QIAamp® UltraSens® Virus Handbook
Contents
1. Step 5 Pelleting the nucleic acid detergent complexes Too high a gforce can lead to a pellet that is highly compressed and difficult to resuspend In rare cases it may be necessary to optimize the gforce used in some microcentrifuge models If the pellet is too solid after centrifugation at 1200 x g decrease the g force in 100 x g steps with each preparation to find the gforce at which a pellet that is easy to resuspend is formed but at which the supernatant is free of debris If debris is present after centrifugation increase the g force in 100 x g steps with each preparation until it is no longer present in the supernatant Once the correct g force has been found for a particular centrifuge it can be used for all plasma and serum samples in that centrifuge no changes need to be made Step 12 Binding nucleic acids to the GlAamp membrane Centrifugation at 3000 5000 x g is necessary at this step to ensure that the complexed nucleic acids have sufficient opportunity to bind to the QlAamp membrane Higher gforces may lead to loss of nucleic acids in the spin column flow through 12 QlAamp UltraSens Virus Handbook 04 2010 Sample storage After collection and centrifugation plasma or serum samples can be stored at 2 8 C for up to 6 hours For longer storage we recommend storing aliquots at 20 C or 80 C Frozen plasma or serum samples should not be thawed more than once Repeated freezing and thawing leads to denaturation and2. A copy of GIAGEN 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 GIAGEN Technical Services or your local distributor see back cover or visit www giagen com QlAamp UltraSens Virus Handbook 04 2010 5 Quality Control In accordance with QIAGEN s ISO certified Quality Management System each lot of QlAamp UltraSens Virus Kit is tested against predetermined specifications to ensure consistent product quality 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 sample and assay technologies and the use of QIAGEN products If you have any questions or experience any difficulties regarding the QlAamp UltraSens Virus Kit or QIAGEN products in general please do not hesitate to contact us QIAGEN customers are a major source of information regarding advanced or special ized 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 see our Technical Support Center at www qiagen com Support or call o
3. 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 e e UK Orders 01293 422 911 Fax 01293 422 922 Technical 01293 422 999 o e o e e USA Orders 800 426 8157 Fax 800 718 2056 Technical 800 DNA PREP 800 362 7737 QIAGEN pou Sample amp Assay Technologies
4. 38 S13 26 36 46 Proteinase K Contains Proteinase K sensitizer irritant Risk and safety phrases R36 37 38 42 43 S23 24 26 36 37 24 hour emergency 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 R10 Flammable R22 Harmful if swallowed R36 38 Irritating to eyes and skin R36 37 38 Irritating to eyes respiratory system and skin R38 Irritating to skin R41 Risk of serious damage to eyes RA2 43 May cause sensitization by inhalation and skin contact S13 Keep away from food drink and animal feedingstuffs S23 Do not breathe vapor 24 Avoid contact with skin S26 In case of contact with eyes rinse immediately with plenty of water and seek medical advice 36 Wear suitable protective clothing S36 37 Wear suitable protective clothing and gloves QlAamp UltraSens Virus Handbook 04 2010 7 Introduction The GlAamp UltraSens Virus Kit is designed for rapid highly sensitive and efficient recovery of viral RNA and DNA from plasma or serum GlAamp UltraSens technology is used to greatly concentrate viral nucleic acids in samples allowing downstream detection of very low viral titers Contaminants and enzyme inhibitors are efficiently removed by the GlAamp purification procedure which is based on advanced silica gel membrane technology and uses no phenol chloroform or other organic solvents
5. 5 more times to completely resuspend each sample Successively vortexing and then incubating each sample in this way aids the proteinase K digestion 0204014 9 Incubate for 10 min at 40 C in mixer incubator with the mixing speed set to maximum Note Ten minutes of digestion with proteinase K at 40 C is sufficient Do not exceed this incubation time If a mixer incubator such as an Eppendorf Thermomixer Compact or similar is not available incubate samples on a heating block or in a water bath for 10 min at 40 vortexing each sample for 5 s after 5 min and again for 5 s after the 10 min incubation Do not allow the temperatures of the samples to decrease during vortexing 10 Briefly centrifuge to remove drops from the inside of the tube lid 11 Add 300 pl Buffer AB mix thoroughly by vortexing and centrifuge briefly to remove drops from the inside of the lid 12 Carefully apply the 700 pl lysate to a QlAamp spin column sitting in a 2 ml collection tube without wetting the rim Close the cap and centrifuge at 3000 5000 x g for 1 min Note Excessive gforces will lead to inefficient binding of nucleic acids to the silica gel membrane resulting in reduced yields 13 Place the GlAamp spin column into new 2 ml collection tube provided and discard the tube containing the filtrate Carefully open the QlAamp spin column and add 500 pl Buffer AW1 Centrifuge at 6000 x g for 1 min Use of a higher gforce during
6. RNA Mini Kit for isolation of viral RNA from cell free body fluids QlAamp Viral RNA Mini Kit 50 For 50 RNA preps 50 GlAamp 52904 Mini Spin Columns Carrier RNA Collection Tubes 2 ml RNase free Buffers QlAcube for fully automated sample prep using spin column kits GlAcube Robotic workstation for automated Inquire purification of DNA RNA or D using QIAGEN spin column its 1 year warranty on parts and labor Fully automatable on the GlAcube t Larger kit sizes available please inquire QlAamp UltraSens Virus Handbook 04 2010 23 Ordering Information Product Contents Cat no QuantiTect Virus Kits for highly sensitive detection of viral RNA and or DNA QuantiTect Virus Kit 200 QuantiTect Virus ROX Vial Kit 200 QIAGEN OneStep RT PCR Kit QIAGEN OneStep RT PCR Kit 25 Accessories Buffer AW 1 concentrate Buffer AW2 concentrate Collection Tubes 2 ml GIAGEN Proteinase K 2 GIAGEN Proteinase K 10 For up to date licensing information and product specific disclaimers see the respective GIAGEN kit handbook or user manual GIAGEN kit handbooks and user manuals are available at www giagen com or can be requested from QIAGEN Technical Services or your local distributor Larger kit sizes available please inquire For 200 x 50 pl reactions 211013 QuantiTect Virus Master Mix contains ROX dye QuantiTect Virus RT Mix RNase Free Water QuantiTect Nucleic Acid
7. The ready touse QIAGEN Proteinase solution is stable for up to one year after delivery when stored at room temperature To prolong the lifetime of QIAGEN Proteinase storage at 2 8 C is recommended Product Use Limitations The QlAamp UltraSens Virus 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 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 GIAGEN 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 or distributor We will credit your account or exchange the product as you wish Separate conditions apply to GIAGEN scientific instruments service products and to products shipped on dry ice Please inquire for more information
8. a complete list of references visit the QIAGEN Reference Database online at www giagen com RefDB search asp or contact QIAGEN Technical Services or your local distributor 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 t Plastics used for some electrophoresis tanks are not resistant to ethanol Take proper care and check supplier s instructions 22 QlAamp UltraSens Virus Handbook 04 2010 Ordering Information Product Contents Cat no QlAamp UltraSens Virus Kit 50 For 50 nucleic acid preps 50 53704 GlAamp Spin Columns Carrier RNA Proteinase K Collection Tubes 2 ml RNase free Buffers QlAamp UltraSens Virus Kit 250 250 nucleic acid preps 250 53706 GlAamp Spin Columns Carrier RNA Proteinase K Collection Tubes 2 ml RNase free Buffers Related products QlAamp MinElute Virus Kits for simultaneous purification of viral DNA and RNA from plasma serum and cell free body fluids QlAamp MinElute For 50 minipreps 50 QlAamp 57704 Virus Spin Kit 50 MinElute Columns QIAGEN Protease Carrier RNA Buffers Collection Tubes 2 ml GlAamp MinElute For 50 minipreps 50 QlAamp 57714 Virus Vacuum Kit 50 MinElute Columns QIAGEN Protease Carrier RNA Buffers Extension Tubes 3 ml Collection Tubes 1 5 ml QlAamp Viral
9. by estoppel This product is for research use only Diagnostic uses require a separate license from Roche Further information on purchasing licenses may be obtained by contacting the Director of Licensing Applied Biosystems 850 Lincoln Centre Drive Foster City California 94404 USA NOTICE TO PURCHASER LIMITED LICENSE The purchase price of this product QuantiTect Virus Kits includes a limited non transferable license under U S Patents Nos 5 407 800 5 322 770 5 310 652 and corresponding patent claims outside the United States owned by Roche Molecular Systems Inc or F Hoffmann La Roche Ltd Roche to use only this amount of product solely for the purchaser s own internal research No right under any other patent claims such as apparatus or system claims and no right to use this product for any other purpose or for commercial services of any kind including without limitation reporting the results of purchaser s activities for a fee or other commercial consideration is hereby granted expressly by implication or by estoppel This product is for research use only Diagnostic uses require a separate license from Roche Further information on purchasing licenses may be obtained by contacting the Director of Licensing Applied Biosystems 850 Lincoln Centre Drive Foster City California 94404 USA 2003 2010 QIAGEN all rights reserved www qiagen com Australia Orders 1 800 243 800 Fax 03 9840 9888 Technical 1 800 243 066 Aust
10. centrifugation at this step will not affect the purification process 14 Place the QlAamp spin column into a new 2 ml collection tube provided and discard the tube containing the filtrate Carefully open the QlAamp spin column and add 500 pl Buffer AW2 Centrifuge at full speed 20 000 x g for 3 min Some centrifuge rotors may vibrate upon deceleration resulting in filtrate contacting QlAamp spin column Removing QlAamp spin column and collection tube from the rotor may also cause the filtrate to come into contact with the bottom of the GlAamp spin column In these cases the additional optional step below should be performed E Place the GlAamp spin column in a clean 2 ml collection tube not provided and discard the old collection tube with the filtrate Centrifuge at full speed for 1 min QlAamp UltraSens Virus Handbook 04 2010 17 o 2 a 15 Place the QlAamp spin column into new 1 5 ml microcentrifuge tube not provided Discard the collection tube containing the filtrate Carefully open the GlAamp spin column To elute viral nucleic acids carefully apply 30 pl Buffer AVE to the membrane of the spin column Centrifuge at 6000 x g for 1 min It is important to moisten all of the QlAamp membrane with Buffer AVE to ensure the highest elution efficiency Repeat the elution by adding a further 30 pl Buffer AVE and centrifuging at 6000 x gfor 1 min A two step elution ensures maximum recovery of vir
11. depends on the type of sample sample types with lower protein contents than plasma or serum may need a greater gforce than the recommended 1200 x g to fully pellet the detergent nucleic acid complexes QlAamp UltraSens technology may not work well with plasma samples that contain unusually high amounts of lipids and recovery of viral nucleic acids from such samples may be reduced Reduced pellet size can be an indication that the reagent in Buffer AC is interacting with lipids in the sample See the Troubleshooting Guide on page 19 for strategies to overcome this problem QlAamp UltraSens Virus Handbook 04 2010 9 The GlAamp UltraSens Virus Procedure 1 ml plasma or serum Lyse and precipitate in Buffer AC and proteinase K and digest proteins Add Buffer AB i Resuspend in Buffer AR lt SS Bind ZB Wash 2x Elute pure viral RNA and DNA 10 QlAamp UltraSens Virus Handbook 04 2010 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 Ethanol 26 100 Sterile RNase free pipet tips with aerosol barrier RNase free microcentrifuge tubes 1 5 ml and 2 ml Phosphate buffered saline PBS may be required for samples smaller than 1 ml Variable speed microcentrifuge adjus
12. precipitation of proteins causing reduced viral titers and subsequently reduced yields of viral nucleic acids In addition cryoprecipitates formed by freeze thawing accumulate after every cycle and may interfere with sample preparation reducing sensitivity However in general cryoprecipitates do not need to be removed since they are lysed in Buffer AC at the start of the protocol Preparation of reagents Carrier RNA Add 310 pl Buffer AVE to each tube of lyophilized carrier RNA to obtain a 1 pg pl solution Dissolve the carrier RNA thoroughly divide it into conveniently sized aliquots and store at 20 C Do not freeze thaw the aliquots of carrier RNA more than three times Note The sample preparation procedure is optimized for 5 6 pg of carrier RNA per sample If less carrier RNA has been shown to be better for your particular amplification system add less carrier RNA to the sample than described in the protocol Use of less than 5 6 pg carrier RNA per sample must be validated for each particular sample type Discard the unused portion of the reconstituted carrier RNA Buffer AB Before use add the appropriate amount of ethanol 96 100 to the Buffer AB concentrate as indicated on the bottle Buffer AB concentrate is viscous Mix thoroughly by inverting the bottle ten times to obtain a homogeneous solution Once the concentrate is dissolved it is stable at room temperature 15 25 C Buffer AB dissolved in ethanol is stable for 7
13. the pellet should retain its shape when the microcentrifuge tube is inverted The pellet may be discolored but this will not affect the yield or quality of the nucleic acid Completely remove and discard the supernatant In order to loosen the pellet it is helpful to do the following after removing the supernatant close the tube lid and flick the bottom of the tube several times with your finger while holding the tube by its lid Vortexing the sample may also help to loosen stubborn pellets Ensure that no pellet debris remains inside the tube lid Add 300 pl Buffer AR warmed to 60 C and 20 pl proteinase K Preheating Buffer AR to 60 C helps to dissolve the pellet and increases the activity of proteinase K In order to reduce the number of pipetting steps a master mix of freshly made warmed Buffer AR and proteinase K can be added to the samples For example for 10 samples mix 3 3 ml of warmed Buffer AR with 220 pl proteinase K both amounts include 10 excess volume to compensate for pipetting errors Mix thoroughly by vortexing for 10 s and add 320 pl master mix to each sample 16 QlAamp UltraSens Virus Handbook 04 2010 8 Vortex thoroughly until the pellet is completely resuspended Note It is vital that pellets are completely resuspended to ensure maximum nucleic acid recovery An efficient way to resuspend multiple samples is to vortex 2 samples simultaneously for 5 10 s and then vortex the next 2 samples repeating 3
14. April 2010 GlAamp UltraSens Virus Handbook For highly efficient purification of viral RNA and DNA from 1 ml plasma and serum samples QIAGEN GIAGEN Sample and Assay Technologies GIAGEN is the leading provider of innovative sample and assay technologies enabling the isolation and detection of contents of any biological sample Our advanced high quality products and services ensure success from sample to result GIAGEN sets standards in EH Purification of DNA RNA and proteins E Nucleic acid and protein assays EB microRNA research and RNAi Automation of sample and assay technologies Our mission is to enable you to achieve outstanding success and breakthroughs For more information visit www giagen com FSC Wwilscorg MIX Papier aus ver antwortungsvollen Quellen GIAGEN is a member of the Forest Stewardship Council FSC For the production of printed materials including handbooks GIAGEN has a policy to select suppliers that comply with FSC standards for print ing processes and well managed forests FSC C009997 Contents Kit Contents 4 Storage 5 Product Use Limitations 5 Product Warranty and Satisfaction Guarantee 5 Quality Control 6 Technical Assistance 6 Safety Information 6 Introduction 8 Principle and procedure 8 Equipment and Reagents to Be Supplied by User 11 Important Notes 12 Preparation of reagents 13 Handling of QlAamp spin columns 14 21 Protocol Purification of Vira
15. Dilution Buffer For 200 x 50 pl reactions 211033 QuantiTect Virus NR Master Mix without ROX dye ROX Dye Solution QuantiTect Virus RT Mix RNase Free Water QuantiTect Nucleic Acid Dilution Buffer For 25 reactions QIAGEN 210210 OneStep RT PCR Enzyme Mix 5x QIAGEN OneStep RT PCR Buffer containing 12 5 mM MgCl dNTP Mix containing 5 mM each dNTP 5x Q Solution RNase free water 242 ml Wash Buffer 1 Concentrate 19081 for 1000 preparations 324 ml Wash Buffer 2 Concentrate 19072 for 1000 preparations 1000 Collection Tubes 2 ml 19201 2 ml gt 600 mAU ml solution 12131 10 ml gt 600 mAU ml solution 19133 24 QlAamp UltraSens Virus Handbook 04 2010 Notes QlAamp UltraSens Virus Handbook 04 2010 25 Notes 26 QlAamp UltraSens Virus Handbook 04 2010 Trademarks QIAGEN QlAamp GlAcube MinEulute QuantiTect UltraSens QIAGEN Group Corex Corning Inc Eppendorf Eppendorf AG Purchase of the GlAamp UltraSens Virus Kit is accompanied by a non transferable limited license under U S Patents 5 674 908 5 834 439 and 6 110 916 and foreign equivalents to use it solely for the internal purposes of the purchaser Purchasers are hereby notified that neither this product nor any components or derivatives thereof may be used in transfection whereby extracellular material is conveyed into one or more cells Limited License Agreement Use of this product signifies the agreem
16. The QlAamp UltraSens procedure is designed to allow the inclusion of internal controls with the samples in the initial step allowing the entire purification process to be accurately monitored QlAamp purified nucleic acids are highly suited for use in all downstream amplification based assays Principle and procedure GlAamp UltraSens technology allows viral RNA and DNA from 1 ml plasma or serum samples to be highly concentrated in a single step prior to purification using GlAamp silica gel membrane technology Ultracentrifugation and specialized laboratory equipment are not required Buffer AC contains a reagent that forms complexes with nucleic acids and these complexes can be sedimented by low gforce centrifugation to form a pellet This pellet can then be resuspended in a small volume of buffer prior to nucleic acid purification using the QlAamp procedure enabling extremely efficient and sensitive viral nucleic acid isolation Buffer AC and carrier RNA are added to a plasma or serum sample After a short incubation step the sample is centrifuged at low speed to pellet the nucleic acid complexes The supernatant is discarded and the pellet is resuspended in Buffer AR and proteinase K and incubated for 10 minutes at 40 C Binding conditions are adjusted by adding Buffer AB and the lysate is applied to a GlAamp spin column During a brief centrifugation RNA and DNA selectively bind to the QlAamp membrane as contaminants pass through Re
17. a RNases are inactivated An internal control can be added at this step Pipet the internal control together with the carrier RNA into the microcentrifuge tube lid If the volume of internal control to be added to the sample is lt 1 pl we recommend preparing a master mix of carrier RNA and the internal control which can then be pipetted into the tube lid For example if the master mix contains 0 5 pl internal control per 5 6 pl carrier RNA pipet 6 1 pl of the mix into the tube lid As well as saving pipetting steps this will reduce sample to sample variability Close the lid and mix thoroughly by first inverting the microcentrifuge tube 3 times and then by vortexing for 10 s Inverting the tube first is essential because it guarantees that the carrier RNA is completely dissolved in the sample Incubate at room temperature 15 25 for 10 min A 10 min incubation is sufficient for optimal recovery of HIV HCV and HBV Do not lyse samples for more than 15 min Centrifuge the sample at 1200 x g for 3 min This centrifugation step is a key step in the purification procedure In rare cases it may be necessary to optimize the centrifugation speed for the microcentrifuge used If either the pellet is difficult to resuspend after the centrifugation or debris remains in the supernatant refer to Optimization of centrifugation on page 12 Following the centrifugation the supernatant should be clear with no debris floating around and
18. al nucleic acids If more eluate is required increase the amount of Buffer AVE used in the two elution steps for instance use 2 x 50 yl instead of 2 x 30 yl rather than diluting the eluate with Buffer AVE after elution 18 QlAamp UltraSens Virus Handbook 04 2010 Troubleshooting Guide This troubleshooting guide may be helpful in solving any problems that may arise For more information see also the Frequently Asked Questions page at our Technical Support Center www giagen com FAQ FAQList aspx The scientists in QIAGEN Technical Services are always happy to answer any questions you may have about either the information and protocols in this handbook or sample and assay technologies for contact information see back cover or visit www giagen com Little or no recovery of nucleic acid a Carrier RNA not added to the sample b Carrier RNA degraded c Ethanol not added to Buffer AB AW1 or AW2 d Inappropriate gforce used to centrifuge the nucleic acid complexes or fo bind nucleic acids to the silica gel membrane e High amount of lipid in the sample Comments and suggestions Repeat the purification procedure with a new sample remembering to add carrier RNA Carrier RNA must not contact plasma before Buffer AC is added Ensure that the inside of the lid is not contaminated with plasma Add the plasma sample first to the microcentrifuge tube followed by Buffer AC before mixing Check that ethanol wa
19. e process for research requires the use of a Licensed 5 Nuclease Kit containing Licensed Probe or the combination of an Authorized 5 Nuclease Core Kit plus Licensed Probe or license rights that may be purchased from Applied Biosystems This product QuantiTect Virus Kits is an Authorized 5 Nuclease Core Kit without Licensed Probe Its purchase price includes a limited non transferable immunity from suit under U S Patents Nos 5 210 015 5 487 972 5 476 774 and 5 219 727 and corresponding patent claims outside the United States owned by Roche Molecular Systems Inc or F Hoffmann La Roche Ltd Roche for using only this amount of the product in the practice of the 5 nuclease process solely for the purchaser s own internal research when used in conjunction with Licensed Probe This product is also an Authorized 5 Nuclease Core Kit for use with service sublicenses available from Applied Biosystems This product conveys no rights under U S Patents Nos 5 804 375 6 214 979 5 538 848 5 723 591 5 876 930 6 030 787 or 6 258 569 or corresponding patents outside the United States expressly by implication or by estoppel No right under any other patent claims such as apparatus or system claims in U S Patent No 6 814 934 and no right to perform commercial services of any kind including without limitation reporting the results of purchaser s activities for a fee or other commercial consideration is hereby granted expressly by implication or
20. edure In case of contact between gloves and sample change the gloves immediately Close the GlAamp spin column before placing it in the microcentrifuge Centrifuge as described in the protocol Remove the GlAamp spin column and collection tube from the microcentrifuge Place the GlAamp spin column in a new collection tube Discard the filtrate and the collection tube Please note that the filtrate may contain hazardous waste and should be disposed of properly Open only one GlAamp spin column at a time and take care to avoid generating aerosols For efficient parallel processing of multiple samples we recommend filling a rack with collection tubes to which the GlAamp spin columns can be transferred after centrifugation Used collection tubes containing the filtrate can be discarded and the new collection tubes containing the GlAamp spin columns placed directly in the microcentrifuge Contains sodium azide See page 6 for safety information 14 QlAamp UltraSens Virus Handbook 04 2010 Protocol Purification of Viral RNA and DNA Important points before starting Read Important Notes on page 12 The use of a shaker incubator is strongly recommended for use in the incubation steps in the protocol All centrifugation steps should be carried out at room temperature 15 25 Things to do before starting E Equilibrate samples to room temperature M Check that Buffers AB AW1 and AW2 and Carrier RNA have been prepared acco
21. ent of any purchaser or user of the QlAamp UltraSens Virus Kit to the following terms 1 The QlAamp UltraSens Virus Kit may be used solely in accordance with the QlAamp UltraSens Virus Handbook and for use with components contained in the Kit only QIAGEN grants no license under any of its intellectual property to use or incorporate the enclosed components of this Kit with any components not included within this Kit except as described in the QlAamp UltraSens Virus Handbook and additional protocols available at www qiagen com 2 Other than expressly stated licenses QIAGEN makes no warranty that this Kit and or its use s do not infringe the rights of third parties 3 This Kit and its components are licensed for one time use and may not be reused refurbished or resold QIAGEN specifically disclaims any other licenses expressed or implied other than those expressly stated 5 The purchaser and user of the Kit agree not to take or permit anyone else to take any steps that could lead to or facilitate any acts prohibited above QIAGEN may enforce the prohibitions of this Limited License Agreement in any Court and shall recover all its investigative and Court costs including attorney fees in any action to enforce this Limited License Agreement or any of its intellectual property rights relating to the Kit and or its components For updated license terms see www qiagen com NOTICE TO PURCHASER LIMITED LICENSE A license to perform the 5 nucleas
22. l RNA and DNA 15 Troubleshooting Guide 19 Appendix General Considerations 21 References 22 Ordering Information 23 QlAamp UltraSens Virus Handbook 04 2010 3 Kit Contents GlAamp UltraSens Virus Kit 50 250 Catalog no 53704 53706 Preps per kit 50 250 GlAamp Spin Columns 50 250 Collection tubes 2 ml 150 750 Buffer AC 46 ml 230 ml Buffer AR 20 ml 90 ml Buffer AB concentrate 5 ml 22 ml Buffer AW1 concentrate 19 ml 95 ml Buffer AW2 concentrate 13 ml 66 ml Buffer AVE 3 2 ml 10 x 2 ml Proteinase K 1 25 ml 6 ml Carrier RNA 310 yg 5 x 310 pg Handbook 1 1 Contains chaotropic salt which is an irritant See page 6 for safety information t Contains sodium azide See page 6 for safety information 4 QlAamp UltraSens Virus Handbook 04 2010 Storage GlAamp spin columns should be stored dry at room temperature 15 25 C storage at higher temperatures should be avoided All buffers and reagents should be stored at room temperature unless otherwise stated After receiving the kit QlAamp spin columns buffers and reagents can be stored for up to 12 months under the above conditions without showing any reduction in performance Lyophilized carrier RNA is stable for up to 1 year when stored at room temperature Carrier RNA dissolved in Buffer AVE supplied with the kit should be aliquoted and stored at 20 C see Preparation of reagents page 13 When dissolved in this manner it is stable for 1 year at 20 C
23. maining contaminants and enzyme inhibitors are efficiently removed by centrifugation in two wash steps and the pure viral nucleic acids are eluted in low salt Buffer AVE Centrifugation GlAamp UltraSens technology requires verylow speed centrifugation The nucleic acid complexes are sedimented by very low gforces 1200 x Lower gforces may reduce yield due to incomplete recovery of the complexes whereas higher gforces can lead to formation of hard pellets that are very difficult to resuspend subsequently It is also important to centrifuge the QlAamp Spin Column at the recommended speed 3000 5000 x g during the nucleic acid binding step This ensures maximum nucleic acid recovery 8 QlAamp UltraSens Virus Handbook 04 2010 Note For best results depending on your microcentrifuge model the gforce required for centrifugation in step 5 of the protocol may need optimization see Important Notes page 11 Sample types The GlAamp UltraSens protocol is designed for isolation of viral nucleic acids e g those of HIV HCV or HBV from 1 ml plasma or serum or from a 1 ml pool of celltree body fluids Such pools can consist of many samples from individual donors and are usually more homogeneous than samples from single donors Note If you use the GlAamp UltraSens Virus Kit to purify nucleic acids from other types of samples you may have to optimize the g force used during centrifugation to ensure maximum yield The optimal gforce
24. make the appropriate buffer 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 Trace amounts of DEPC will modify purine residues in RNA by carbethoxylation Carbethoxylated RNA is translated 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 removed from solutions or vessels by autoclaving or heating to 100 C for 15 minutes Add 0 1 ml DEPC to 100 ml of the solution to be treated and shake vigorously to bring the DEPC into solution or let the solution bake for at 37 C 12 hours Autoclave for 15 minutes to remove any trace of DEPC It may be desirable to test water sources for the presence of contaminating RNases since many sources of distilled water are free of RNase activity Note GlAamp UltraSens buffers are not rendered RNase free by DEPC treatment and are therefore free of any DEPC contamination References QIAGEN maintains a large up to date online database of scientific publications utiliz ing QIAGEN products Comprehensive search options allow you to find the articles you need either by a simple keyword search or by specifying the application research area title etc For
25. months when stored closed at room temperature Buffer AW1 Add ethanol 96 100 to Buffer AW1 concentrate before use as indicated on the bottle Buffer AW1 is stable for 1 year when stored closed at room temperature 15 25 C When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles g 9 gogg For more information consult the appropriate material safety data sheets MSDSs available from the product supplier Not compatible with disinfecting agents that contain bleach see page 7 for safety information QlAamp UltraSens Virus Handbook 04 2010 13 Buffer AW2 Add ethanol 96 100 to Buffer AW2 concentrate before use as indicated on the bottle Buffer AW2 is stable for 1 year when stored closed at room temperature 15 25 C Handling of QlAamp spin columns Owing to the sensitivity of nucleic acid amplification technologies the following precautions are necessary when handling QlAamp spin columns to avoid cross contamination Carefully apply the sample to the QlAamp spin column Pipet the sample into the GlAamp spin column without moistening the rim of the column Change pipet tips between all liquid transfer steps The use of aerosol barrier tips is recommended Avoid touching the GlAamp membrane with the pipet tip After all vortexing steps briefly centrifuge 1 5 ml microcentrifuge tubes to remove drops from the insides of the lids Wear gloves throughout the proc
26. ne of the QIAGEN Technical Service Departments or local distributors see back cover or visit www giagen com 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 qiagen com Support MSDS aspx where you can find view and print the MSDS for each QIAGEN kit and kit component CAUTION DO NOT add bleach or acidic solutions directly to the sample preparation waste Buffers AR and AW1 contain guanidine hydrochloride 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 6 QlAamp UltraSens Virus Handbook 04 2010 The following risk and safety phrases apply to the components of the QlAamp UltraSens Virus Kit Buffer AC Contains isopropanol flammable Risk and safety phrases R10 Buffer AB Contains ethanol and Nonidet P40 Substitute irritant flammable Risk and safety phrases R10 38 41 13 26 36 46 Buffers AR and AW1 Contains guanidine hydrochloride harmful irritant Risk and safety phrases R22 36
27. osable 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 During the procedure work quickly to avoid degradation of RNA by endogenous or residual RNases 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 Non disposable plasticware Nondisposable 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 RNasefree water 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 detergent thoroughly rinsed and oven baked at gt 240 C for four or more hours overnight if more convenient before use Autoclaving alone will not fully inactivate many RNases Oven baking will both inac
28. rding to the instructions on pages 13 and 14 Mix Buffer AB thoroughly with ethanol by inverting ten times to ensure that the viscous buffer concentrate is com pletely dissolved Eqyilibrate Buffer AR to 60 C in a water bath Procedure 1 Pipet 1 ml plasma or serum equilibrated to room temperature 15 25 C into a 2 ml microcentrifuge tube not provided It is important that plasma is added before Buffer AC If the sample is smaller than 1 ml adjust to 1 ml using phosphate buffered saline At least 200 yl of plasma or serum should be processed Ensure that the microcentrifuge tube lid is not contaminated with plasma since RNases contained in the plasma will immediately degrade the carrier RNA added to the lid in the following step 2 0 8 ml Buffer AC on top of the sample in the microcentrifuge tube Pipet 5 6 yl carrier RNA solution into the tube lid This protocol will also work with smaller amounts of carrier RNA Using less than 5 6 yg carrier RNA is recommended only when it is known that a higher amount of carrier RNA will interfere with downstream applications The use of less than 5 6 yg carrier RNA per sample must be validated for each particular sample type QlAamp UltraSens Virus Handbook 04 2010 15 0204014 o ej a Note Do not mix Buffer AC and carrier RNA together before adding them to the sample as this can lead to variable RNA recovery After Buffer AC is added to the plasm
29. ria 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 1 1 5079 4001 Technical 0800 557779 Canada 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 1122 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
30. s added to Buffers AB AW1 and AW2 see Preparation of reagents on page 13 Repeot the purification procedure with a new sample Ensure that the gforce for each centrifugation step is within the recommended range Read the comments on page 9 and for protocol steps 5 and 12 IF there is a high amount of lipid in a plasma sample it will appear milky and the pellet will be small or not present at all Take a fresh 1 5 ml aliquot of the sample and centrifuge it for 2 min at 10 000 x g ina microcentrifuge The lipid fraction will form a pellet and a thin layer on top of the sample Pipet 1 ml of the cleared plasma into a new 2 ml microcentrifuge tube not provided and process the sample according to the standard protocol QlAamp UltraSens Virus Handbook 04 2010 19 High amount of lipid in the sample continued Comments and suggestions Note This procedure must be validated for each virus type e g by spiking a known amount of virus into virus free plasma with a high lipid content and quantifying the amount of viral nucleic acids recovered Nucleic acid does not perform well in downstream enzymatic reactions a Reduced sensitivity b Buffers AW1 and AW2 used in the wrong order c Too much carrier RNA in the eluate d Human genomic DNA interfered with the downstream amplification reaction Determine the maximum volume of eluate suitable for your amplification reaction Reduce the volume of eluate added to
31. table between 250 and 1200 x g with a rotor for 2 ml microcentrifuge tubes A shaker incubator for example the Eppendorf Thermomixer Compact or a heating block QlAamp UltraSens Virus Handbook 04 2010 11 Important Notes If preparing RNA for the first time read Handling RNA in the Appendix page 21 before starting All steps of the protocol should be performed quickly and at room temperature 15 25 Internal controls An internal control can be added to the sample together with the carrier RNA and Buffer AC in step 2 If only a small volume of internal control is added to the sample it may be worthwhile to prepare a master mix of carrier RNA and internal control Thus a larger volume which can be more accurately pipetted reducing sample to sample variability can be added to each sample in only a single pipetting step Note Plasma and serum are rich in RNases Do not add carrier RNA or an internal control RNA directly to untreated samples Optimization of centrifugation The centrifugation steps 5 and 12 in the protocol are key to optimal nucleic acid recovery A centrifuge with adjustable gforce is highly recommended for use with this protocol If no such centrifuge is available the g force can be calculated as follows rcf 11 2 x r x rpm 1000 Where rcf is the relative centrifugal force in g r is the radius of the rotor in centimeters and rpm is the speed of the centrifuge in revolutions per minute
32. the amplification reaction if necessary Ensure that Buffer AW1 and AW2 are used in the correct order in the protocol Repeat the purification procedure with a new sample Determine the maximum amount of carrier RNA suitable for your amplification reaction Adjust the amount of carrier RNA added to the sample accordingly As little as 1 pg carrier RNA may be sufficient for the purification procedure Human genomic DNA contained in the plasma may be copurified with the viral nucleic acids For DNA viruses optimize the PCR conditions to increase specificity For RNA viruses optimize RT PCR conditions or DNase digest the eluate Note Do not perform a DNase digestion if you wish to detect DNA viruses in the sample Always use 5 6 pg of carrier RNA if a DNase digestion is performed 20 QlAamp UltraSens Virus Handbook 04 2010 Appendix General Considerations 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 only 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 disp
33. tivate ribonucleases and ensure that no other nucleic acids such as plasmid DNA are left on the surface of the glassware Alternatively glassware can be treated with DEPC diethyl pyrocarbonate Rinse the glassware with 0 1 DEPC 0 1 in water at 37 C overnight and then autoclave or heat to 100 C for 15 minutes to remove residual DEPC 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 QlAamp UltraSens Virus Handbook 04 2010 21 Note Corex tubes should be rendered RNase free by treatment with DEPC and not by baking This will reduce the failure rate of this type of tube during centrifugation Electrophoresis tanks Electrophoresis tanks should be cleaned with detergent solution e g 0 5 SDS rinsed with water dried with ethanol and then filled with a solution of 3 H O After 10 minutes at room temperature 15 25 the electrophoresis tanks should be rinsed thoroughly with RNase free walter Solutions Solutions water and other solutions should be treated with 0 1 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 When preparing Tris buffers treat water with DEPC first and then dissolve Tris to
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