Viral RNA/DNA Isolation
Contents
1. Viral RNA DNA Isolation 1 2 Material to be supplied by user Product Cat No Pack of Separation plate for magnetic beads separa tion 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 e g Rack of Tubes Strips 1 set consists of sd 24 a 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 740673 20 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 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 King Fisher 96 platform MACHEREY NAGEL 07 2009 Rev 02 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 appropri ate buffer conditions Sample lysis is achieved by incubation in a solution containing chaotropic ions supported by2. 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 con taminants and salts using the Wash Buffers MV3 and MV4 Residual ethanol from pre vious 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 wa ter 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 prepa ration 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 order ing 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 con figuration of the instrument and the magnetic separation system used Typically 96 samples can be
3. procedure e Useonlythe appropriate combinations of separator and plate e g MN Square well Block in combination with NucleoMag SEP e 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 per formance of RNA in downstream applications Carry over of ethanol from wash buffers e Be sure to remove all of the ethanolic wash solution Buffer MV4 as residual ethanol interferes with downstream applications Ethanol evaporation from wash buffers e Close buffer bottles tightly avoid ethanol evaporation from buf fer bottles as well as from buffer filled in reservoirs Do not reuse buffers from buffer reservoirs MACHEREY NAGEL 07 2009 Rev 02 17 Viral RNA DNA Isolation Time for magnetic separation too short e 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 e High aspiration speed during the elution step may cause bead carry over Reduce aspiration speed for elution step 6 2 Ordering information Product Cat No 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. purified in less than 120 minutes using the NucleoMag SEP on the automation platform 6 MACHEREY NAGEL 07 2009 Rev 02 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 informa tion The kit can also be used with other common separators Static magnetic pins Separators with static magnetic pins e g NucleoMag SEP for manual use and for use on liquid handling workstations This type of separator is recommended in combina tion 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 work stations 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 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
5. 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 18 MACHEREY NAGEL 07 2009 Rev 02 Viral RNA DNA Isolation 6 3 Product use restriction warranty NucleoMag 96 Virus kit components were developed designed distributed and sold FOR RESEARCH PURPOSES ONLY They are suitable FOR IN VITRO USES ONLY No claim or representation is intended for its use to identify any specific organism or for Clinical use diagnostic prognostic therapeutic or blood banking It is rather the responsibility of the user to verify the use of the NucleoMag 96 Virus kit for a specific application range as the performance characteristic of this kit has not been verified to a specific organism This MACHEREY NAGEL product is shipped with documentation stating specifica tions 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 conta
6. Beads are resuspended from the rod covered magnets Following binding washing or elution beads are collected again with the rod covered magnets and trans ferred to the next plate or tube MACHEREY NAGEL 07 2009 Rev 02 7 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 e Load 1000 ul for checking the settings for the binding step or 600 ul for check ing 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 e Increase speed setting shake for an additional 30 seconds and check the plate surface for droplets again e 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 water to the wells of the collection plate and proceed as de scribed above 2 5 Handling of beads Distribution of beads Ahomogeneous distribution of the magnetic beads to the in
7. Viral RNA DNA Isolation User Manual NucleoMag 96 Virus July 2009 Rev 02 MACHEREY NAGEL MN Viral RNA DNA Isolation Table of contents 1 Components 4 1 1 Kit contents 4 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 risk and safety phrases 11 5 Procedure 13 5 1 General procedure 13 5 2 Protocol for the isolation of viral RNA DNA from cell free body fluids 15 6 Appendix 17 6 1 Troubleshooting 17 6 2 Ordering information 18 6 3 Product use restriction warranty 19 MACHEREY NAGEL 07 2009 Rev 02 3 Viral RNA DNA Isolation 1 Components 1 1 Kit contents NucleoMag 96 Virus 1x 96 preps 4 x 96 preps Cat No 744800 1 744800 4 NucleoMag V Beads 3 ml 4x3ml Lysis Buffer MV1 20 ml 80 ml Binding Buffer MV2 60 ml 2x 120 ml Wash Buffer MV3 50 ml 2 x 100 ml Wash Buffer MV4 50 ml 2 x 100 ml Wash Buffer MV5 55 ml 2x 110ml Elution Buffer MV6 10 ml 40 ml Carrier RNA 400 ug 4 x 400 ug Carrier RNA Buffer 500 ul 4x 500 ul Proteinase K lyophilized 22 mg 4x 22 mg 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 07 2009 Rev 02
8. 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 07 2009 Rev 02 15 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 pipetting up and down Incubate for 1 min 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 pipet ting MV4 wash Remove the Square well Block from the NucleoMag SEP magnetic separator Add 500 ul Buffer MV4 and resuspend the beads by pipetting up and down Incubate for 1 min 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 pipet ting MV5 wash Leave the Square well Block on the NucleoMag SEP magnetic separator Gently add 550 pl 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 incu
9. bation time of max 1 min Elution Add desired volume of Buffer MV6 50 100 pl to each well of the Square well Block and resuspend the beads by pipetting up and down Incubate the suspension 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 infor mation 16 MACHEREY NAGEL 07 2009 Rev 02 Viral RNA DNA Isolation 6 Appendix 6 1 Troubleshooting Problem Possible cause and suggestions Elution buffer volume insufficient e Beads pellet must be covered completely with elution buffer Insufficient performance of elution buffer during elution step e Remove residual buffers during the separation steps complete ly Remaining buffers decrease the efficiency of following wash and elution steps Beads dried out Poor yield e Do not let the beads dry as this might result in lower elution low sensitivity efficiencies Aspiration of attracted bead pellet e Do not disturb the attracted beads while aspirating the super natant especially when the magnetic bead pellet is not visible in the lysate Aspiration and loss of beads e Time for magnetic separation too short or aspiration speed too high Low purity low sensitivity Insufficient washing
10. ct us if you wish an extra copy MACHEREY NAGEL does not warrant against damages or defects arising in shipping and handling transport insurance for customers excluded or out of accident or im proper or abnormal use of this product against defects in products or components not manufactured by MACHEREY NAGEL or against 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 spe cial 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 appea
11. dividual wells of the separa tion 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 pi petting up and down mixing by shaker or magnetic mixing allows simultaneous mixing of all samples This reduces the time and number of tips needed for the preparation 8 MACHEREY NAGEL 07 2009 Rev 02 Viral RNA DNA Isolation Resuspension by pipetting up and down however is more efficient than mixing by a shaker
12. erformed 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 10 pl Proteinase K and 200 pl of sample to a suitable reaction tube Add 200 pl Buffer MV1 with added Carrier RNA to the reaction tube lf Carrier RNA is not premixed with the Buffer MV1 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 MV1 and Carrier RNA can be prepared This premix should be added to the sample immediately with in 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 pl resuspended V Beads and 600 ul Buffer MV2 to the lysed sample Mix by pipetting up and down 6 times and incubate for 5 min at room tempera ture NucleoMag V Beads and Buffer MV2 can be pre mixed Be sure to resuspend the NucleoMag V Beads before removing them from the stor age bottle Vortex storage bottle briefly until a homogenous suspension has formed Separate the magnetic beads against the side of the wells by placing the Square well Block on the NucleoMag SEP
13. ng Do not breathe dust Avoid contact with the skin In case of contact with eyes rinse immediately with plenty of water and seek medical advice Wear suitable protective clothing and gloves 12 MACHEREY NAGEL 07 2009 Rev 02 NucleoMag 96 Virus 5 Procedure 5 1 General procedure 1 Lyse sample 200 pl sample 10 pl Proteinase K 4 ul Carrier RNA 200 pl MV1 Mix 56 C 10 min 2 Bind viral RNA DNA to 600 pl MV2 NucleoMag V Beads 30 pl V Beads Mix RT 5 min Optional Mix by shaking Separate 2 min and remove supernatant 3 MV3 wash 500 pl MV3 Resuspend separate 2 min Aspirate and discard supernatant 4 MV4 wash 500 pl MV4 Resuspend separate 2 min Aspirate and discard supernatant 5 MV5 wash 550 pl MV5 RT 45s Aspirate and discard supernatant MACHEREY NAGEL 07 2009 Rev 02 13 NucleoMag 96 Virus 6 Elution 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 14 MACHEREY NAGEL 07 2009 Rev 02 NucleoMag 96 Virus 5 2 Protocol for the isolation of viral RNA DNA from cell free body fluids 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 p
14. om 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 MV1 containing Carrier RNA more than 6 times Before starting any NucleoMag 96 Virus protocol prepare the following e 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 e 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 Cat No 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 pg 4 vials 400 pg vial Add 500 ul Add 500 ul Carrier RNA Carrier RNA Buffer Buffer to each vial 10 MACHEREY NAGEL 07 2009 Rev 02 Viral RNA DNA Isolation 4 Safety instructions risk and safety phrases The following components of the NucleoMag 96 Virus kits contain hazardous con tents Wear gloves and goggles and follow the safety instructions given in this section Component Hazard Haza
15. 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 MV6 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 pel let inside the separation plate For efficient elution the magnetic bead pellet should be resuspended completely in the elution buffer For some separators high elution vol umes might be necessary to cover the whole pellet 8 channel pipetting device MACHEREY NAGEL 07 2009 Rev 02 9 Viral RNA DNA Isolation 3 Storage conditions and preparation of working solutions Attention Buffers MV1 MV2 and MV3 contain chaotropic salt Wear gloves and goggles e All components of the NucleoMag 96 Virus kit should be stored at room tem perature 20 25 C and are stable for up to one year e All buffers are delivered ready to use e Lysis Buffer MV1 Lysis Buffer MV1 may form a salt precipitate 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 MV1 with Carrier RNA Lysis Buffer MV1 with added Carrier RNA can be stored at ro
16. rd Risk Safety contents symbol phrases phrases MV1 Guanidine x Xn Harmful by inhalation R 20 21 22 thiocyanate in contact with the skin and if swallowed MV2 Sodium per Flammable R10 chlorate lt 15 ethanol lt 50 Sodium per Flammable R 10 chlorate lt 15 ethanol lt 24 MV4 Ethanol lt 60 4 F Highly flammable R11 S 7 16 Carrier RNA Guanidine x Xn Harmful by inhalation R 20 21 22 S13 Buffer thiocyanate in contact with the skin and if swallowed Proteinase K Proteinase K x Xn Irritating to eyes R 36 37 38 S 22 24 lyophilized Xi respiratory system 42 26 36 37 and skin May cause senzitization by inhalation Risk phrases R 10 Flammable R11 Highly flammable R 20 21 22 Harmful by inhalation in contact with the skin and if swallowed R 36 37 38 Irritating to eyes respiratory system and skin R 42 May cause senzitization by inhalation 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 MACHEREY NAGEL 07 2009 Rev 02 11 Viral RNA DNA Isolation Safety phrases S7 S13 S16 S22 S24 S 26 S 36 37 Keep container tightly closed Keep away from food drink and animal feedstuffs Keep away from sources of ignition No smoki
17. ring 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 state ments 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 MACHEREY NAGEL 07 2009 Rev 02 19 Viral RNA DNA Isolation 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 Please contact MACHEREY NAGEL Germany Tel 49 0 24 21 969 270 e mail TECH BIO mn net com Last updated 12 2006 Rev 02 20 MACHEREY NAGEL 07 2009 Rev 02
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