Viral RNA / DNA isolation - MACHEREY
Contents
1. 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 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 TRIzol is a registered trademark of Molecular Research Center Inc All used names and denotations can be brands trademarks or registered labels of their respective owner also if they are no2. each well of the 96 well deep well block mix by pipetting up and down Add 180 uL Buffer VL1 and mix by pipetting up and down 3 times Optional Shake at 1000 rpm for 15 min at room temperature Continue with the preparation of the wash and elution plates before adding magnetic beads and binding buffer to the sample plate Prepare wash and elution plates Wash plates Fill 600 pL Buffer VEW1 to each well of an empty Thermo 96 well deep well plate Fill 600 pL Buffer VEW2 to each well of an empty Thermo 96 well deep well plate Fill 600 uL 80 ethanol to each well of an empty Thermo 96 well deep well plate Elution plate Fill 100 pL Buffer VEL to each well of an empty Thermo 200 uL 96 well plate Prepare sample lysis plate part Il Add 20 uL B Beads and 600 uL Buffer VEB to each well of the sample lysis plate MACHEREY NAGEL 06 2015 Rev 03 19 NucleoMag VET Run purification protocol on instrument Start the isolation of viral RNA DNA on the KingFisher Flex 96 instrument Start the method file NucleoMag VET Insert plates as indicated on the KingFisher instrument display Method starts with a mixing step combined lysis and binding step after setting up the last plate to the instrument Remove eluted viral RNA DNA The instrument stops after the final elution step Follow the instructions on the instrument s display and unload the plates from the instrument Purified vira
3. 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 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 VEL Elution can be carried out in a volume of 50 UL It is 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 8 MACHEREY NAGEL 06 201 Rev 03 Viral RNA DNA isolation 3 Storage conditions and preparation of working solutions Attention Buffers VL1 VEB VEW1 and VEW2 contain chaotropic salt Wear gloves and goggles All components of the NucleoMag VET 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 Before starting any NucleoMag VET protocol prepare the following Proteinase K Before first use of the kit add 3 35 mL
4. pL VEW1 14 MACHEREY NAGEL 06 201 Rev 03 NucleoMag VET Resuspend Shake 1 min at RT Remove supernatant after 2 min separation Wash with VEW2 Remove Square well Block from NucleoMag SEP 600 pL VEW2 Resuspend Shake 1 min at RT Remove supernatant after 2 min separation Wash with 80 Remove Square well Block ethanol from NucleoMag SEP 600 uL 80 ethanol Resuspend Shake 1 min at RT Remove supernatant after 2 min separation Air dry magnetic beads Air dry 10 min at RT Elute RNA DNA Remove Square well Block from NucleoMag SEP 50 100 pL VEL MACHEREY NAGEL 06 2015 Rev 03 15 NucleoMag VET Shake 5 min at RT Optional Mix by pipetting up and down Separate 2 min and transfer viral RNA DNA into elution plate tubes 16 MACHEREY NAGEL 06 201 Rev 03 NucleoMag VET 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 RNA DNA can be performed in reaction tubes with suitable magnetic separators This protocol is for manual use and serves as a guideline for adapting the kit to robotic instruments 1 Lyse sample Pre dispense 20 uL Proteinase K and 200 uL of sample to a suitable reaction tube Add 180 uL
5. Buffer VL1 to the reaction tube Optional add 4 uL of the Carrier RNA stock solution to the reaction tube Mix well by repeated pipetting up and down and incubate at room temperature for 15 min with shaking Alternatively Iysis step can be performed in Tube Strips see ordering information Following the Iysis incubation spin down to collect any sample from the Iysis tube lids and transfer each Iysate to the wells of a Square well Block 2 Bind nucleic acid to magnetic beads Add 20 uL resuspended B Beads and 600 uL Buffer VEB to the lysed sample Mix by pipetting up and down 6 times and shake for 5 min atroom temperature Alternatively when processing the kit without a shaker pipette up and down 10 times and incubate for 5 min at room temperature NucleoMag B Beads and Buffer VEB can be pre mixed Be sure to resuspend the NucleoMag B Beads before removing them from the storage bottle Vortex storage bottle briefly until a homogenous suspension has been formed Separate the magnetic beads against 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 3 Wash with VEW1 Remove the Square well Block from the NucleoMag SEP magnetic separator Add 600 pL Buffer VEW1 and resuspend the beads b
6. DK A Viral RNA DNA isolation User manual NucleoMag VET June 2015 Rev 03 MACHEREY NAGEL 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 6 2 4 Adjusting the shaker settings 7 2 5 Handling of beads 8 2 6 Elution procedures 8 3 Storage conditions and preparation of working solutions 9 4 Safety instructions 10 5 Protocols 13 5 1 Preparation of sample materials 13 5 2 Isolation of viral RNA DNA and bacterial DNA from blood tissue homogenates serum plasma other body fluids and washes 14 5 3 Detailed protocol for KingFisher Flex 96 19 6 Appendix 21 6 1 Troubleshooting 21 6 2 Ordering information 22 6 3 Product use restriction warranty 23 MACHEREY NAGEL 06 2015 Rev 03 3 Viral RNA DNA isolation 1 Components 1 1 Kit contents NucleoMag VET 1x 96 preps 4 x 96 preps REF 744200 1 744200 4 NucleoMag B Beads 2 5 mL 10 mL Lysis Buffer VL1 30 mL 100 mL Binding Buffer VEB 110 mL 3x 110 mL Wash Buffer VEW1 75 mL 300 mL Wash Buffer VEW2 75 mL 300 mL Elution Buffer VEL 30 mL 125 mL Carrier RNA 400 ug 4 x 400 ug Carrier RNA Buffer 500 uL 4 x 500 uL Proteinase K lyophilized 75 mg 3x 75 mg Proteinase Buffer PB 8 mL 15 mL User manual 1 1 For preparation of working solutions and sto
7. Danger 226 302 210 233 301 312 40 ethanol 35 55 330 403 235 Natriumperchlorat 20 40 Gefahr Ethanol 35 55 VEW1 Sodium perchlorate Warning 226 210 233 403 235 VEW2 5 20 ethanol 20 35 Natriumperchlorat 5 20 Achtung Ethanol 20 35 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 D Danger 315 317 261 280 302 352 Proteinase K lyophilisiert Gefahr 319 334 304 340 335 305 351 338 312 lt gt 333 313 337 313 342 311 363 403 233 10 MACHEREY NAGEL 06 201 Rev 03 Viral RNA DNA isolation Hazard phrases H 226 H 302 H315 H317 H 319 H 334 H 335 H 412 EUH 031 Flammable liquid and vapour Fl ssigkeit und Dampf entz ndbar Harmful if swallowed Gesundheitssch dlich bei Verschlucken Causes skin irritation Verursacht Hautreizungen May cause an allergic skin reaction Kann allergische Hautreaktionen verursachen 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 verursa chen May cause respiratory irritation Kann die Atemwege reizen Harmful to aquatic life with long lasting effects Sch dlich f r Wasserorganismen mit langfristiger Wi
8. 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 VET 1 x 96 preps 4 x 96 preps REF 744200 1 744200 4 Proteinase K 1 vial 75 mg 3 vials 75 mg vial f yoptllzed Add 3 35 mL Add 3 35 mL Proteinase Proteinase Buffer Buffer to each vial Carrier RNA 1 vial 400 ug 4 vials 400 ug vial lyophilized Add 500 pL Add 500 uL Carrier RNA Carrier RNA Buffer Buffer to each vial MACHEREY NAGEL 06 2015 Rev 03 9 Viral RNA DNA isolation 4 Safety instructions The following components of the NucleoMag VET 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 Hazard Precaution symbol phrases phrases Inhalt Gefahrstoff GHS Symbol H Sdtze P S tze VL1 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 VEB Sodium perchlorate 20
9. R 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 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 tra
10. e Kontaktlinsen nach M glichkeit entfernen Weiter aussp len Rinse mouth Mund aussp len MACHEREY NAGEL 06 2015 Rev 03 11 Viral RNA DNA isolation P 333 313 P 342 311 P 337 313 P 363 P 403 235 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 doctor Bei Symptomen der Atemwege GIFTINFORMATIONSZENTRUM 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 A The symbol shown on labels refers to the precaution phrases of this section Das auf Etiketten dargestellte Symbol weist auf die P S tzen dieses Kapitels hin 12 MACHEREY NAGEL 06 201 Rev 03 NucleoMag VET 5 Protocols 5 1 Preparation of sample materials a Blood and serum plasma samples A sample volume of 100 200 uL blood can be used Do not use higher volumes When using less than 200 uL samples adjus
11. ed product information 22 MACHEREY NAGEL 06 201 Rev 03 Viral RNA DNA isolation 6 3 Product use restriction warranty NucleoMag VET 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 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 OTHE
12. er to the wells of the collection plate and proceed as described above MACHEREY NAGEL 06 2015 Rev 03 7 Viral RNA DNA isolation 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 simultaneous mixing
13. ing magnetic pins Magnetic pins rods are moved from one side of the well to the other and vice 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 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 600 uL 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 washing step Adjusting shaker speed for the elution step e Load 100 uL dyed wat
14. l RNA DNA can be used for further PCR based analysis 20 MACHEREY NAGEL 06 201 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 Aspiration 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 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 80 ethanolic wash
15. magnetic beads Air dry the magnetic bead pellet for 10 min at room temperature Elute RNA DNA Remove the Square well Block from the NucleoMag SEP magnetic separator Add desired volume of Buffer VEL 50 100 uL to each well of the Square well Block and resuspend the beads by shaking 5 min at room temperature 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 elution plates or tube strips see ordering information 18 MACHEREY NAGEL 06 201 Rev 03 NucleoMag VET 5 3 Detailed protocol for KingFisher Flex 96 Note The required method file NucleoMag VET for the instrument is available at Technical Support Bioanalysis tech bio mn net com Important Always prepare deep well block with samples first and add reagents exactly in the order as given below Before starting the preparation Check that Proteinase K and Carrier RNA were prepared according to section 3 KingFisher Accessory Kit A see ordering information Prepare sample lysis plate part I Dispense 20 uL Proteinase K solution to each well of the 96 well deep well block Add 200 uL uL blood sample homogenized tissue sample to
16. n of nucleic acids to paramagnetic beads under appropriate buffer conditions Sample lysis is achieved by incubation with a Lysis Buffer VL1 containing chaotropic ions supported by Proteinase K digestion For binding of nucleic acids to the paramagnetic beads Binding Buffer VEB and the NucleoMag B Beads are added to the lysate After magnetic separation the paramagnetic beads are washed to remove contaminants and salts using Wash Buffers VEW1 and VEW2 and 80 ethanol Residual ethanol from previous wash steps is removed by airdrying Finally highly pure viral RNA DNA is eluted with low salt Elution Buffer VEL or water Purified viral RNA DNA can directly be used for downstream applications The NucleoMag VET kit can be used either manually or automated on standard liquid handling instruments or automated magnetic separators 2 2 Kit specifications NucleoMag VET 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 blood samples or homogenized tissue suspensions 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 VET allows easy automation on common liquid handling i
17. nsport insurance for customers excluded or out of accident or improper or abnormal use of this product defects in products or MACHEREY NAGEL 06 2015 Rev 03 23 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 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
18. nstruments or automated magnetic separators The actual processing time depends on the configuration 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 2 3 Magnetic separation systems For use of NucleoMag VET 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 6 MACHEREY NAGEL 06 201 Rev 03 Viral RNA DNA isolation 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 mov
19. rage conditions see section 3 4 MACHEREY NAGEL 06 201 Rev 03 Viral RNA DNA isolation 1 2 Material to be supplied by user Product REF Pack of Separation plate for magnetic beads separation 740481 4 e g Square well Block 96 well block with 740481 24 24 2 1 mL square wells Lysis tubes for incubation of samples and lysis y l 740477 4 sets e g Rack of Tubes Strips 1 set consists 740477 24 24 sets of 1 Rack 12 Strips with 8 tubes 1 2 mL wells each and 12 Cap Strips Elution plate for collecting purified nucleic acids 740486 24 24 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 740673 20 For use of kit on KingFisher 96 instrument e g KingFisher 96 Accessory Kit A Square well Blocks Deep well tip combs 744950 1 set Elution Plates for 4 x 96 NucleoMag 96 Virus preps using KingFisher 96 platform Reagents 80 ethanol MACHEREY NAGEL 06 2015 Rev 03 Viral RNA DNA isolation 2 Product description 2 1 The basic principle The NucleoMag VET kit is designed for the isolation of viral DNA or RNA from cell free body fluids such as serum or plasma blood or homogenized tissue sample suspensions This kit provides reagents and magnetic beads for isolation of 96 samples from 100 200 uL The procedure is based on the reversible adsorptio
20. rkung 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 304 340 P 305 351 313 P 330 Keep away from heat hot surfaces sparks open flames and other ignition sources No smoking Von Hitze heiBen Oberflachen Funken offenen Flammen sowie anderen Ztindquellenarten 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 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 com fortable for breathing BEI EINATMEN An die frische Luft bringen und in einer Position ruhigstellen die das Atmen erleichtert IF IN EYES Rinse continuously 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 Vorhanden
21. solution from the final wash as residual ethanol interferes with downstream applications MACHEREY NAGEL 06 2015 Rev 03 21 Viral RNA DNA isolation Poor Ethanol evaporation from wash buffers performance e 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 high 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 VET 744200 1 1 x 96 preps 744200 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 with 8 740477 24 24 sets tubes each and 12 Cap Strips Elution Plate U bottom 740486 24 24 KingFisher 96 Accessory Kit A 744950 1 set Square well Blocks Deep well tip combs Elution Plates for 4 x 96 NucleoMag VET preps using KingFisher 96 platform Buffer RA1 50 mL 740961 50 mL Reducing Agent TCEP 740395 107 107 mg Visit www mn net com for more detail
22. t 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 24 MACHEREY NAGEL 06 201 Rev 03
23. t with PBS buffer to 200 yL b Tissue samples Homogenize tissue samples Typically 5 10 mg sample material can be homogenized in 400 uL PBS buffer using a bead based homogenizer If necessary higher ammounts of sample material can be used up to 25 mg It should be considered that the copurified total nucleic acid may cause inhibition in the subsequent PCR assays After homogenization of the tissue centrifuge and use up to 200 uL clear supernatant for the purification protocol If using less than 200 uL adjust with PBS buffer to a final volume of 200 pL For isolation of viral RNA Tissue samples can be also disrupted in a buffer containing chaotropic salt e g Buffer RA1 see ordering information and beta mercaptoethanol or TCEP reducing agent see ordering information c Swab samples Incubate the swabs with PBS sodium chloride or cell culture medium for 30 min with shaking Remove and sqeeze out the swab Proceed with 200 uL of the particle free buffer or medium for purification protocol d Feces Mix 1 volume of feces e g 500 uL with an equal volume of PBS buffer Mix vigorously by vortexing for 1 min Allow the particles to settle down or centrifuge with low speed e g at 500 x g For difficult to lyse bacteria mechanical disruption or treatment using suitable glas beads may be required Take the supernatant and use 200 uL for the purification protocol e TRIzol lysis For sample materials such as semen a TRI
24. y shaking until the beads are resuspended completely 1 3 min Alternatively resuspend beads completely by repeated pipetting up and down MACHEREY NAGEL 06 2015 Rev 03 17 NucleoMag VET 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 Wash with VEW2 Remove the Square well Block from the NucleoMag SEP magnetic separator Add 600 pL Buffer VEW2 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 Wash with 80 ethanol Remove the Square well Block from the NucleoMag SEP magnetic separator Add 600 pL 80 ethanol 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 Air dry
25. zol lysis may be required Homogenize 10 30 mg tissue or up to 250 uL blood with 1 mL TRizol reagent to manufacturer s instructions After phase separation by centrifugation remove aqueous colorless upper phase approximately 400 uL For further processing start with step 2 of the purification protocol by mixing 400 uL of the aqueous phase with 600 uL Buffer VEB and 20 uL NucleoMag B Beads MACHEREY NAGEL 06 2015 Rev 03 13 NucleoMag VET 5 2 Isolation of viral RNA DNA and bacterial DNA from blood tissue homogenates serum plasma other body fluids and washes Preparation of sample material The standard protocol is related to a volume of 200 uL homogenized sample For the preparation of different sample materials e g tissue swabs feces please see the indications at section 5 1 Protocol at a glance For hardware requirements refer to section 2 3 For detailed information on each step see page 18 Before starting the preparation Check that Proteinase K and Carrier RNA were prepared according to section 3 1 Lyse sample 200 uL homogenized sample 20 uL Proteinase K 4 uL Carrier RNA 180 pL VL1 Mix SS RT 15 min 2 Bind nucleic acid to 600 pL VEB NucleoMag B Beads 20 uL B Beads Mix by shaking for 5 10 min at RT Optional Mix by pipetting up and down Remove supernatant after 2 min separation 3 Wash with VEW1 Remove Square well Block from NucleoMag SEP 600
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