MagAttract Viral RNA M48 Handbook
Contents
1. 2010 5 Safety Information When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information please consult the appropriate material safety data sheets MSDSs These are available online in convenient and compact PDF format at www giagen com ts msds asp where you can find view and print the MSDS for each GIAGEN kit and kit component CAUTION DO NOT add bleach or acidic solutions directly to the sample preparation waste Buffers MFL and MFW1 contain guanidine hydrochloride or guanidine thiocyanate which can form highly reactive compounds when combined with bleach If liquid containing these buffers is spilt clean with suitable laboratory detergent and water If the spilt liquid contains potentially infectious agents clean the affected area first with laboratory detergent and water and then with 1 v v sodium hypochlorite If liquid containing potentially infectious agents is spilt on the BioRobot M48 clean the affected area first with laboratory detergent and water and then with 1 v v sodium hypochlorite followed by water The following risk and safety phrases apply to the components of the MagAttract Viral RNA M48 Kit Buffer MFL Contains guanidine thiocyanate harmful Risk and safety phrases R20 21 22 32 13 26 36 46 Buffer MFW1 Contains guanidine hydrochloride harmful irritant Risk and safety phrases R22 36 38 13 26 36 46 24 hour em2. DEPC is a strong but not absolute inhibitor of RNases It is commonly used at a concentration of 0 1 to inactivate RNases on glass or plasticware or to create RNase free solutions and water DEPC inactivates RNases by covalent modification Add 0 1 ml DEPC to 100 ml of the solution to be treated and shake vigorously to bring the DEPC into solution Let the solution incubate for 12 hours at 37 C Autoclave for 15 minutes to remove any trace of DEPC DEPC will react with primary amines and cannot be used directly to treat Tris buffers DEPC is highly unstable in the presence of Tris buffers and decomposes rapidly into ethanol and CO When preparing Tris buffers treat water with DEPC first and then dissolve Tris to make the appropriate buffer Trace amounts of DEPC will modify purine residues in RNA by carbethoxylation Carbethoxylated RNA is translat ed with very low efficiency in cell free systems However its ability to form DNA RNA or RNA RNA hybrids is not seriously affected unless a large fraction of the purine residues have been modified Residual DEPC must always be eliminated from solutions or vessels by autoclaving or heating to 100 C for 15 minutes Note MagAttract Viral RNA M48 Kit buffers are guaranteed RNase free without using DEPC treatment and are therefore free of any DEPC contamination When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information consult the ap
3. High quality viral RNA is eluted at 65 C in an RNase free elution buffer The MagAttract Viral RNA M48 Procedure Serum or blood plasma iieereree Lyse with Buffer MFL l MagAttract Suspension F added to samples RNA binds to magnetic particles i i Wash with Buffer MFW1 then with Buffer MFW2 Magnetic separation FGF ee vh eter mr Pure high quality viral RNA MagAttract Viral RNA M48 Handbook 04 2010 7 Equipment and Reagents to Be Supplied by User When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information consult the appropriate material safety data sheets MSDSs available from the product supplier E BioRobot M48 workstation cat no 9000708 App Package M48 Inf Dis cat no 9016145 Filter Tips 1000 pl M48 1000 cat no 995652 Reagent Containers small M48 100 cat no 995902 Reagent Containers large M48 50 cat no 995904 Reagent Container Seals M48 50 cat no 995906 Sample Prep Plates 42 well M48 100 cat no 995908 Cooling Block 48 tube 0 2 ml M48 cat no 9015178 Sample tubes 1 5 ml without lids Sarstedt cat no 72 696 or with screw caps Sarstedt cat no 72 692 Tubes for cooling block 1 5 ml without lids Sarstedt cat no 72 696 Elution tubes with screw caps 1 5 ml Sarstedt cat no 72 692 or 2 ml Sarstedt cat no 72 693 BW PCR tubes 0 2 ml thin walled MH Sterile
4. RNase free pipet tips E Optional 14 3 M f mercaptoethanol B ME commercially available solutions are usually 14 3 M Required if eluting viral RNA into 0 2 ml tubes t This is not a complete list of suppliers and does not include many important vendors of biological supplies however use of other tubes may result in an instrument crash The addition of B ME to Buffer MFL will slightly improve the yield and quality of viral RNA Dispense in a fume hood and wear appropriate protective clothing Add 10 pl of p ME per 1 ml of Buffer MFL just before use Discard unused solution at the end of the day 8 MagAttract Viral RNA M48 Handbook 04 2010 Important Notes Preparing serum and plasma samples The purification procedure is optimized for use with 300 yl serum or plasma samples Blood samples treated with EDTA or citrate as anticoagulant can be used for plasma preparation Samples can be either fresh or frozen provided that they have not been frozen and thawed more than once After collection and centrifugation plasma or serum can be stored at 2 8 C for up to 6 hours For longer storage we recommend freezing aliquots at 20 C or 80 C Frozen plasma or serum must not be thawed more than once Repeated freeze thaw ing leads to denaturation and precipitation of proteins resulting in reduced viral titers and therefore reduced yields of viral RNA If cryoprecipitates are visible in the samples centrifuge at 6800 x g for 3 minutes tra
5. or distributor We will credit your account or exchange the product as you wish Separate conditions apply to QIAGEN scientific instruments service products and to products shipped on dry ice Please inquire for more information A copy of QIAGEN terms and conditions can be obtained on request and is also provided on the back of our invoices If you have questions about product specifications or performance please call QIAGEN Technical Services or your local distributor see back cover Technical Assistance At QIAGEN we pride ourselves on the quality and availability of our technical support Our Technical Service Departments are staffed by experienced scientists with extensive practical and theoretical expertise in molecular biology and the use of QIAGEN products If you have any questions or experience any difficulties regarding the MagAttract Viral RNA M48 Kit or QIAGEN products in general please do not hesitate to contact us QIAGEN customers are a major source of information regarding advanced or specialized uses of our products This information is helpful to other scientists as well as to the researchers at QIAGEN We therefore encourage you to contact us if you have any suggestions about product performance or new applications and techniques For technical assistance and more information please call one of the QIAGEN Technical Service Departments or local distributors see back cover MagAttract Viral RNA M48 Handbook 04
6. pl elution buffer Buffer MFE Elution in smaller volumes increases the final RNA concentration in the eluate but slightly reduces overall RNA yield We recommend using an elution volume appropriate for the intended downstream application This protocol purifies both RNA and DNA Use RNase free DNase to remove DNA if performing sensitive downstream applications E At the end of a protocol run residual reagents should either be removed immediately from the workstation and transferred to an airtight container for later use or discarded Things to do before starting E Optional The addition of B mercaptoethanol B ME to Buffer MFL will slightly improve the yield and quality of viral RNA Add 10 pl B ME per 1 ml Buffer MFL just before use Dispense in a fume hood and wear appropriate protective clothing Buffer MFL is stable at 4 C for 3 months after opening After addition of B ME to an aliquot of Buffer MFL the solution must be used up on the same day Procedure 1 Distribute 300 pl of serum or plasma into 1 5 ml sample tubes Proceed to step 2 immediately or freeze the samples and store at 70 C for long term storage If using frozen samples thaw at room temperature 15 20 C and mix well by inverting sample tubes before proceeding to step 2 See Preparing serum and plasma samples on page 9 Note Avoid repeated freezing and thawing of the samples and do not store the samples for more than 6 h at 2 8 C otherwise aggregation of m
7. 293 422 922 Technical 01293 422 999 USA Orders 800 426 8157 Fax 800 718 2056 Technical 800 DNA PREP 800 362 7737 WWW QIAGEN COM 1063013 04 2010
8. Orders 800 572 9613 Fax 800 713 5951 Technical 800 DNA PREP 800 362 7737 China Orders 86 21 3865 3865 Fax 86 21 3865 3965 Technical 800 988 0325 Denmark Orders 80 885945 Fax 80 885944 Technical 80 885942 Finland Orders 0800 914416 Fax 0800 914415 Technical 0800 914413 France Orders 01 60 920 926 Fax 01 60 920 925 Technical 01 60 920 930 Offers 01 60 920 928 Germany Orders 02103 29 12000 Fax 02103 29 22000 Technical 02103 29 12400 Hong Kong Orders 800 933 965 Fax 800 930 439 Technical 800 930 425 Ireland Orders 1800 555 049 Fax 1800 555 048 Technical 1800 555 061 Italy Orders 800 789 544 Fax 02 334304 826 Technical 800 787980 Japan Telephone 03 6890 7300 Fax 03 5547 0818 Technical 03 6890 7300 Korea South Orders 080 000 7146 Fax 02 2626 5703 Technical 080 000 7145 Luxembourg Orders 8002 2076 Fax 8002 2073 Technical 8002 2067 Mexico Orders 01 800 7742 639 Fax 01 800 1 122 330 Technical 01 800 7742 639 The Netherlands Orders 0800 0229592 Fax 0800 0229593 Technical 0800 0229602 Norway Orders 800 18859 Fax 800 18817 Technical 800 18712 Singapore Orders 1800 742 4362 Fax 65 6854 8184 Technical 1800 742 4368 Spain Orders 91 630 7050 Fax 91 630 5145 Technical 91 630 7050 Sweden Orders 020 790282 Fax 020 790582 Technical 020 798328 Switzerland Orders 055 254 22 11 Fax 055 254 22 13 Technical 055 254 22 12 UK Orders 01293 422 911 Fax 01
9. Second Edition April 2010 MagAttract Viral RNA M48 Handbook For purification of viral RNA from serum and plasma using the BioRobot M48 workstation 8098 88690 QIAGEN Trademarks GIAGEN BioRobot MagAttract QIAGEN Group Microsoft Windows Microsoft Corporation The PCR process is covered by the foreign counterparts of U S Patents Nos 4 683 202 and 4 683 195 owned by F Hoffmann La Roche Ltd 2003 2010 QIAGEN all rights reserved QIAGEN is a member of the Forest Stewardship Council FSC For the production of printed materials including handbooks QIAGEN has a policy to select suppliers that comply with FSC standards for printing processes and well managed forests FSC C009997 Contents Kit Contents Storage Quality Control Product Use Limitations Product Warranty and Satisfaction Guarantee Technical Assistance Safety Information Introduction Principle and procedure Equipment and Reagents to Be Supplied by User Important Notes Preparing serum and plasma samples Yields of viral RNA Storing viral RNA Protocol Bi Purification of Viral RNA from Serum or Plasma Troubleshooting Guide Appendix General Remarks on Handling RNA Ordering Information GIAGEN Distributors MagAttract Viral RNA M48 Handbook 04 2010 0 ODO DANN AO aa 3 BP RB Kit Contents MagAttract Viral RNA M48 Kit 96 Catalog no 955235 Number of preps 96 MagAttract Suspension F 10 ml Buffer MFL 77 ml Bu
10. agnetic particles may occur during the purification procedure leading to reduced viral RNA yields 2 Switch on the BioRobot M48 The power switch is on the left side of the instrument 3 Switch on the computer and monitor 10 MagAttract Viral RNA M48 Handbook 04 2010 4 Launch the QIAsoft M Operating System Upon startup the computer displays the QIAsoft M startup window Click Start to continue If the QIAsoft M startup window does not appear either double click the QIAsoft M icon on the desktop or click the Microsoft Windows Start menu and select QIAsoft M Operating System QIAsoft M V2 0 for BioRobot M48 5 Select the protocol group Infectious Disease from the drop down menu by click ing the dark green arrow then select Viral NA 6 Select the protocol Viral RNA and click the Select button to choose the type of elution tube Input the number of samples and sample and elution volumes into the software The GlAsoft M software will now guide you through the remaining steps required to set up the BioRobot M48 for the protocol selected these steps include the option of entering names for your samples Be sure to follow all instructions that appear Wear gloves when loading the required items on the worktable JO20JO1g Note Ensure that the appropriate cooling block is installed at the Heat Cool Block 2 slot of the worktable For details refer to the BioRobot M48 User Manual 7 Close the works
11. al RNA M48 Handbook 04 2010 13 Appendix General Remarks on Handling RNA Handling RNA Ribonucleases RNases are very stable and active enzymes that generally do not require cofactors to function Since RNases are difficult to inactivate and even minute amounts are sufficient to destroy RNA do not use any plasticware or glassware without first eliminating possible RNase contamination Great care should be taken to avoid inadvertently introducing RNases into the RNA sample during or after the isolation procedure In order to create and maintain an RNase free environment the following precautions must be taken during pretreatment and use of disposable and non disposable vessels and solutions while working with RNA General handling Proper microbiological aseptic technique should always be used when working with RNA Hands and dust particles may carry bacteria and molds and are the most common sources of RNase contamination Always wear latex or vinyl gloves while handling reagents and RNA samples to prevent RNase contamination from the surface of the skin or from dusty laboratory equipment Change gloves frequently and keep tubes closed whenever possible Keep isolated RNA on ice when aliquots are pipetted for downstream applications Disposable plasticware The use of sterile disposable polypropylene tubes is recommended throughout the procedure These tubes are generally RNase free and do not require pretreatment to inactivate RNases N
12. dialog box error dialog box during a protocol run refer to the Troubleshooting Guide in the BioRobot M48 User Manual Low viral RNA yield a MagAttract Suspension F Before starting the procedure ensure that MagAttract was not completely Suspension F is fully resuspended Vortex for at least resuspended 3 min before first use and for 1 min before subsequent uses b Reagents were loaded Ensure that all reagents are loaded onto the worktable onto worktable in in the correct order Repeat the purification procedure wrong order with new samples c RNA degraded RNA may have been degraded by RNases in the orig inal plasma or serum samples Ensure that the samples are processed immediately after collection or removal from storages Although all buffers have been tested and are guaran teed RNase free RNases can be introduced during use Take care not to introduce any RNases during the purifi cation procedure or later handling see appendix page 14 Use Buffer MFE for RNA elution d RNase contamination IF opening and closing a Buffer MFE vial many times in Buffer MFE take care not to introduce RNases In case of RNase contamination replace the contaminated vial with a new Buffer MFE vial Repeat the purification procedure with new samples 12 MagAttract Viral RNA M48 Handbook 04 2010 Comments and suggestions Viral RNA does not perform well in downstream enzymatic reactions a Little or no RNA in the eluate b Froze
13. ergency information Emergency medical information in English French and German can be obtained 24 hours a day from Poison Information Center Mainz Germany Tel 49 6131 19240 R20 21 22 Harmful by inhalation in contact with skin and if swallowed R22 Harmful if swallowed R32 Contact with acids liberates very toxic gas R36 38 Irritating to eyes and skin 13 Keep away from food drink and animal feedingstuffs 526 In case of contact with eyes rinse immediately with plenty of water and seek medical advice 36 Wear suitable protective clothing 46 If swallowed seek medical advice 6 MagAttract Viral RNA M48 Handbook 04 2010 Introduction The MagAttract Viral RNA M48 Kit provides fully automated purification of viral RNA from serum and plasma MagAttract technology provides high quality RNA which is suitable for direct use in downstream applications such as amplification or other enzymatic reactions The BioRobot M48 performs all steps of the sample preparation procedure Up to 48 samples in multiples of 6 are processed in a single run Principle and procedure MagAttract technology combines the speed and efficiency of silica based RNA purification with the convenient handling of magnetic particles Viral RNA binds to the silica surface of the magnetic particles in the presence of a chaotropic salt see flowchart below Viral RNA bound to the magnetic particles is then efficiently washed using two different wash buffers
14. ffer MFW1 2 x 77 ml Buffer MFW2 2 x 100 ml Buffer MFE 10 ml Handbook il Storage All buffers and reagents should be stored at 2 8 C Do not freeze MagAttract Suspension F Quality Control In accordance with QIAGEN s ISO certified Quality Management System each lot of MagAttract Viral RNA M48 Kits is tested against predetermined specifications to ensure consistent product quality Product Use Limitations The MagAttract Viral RNA M48 Kit is intended for molecular biology applications This product is not intended for the diagnosis prevention or treatment of a disease All due care and attention should be exercised in the handling of the products We recommend all users of QIAGEN products to adhere to the NIH guidelines that have been developed for recombinant DNA experiments or to other applicable guidelines 4 MagAttract Viral RNA M48 Handbook 04 2010 Product Warranty and Satisfaction Guarantee GIAGEN guarantees the performance of all products in the manner described in our product literature The purchaser must determine the suitability of the product for its particular use Should any product fail to perform satisfactorily due to any reason other than misuse QIAGEN will replace it free of charge or refund the purchase price We reserve the right to change alter or modify any product to enhance its performance and design If a QIAGEN product does not meet your expectations simply call your local Technical Service Department
15. k of 50 16 MagAttract Viral RNA M48 Handbook 04 2010 Ordering Information Product Contents Cat no Sample Prep Plates 42 well Disposable polypropylene plates 995908 M48 100 for sample preparation including nucleic acid binding and washing steps pack of 100 Cooling Block 48 tube Holder for accommodating 9015178 0 2 ml M48 A8 x 0 2 ml PCR tubes on the cooling and heating system of the BioRobot M48 worktable Accessories 12 Tube Magnet Magnet for separating magnetic 36912 particles in 12 x 1 5 ml or 2 ml tubes Related product MagAttract Virus Mini MagAttract Suspension B and 955336 M48 Kit 192 RNase Free Reagents and Buffers for up to 192 virus nucleic acid preps For up to date licensing information and productspecific disclaimers see the respective QIAGEN kit handbook or user manual QIAGEN kit handbooks and user manuals are available at www giagen com or can be requested from QIAGEN Technical Services or your local distributor MagAttract Viral RNA M48 Handbook 04 2010 17 Notes 18 MagAttract Viral RNA M48 Handbook 04 2010 Notes MagAttract Viral RNA M48 Handbook 04 2010 19 www qiagen com Australia Orders 1 800 243 800 Fax 03 9840 9888 Technical 1 800 243 066 Austria Orders 0800 28 10 10 Fax 0800 28 10 19 Technical 0800 28 10 11 Belgium Orders 0800 79612 Fax 0800 79611 Technical 0800 79556 Brazil Orders 0800 557779 Fax 55 11 5079 4001 Technical 0800 557779 Canada
16. n serum or plasma samples not mixed properly after thawing c RNA in samples already degraded prior to purification d Too much eluate in the amplification reaction e Carryover of magnetic particles See Low viral RNA yield page 12 for possible reasons Increase the amount of eluate added to the reaction if possible Thaw frozen samples at room temperature 15 25 C and mix by pulse vortexing for 15 s Samples were frozen and thawed more than once or stored at room temperature for too long Always use fresh samples or samples thawed only once see Preparing serum and plasma samples page 9 Repeat the purification procedure with new samples Determine the maximum volume of eluate suitable for your amplification reaction Reduce or increase the volume of eluate added to the amplification reaction accordingly Carryover of magnetic particles in the eluates will not affect most downstream applications including RT PCR If the risk of magnetic particle carryover needs to be minimized e g for applications such as realtime RT PCR first place the tubes containing eluate in a suitable magnet e g 12 Tube Magnet cat no 36912 for 1 min and then transfer the eluates to clean tubes If a suitable magnet is not available centrifuge the tubes containing eluates in a microcentrifuge at full speed for 1 min to pellet any remaining magnetic particles and transfer the supernatants to clean tubes MagAttract Vir
17. nsfer the supernatants to fresh tubes without disturbing the pellets and start the purification procedure immediately Removing cry oprecipitates does not reduce viral titers Yields of viral RNA The yield of purified viral RNA depends on the sample type and virus titer Using this kit the yield of viral RNA per sample is normally below 1 pg and is therefore difficult to determine with a spectrophotometer Quantitative amplification methods are recom mended for determination of yields The size distribution of viral RNA purified with this procedure can be checked by agarose gel electrophoresis and hybridization to a virus specific labeled probe fol lowed by autoradiography Sambrook J Russell D W eds 2001 Molecular Cloning A Laboratory Manual 3rd ed Cold Spring Harbor Laboratory Press Storing viral RNA For short term storage of up to 24 hours we recommend storing the purified viral RNA at 2 8 C For long term storage of over 24 hours we recommend storage at 20 C to 70 C MagAttract Viral RNA M48 Handbook 04 2010 9 Y o 2 b a Protocol Purification of Viral RNA from Serum or Plasma Important points before starting E df working with RNA for the first time read the appendix page 14 E Ensure that you are familiar with operating the BioRobot M48 workstation Refer to the BioRobot M48 User Manual for operating instructions E The purified viral RNA can be eluted in 50 pl 65 yl 80 pl or 100
18. on disposable plasticware Non disposable plasticware should be treated before use to ensure that it is RNase free Plasticware should be thoroughly rinsed with 0 1 M NaOH 1 mM EDTA followed by RNase free water see Solutions page 15 Alternatively chloroform resistant plasticware can be rinsed with chloroform to inactivate RNases Glassware Glassware should be treated before use to ensure that it is RNase free Glassware used for RNA work should be cleaned with a detergent thoroughly rinsed and oven baked at 240 C for four or more hours overnight if more convenient before use Autoclaving When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information consult the appropriate material safety data sheets MSDSs available from the product supplier 14 MagAttract Viral RNA M48 Handbook 04 2010 alone will not fully inactivate many RNases Alternatively glassware can be treated with DEPC diethyl pyrocarbonate Fill glassware with 0 1 DEPC 0 1 in water allow to stand overnight 12 hours at 37 C and then autoclave or heat to 100 C for 15 minutes to eliminate residual DEPC Electrophoresis tanks Electrophoresis tanks should be cleaned with detergent solution e g 0 5 SDS thoroughly rinsed with RNase free water and then rinsed with ethanol and allowed to dry Solutions Solutions water and other solutions should be treated with 0 1 DEPC
19. propriate material safety data sheets MSDSs available from the product supplier t Plastics used for some electrophoresis tanks are not resistant to ethanol Take proper care and check the supplier s instructions MagAttract Viral RNA M48 Handbook 04 2010 15 Ordering Information Product Contents Cat no MagAttract Viral RNA MagAttract Suspension F and 255235 M48 Kit 96 buffers for up to 96 preps BioRobot M48 Robotic workstation for automation 9000708 of magnetic particle purification technology App Package Software protocol package for 9016145 M48 Inf Dis CD infectious disease applications on the BioRobot M48 workstation Starter Pack M48 Pack includes sterile filter tips 600 995999 sample prep plates 40 large reagent containers 8 small reagent containers 8 silicon seals 8 sample tubes 1 5 ml 250 sample tubes 2 ml 250 elution tubes screw cap 1 5 ml 250 tip waste bags 2 Filter Tips 1000 pl M48 1000 Sterile disposable filter tips 995652 bagged pack of 1000 Reagent Containers small Reagent containers 20 ml with lids 995902 M48 100 To be used with the Reagent Container Rack M48 pack of 100 Reagent Containers large Reagent containers 110 ml with 995904 M48 50 lids To be used with the Reagent Container Rack M48 pack of 50 Reagent Container Seals Lid sealing sheets for small and 995906 M48 50 large Reagent Containers allowing storage of unused reagents pac
20. tation door and start the protocol when instructed by the software All subsequent steps are automated The software displays a table of results when the protocol is finished 8 Retrieve the elution tubes containing the purified viral RNA from the cooling block The viral RNA is ready to use or can be stored at 2 8 C for a few hours or at 20 C to 70 C for long term storge Carryover of magnetic particles in eluates will not affect most downstream applications including RT PCR If the risk of magnetic particle carryover needs to be minimized e g for applications such as realtime PCR the elution tubes containing eluate should first be applied to a suitable magnetic separator e g QIAGEN 12 Tube Magnet cat no 36912 for 1 min and the eluates transferred to clean tubes If a suitable magnetic separator is not available centrifuge the tube containing the RNA for 1 min at full speed in a microcentrifuge to pellet any remaining magnetic particles MagAttract Viral RNA M48 Handbook 04 2010 11 Troubleshooting Guide This troubleshooting guide may be helpful in solving any problems that may arise The scientists in QIAGEN Technical Services are always happy to answer any questions you may have about either the information and protocol in this handbook or molecular biology applications see back cover for contact information Comments and suggestions Software error QIAsoft M software If the QIAsoft M software displays an error
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