dyed water to the wells of the collection plate and proceed as described above 2 5 Handling of beads Distribution of beads A homogeneous distribution of the magnetic beads to the individual wells of the separation plate is essential for a high well to well consistency Therefore before distributing the beads make sure that the beads are completely resuspended Shake the storage bottle well or place it on a vortexer shortly Premixing magnetic beads with the binding buffer allows easier homogenous distribution of the beads to the individual wells of the separation plate During automation a premix step before aspirating the beads binding buffer mixture from the reservoir is recommended to keep the beads resuspended Magnetic separation time Attraction of the magnetic beads to the magnetic pins depends on the magnetic strength of the magnetic pins the selected separation plate distance of the separation plate from the magnetic pins and the volume to be processed The individual times for complete attraction of the beads to the magnetic pins should be checked and adjusted on each system It is recommended using the separation plates or tubes specified by the supplier of the magnetic separator Washing the beads Washing the beads can be achieved by shaking or mixing In contrast to mixing by pipetting up and down mixing by shaker or magnetic mixing allows simultaneo
16. inst the side of the wells by placing the Square well Block on the NucleoMag SEP a magnetic separator Wait at least 2 min until all the beads have been attracted to the magnets Remove and discard supernatant by pipetting Do not disturb the attracted beads while aspirating the supernatant MACHEREY NAGEL 11 2012 Rev 03 17 NucleoMag 96 Virus MV3 wash Remove the Square well Block from the NucleoMag SEP magnetic separator Add 500 uL Buffer MV3 and resuspend the beads by shaking until the beads are resuspended completely 1 3 min Alternatively resuspend beads completely by repeated pipetting up and down Separate the magnetic beads by placing the Square well Block on the NucleoMag SEP magnetic separator Wait at least 2 min until all the beads have been attracted to the magnet Remove and discard supernatant by pipetting MV4 wash Remove the Square well Block from the NucleoMag SEP magnetic separator Add 500 uL Buffer MV4 and resuspend the beads by shaking until the beads are resuspended completely 1 3 min Alternatively resuspend beads completely by repeated pipetting up and down Separate the magnetic beads by placing the Square well Block on the NucleoMag SEP magnetic separator Wait at least 2 min until all the beads have been attracted to the magnet Remove and discard supernatant by pipetting MV5 wash Leave the Square well Block on the NucleoMag SEP magnetic separator Gently add 550
17. ion of attracted bead pellet Do not disturb the attracted beads while aspirating the supernatant especially when the magnetic bead pellet is not visible in the lysate Aspiration and loss of beads Time for magnetic separation too short or aspiration speed too high Low purity low sensitivity Insufficient washing procedure e Use only the appropriate combinations of separator and plate for example Square well Block in combination with NucleoMag SEP Make sure that beads are resuspended completely during the washing procedure If shaking is not sufficient to resuspend the beads completely mix by repeated pipetting up and down Poor performance of RNA in downstream applications Carry over of ethanol from wash buffers Be sure to remove all of the ethanolic wash solution Buffer MV4 as residual ethanol interferes with downstream applications MACHEREY NAGEL 11 2012 Rev 03 19 Viral RNA DNA isolation Poor Ethanol evaporation from wash buffers performance Close buffer bottles tightly avoid ethanol evaporation from of RNA in buffer bottles as well as from buffer filled in reservoirs Do not downstream reuse buffers from buffer reservoirs applications continued Time for magnetic separation too short Increase separation time to allow the beads to be completely attracted to the magnetic pins before aspirating any liquid from Carry over the well of beads Aspiration speed too h
18. n of the instrument and the magnetic separation system used Typically 96 samples can be purified in less than 120 minutes using the NucleoMag SEP on the automation platform 6 MACHEREY NAGEL 11 2012 Rev 03 Viral RNA DNA isolation 2 3 Magnetic separation systems For use of NucleoMag 96 Virus the use of the magnetic separator NucleoMag SEP is recommended Separation is carried out in a Square well Block see ordering information The kit can also be used with other common separators Magnetic separator Separation plate or tube NucleoMag SEP MN REF 744900 Square well Block MN REF 740481 Tecan Te MagS 1 5 mL tubes without lid Sarstedt Static magnetic pins Separators with static magnetic pins for example NucleoMag SEP for manual use and for use on liquid handling workstations This type of separator is recommended in combination with a suitable microplate shaker for optimal resuspension of the beads during the washing and elution steps Alternatively beads can be resuspended in the buffer by pipetting up and down several times For fully automated use on liquid handling workstations a gripper tool is required the plate is transferred to the magnetic separator for separation of the beads and transferred to the shaker module for resuspension of the beads Movable magnetic systems Separators with moving magnetic pins Magnetic pins rods are moved from one side of the well to the other and vice
19. on see Material Safety Data Sheet MACHEREY NAGEL 11 2012 Rev 03 11 Viral RNA DNA isolation Risk phrases R10 R 11 R22 R 20 21 22 R 32 R 36 38 R 36 37 38 R42 R 42 43 R 52 53 Flammable Entztindlich Highly flammable Leichtentz ndlich Harmful by inhalation Gesundheitssch dlich beim Verschlucken Harmful by inhalation in contact with skin and if swallowed Gesundheitssch dlich beim Einatmen Verschlucken und Ber hrung mit der Haut Contact with acids liberates very toxic gas Entwickelt bei Ber hrung mit S ure sehr giftige Gase Irritating to eyes and skin Reizt die Augen und die Haut Irritating to eyes respiratory system and skin Reizt die Augen Atmungsorgane und die Haut May cause sensitization by inhalation Sensibilisierung durch Einatmen m glich May cause sensitization by inhalation and skin contact Sensibilisierung durch Einatmen und Hautkontakt m glich Harmful to aquatic organisms may cause long term adverse effects in the aquatic environment Sch dlich f r Wasserorganismen kann in Gew ssern l ngerfristig sch dliche Wirkungen haben Safety phrases S S13 S16 S22 S24 S26 S 36 37 S 37 39 S 61 Keep container tightly closed Beh lter dicht geschlossen halten Keep away from food drink and animal feedstuffs Von Nahrungsmitteln Getr nken und Futtermitteln fernhalten Keep away from sources of ignition No Smoking
20. rators This protocol is for manual use and serves as a guideline for adapting the kit to robotic instruments 1 Lyse sample Pre dispense 10 uL Proteinase K and 200 uL of sample to a suitable reaction tube Add 200 uL Buffer MVL with added Carrier RNA to the reaction tube If Carrier RNA is not premixed with the Buffer MVL add 4 uL of the stock solution to the reaction tube Mix well by repeated pipetting up and down and incubate at 56 C for 10 min Alternatively lysis step can be performed in Tube Strips see ordering information For higher convenience a premix of Proteinase K Buffer MVL and Carrier RNA can be prepared This premix should be added to the sample immediately within 15 min after preparation Following the lysis incubation spin down to collect any sample from the lysis tube lids and transfer each lysate to the wells of a Square well Block 2 Bind viral RNA DNA to magnetic beads Add 30 uL resuspended V Beads and 600 uL Buffer MV2 to the lysed sample Mix by pipetting up and down 6 times and shake for 5 min at room temperature Alternatively when processing the kit without a shaker pipette up and down 10 times and incubate for 5 min at room temperature NucleoMag V Beads and Buffer MV2 can be pre mixed Be sure to resuspend the NucleoMag V Beads before removing them from the storage bottle Vortex storage bottle briefly until a homogenous suspension has been formed Separate the magnetic beads aga
21. t 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 Trademarks KingFisher is a registered trademark of Thermo Fisher Scientific NucleoMag is a registered trademark of MACHEREY NAGEL GmbH amp Co KG Te MagS is a trademark of Tecan Group Ltd Switzerland 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 22 MACHEREY NAGEL 11 2012 Rev 03
22. 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 P 273 P 280 P 301 312 P 302 352 P 304 341 P 305 351 313 P 330 P 333 313 P 342 311 P 337 313 P 363 P 403 235 Keep away from heat sparks open flames hot surfaces No smoking Von Hitze Funken offener Flamme heiBen Oberfl chen fernhalten Nicht rauchen Keep container tightly closed Beh lter dicht verschlossen halten Do not breathe dust fume gas mist vapours spray 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 or doctor physician if you feel unwell Bei Verschlucken Bei Unwohlsein Giftinformationszentrum oder Arzt anrufen IF ON SKIN Wash with plenty of soap and water Bei Kontakt mit der Haut Mit viel Wasser und Seife waschen IF INHALED If breathing is difficult remove to fresh air and keep at rest ina position comfortable for breathing Bei Einatmen Bei Atembeschwerden an die frische Luft bringen und in einer Position ruhigstellen die das Atmen erleichtert IF IN EYES Rinse continuously with water for several minutes Remo
23. us mixing 8 MACHEREY NAGEL 11 2012 Rev 03 Viral RNA DNA isolation of all samples This reduces the time and number of tips needed for the preparation Resuspension by pipetting up and down however is more efficient than mixing by a shaker or magnetic mix Method Resuspension Number of tips efficiency needed Magnetic mix Low Shaker Low Pipetting High 2 6 Elution procedures Purified viral RNA DNA can be eluted directly with the supplied Elution Buffer MV6 Elution can be carried out in a volume of gt 50 UL Itis essential to cover the NucleoMag Beads completely with elution buffer during the elution step The volume of dispensed elution buffer depends on the magnetic separation system e g the position of the pellet inside the separation plate For efficient elution the magnetic bead pellet should be resuspended completely in the elution buffer For some separators high elution volumes might be necessary to cover the whole pellet 8 channel pipetting device MACHEREY NAGEL 11 2012 Rev 03 9 Viral RNA DNA isolation 3 Storage conditions and preparation of working solutions Attention Buffers MVL MV2 and MV3 contain chaotropic salt Wear gloves and goggles All components of the NucleoMag 96 Virus kit should be stored at room temperature 18 25 C and are stable for up to one year All buffers are delivered ready to use Lysis Buffer MVL Lysis Buffer M
24. ve 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 aussp len Rinse mouth Mund aussp len If skin irritation or a rash occurs Get medical advice attention Bei Hautreizung oder ausschlag Arztlichen Rat einholen rztliche Hilfe hinzuziehen If experiencing respiratory symptoms Call a POISON CENTER or doc tor physician Bei Symptomen der Atemwege Giftinformationszentrum oder Arzt anrufen Get medical advice attention Bei anhaltender Augenreizung Arztliche Rat einholen rztliche Hilfe hinzuziehen Wash contaminated clothing before reuse Kontaminierte Kleidung vor erneutem Tragen waschen Store in a well ventilated place Keep cool Beh lter dicht verschlossen 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 11 2012 Rev 03 NucleoMag 96 Virus 5 Protocol for the isolation of viral RNA DNA from cell free body fluids Protocol at a glance For hardware requirements refer to section 2 3 For detailed information on each step see page 17 Before starting the preparation Check if Proteinase K and Carrier RNA were prepared according to section 3 1 Lyse sample 2
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