QIAamp MinElute Media Handbook
Contents
1. 3 0 9 9 4 1 2 12 5 1 5 15 6 1 8 18 7 2 1 21 8 2 4 24 9 2 7 27 10 3 0 30 QlAamp MinElute Media Handbook 04 2010 15 Table 1 Continued No samples Vol Buffer AL ml Vol Carrier RNA AVE ul 11 12 13 14 15 16 17 18 19 20 21 22 23 24 3 3 3 6 3 9 4 2 4 5 4 8 5 1 5 4 5 7 6 0 6 3 6 6 6 9 72 33 36 39 42 45 48 51 54 57 60 63 66 69 72 Note The sample preparation procedure is optimized for 2 5 ug of carrier RNA per sample If less carrier RNA has been shown to be better for your amplification system transfer only the required amount of dissolved carrier RNA to the tubes containing Buffer AL For each microgram of carrier RNA required per preparation add 4 ul Buffer AVE dissolved carrier RNA per milliliter of Buffer AL Use of less than 2 5 ug carrier RNA per sample must be validated for each particular sample type and downstream assay Buffer AW2 Add 30 ml of ethanol 96 10096 to a bottle containing 13 ml of Buffer AW2 concentrate as described on the bottle Tick the check box on the label to indicate that ethanol has been added Store reconstituted Buffer AW2 at room temperature 15 25 C Reconstituted Buffer AW2 is stable for 1 year when stored at room temperature Note Always mix reconstituted Buffer AW2 by shaking before starting the procedure QlAamp MinElute Media Handbook 04 2010 Protocol Purification of Nucleic Acids from Media For isol2. CAUTION DO NOT add bleach or acidic solutions directly to waste containing Buffer AL Buffer AL contains guanidine hydrochloride which can form highly reactive compounds when combined with bleach If liquid containing this buffer 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 196 v v sodium hypochlorite 6 QlAamp MinElute Media Handbook 04 2010 The following risk and safety phrases apply to components of the QlAamp MinElute Media Kit Buffer AL Contains guanidine hydrochloride harmful irritant Risk and safety phrases R22 36 38 S13 26 36 46 QIAGEN proteinase K Contains proteinase K sensitizer irritant Risk and safety phrases R36 37 38 42 43 23 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 R22 Harmful if swallowed R36 38 Irritating to eyes and skin R36 37 38 Irritating to eyes respiratory system and skin R42 43 May cause sensitization by inhalation and skin contact S13 Keep away from food drink and animal feeding stuffs S23 Do not breathe spray 24 Avoid contact with skin S26 In case of contact with eyes rinse immediately with plenty of water and seek medical advice S36 Wea
3. M Vacuum Regulator cat no 19530 for easy monitoring of vacuum pressures and easy release of vacuum If using the QlAvac 24 Plus M QlAvac Connecting System cat no 19419 for connection of the QlAvac 24 Plus with the Vacuum Pump cat no 84000 84010 or 840203 If using the QlAvac 24 or QlAvac 24 Plus E Optional QlAamp Vac Accessory Set cat no 19409 contains 12 VacValves for vacuum regulation of each individual column if sample flow rates differ significantly and 500 VacConnectors for prevention of cross contamination caused by direct contact between QlAamp MinElute column and QlAvac 24 or QlAvac 24 Plus Available by mid 2004 please check www qiagen com products accessories t Japan 110 V 60 Hz Canada and USA 115 V 60 Hz Australia and Europe 230 V 50 Hz QlAamp MinElute Media Handbook 04 2010 11 Important Notes Elution of pure nucleic acids DNA is eluted into Buffer AVE QlAamp MinElute columns allow minimal elution volumes of only 20 ul Low elution volume leads to highly concentrated nucleic acid eluates For downstream applications that require small starting volumes e g some PCR and RT PCR assays a more concentrated eluate may increase assay sensitivity For downstream applications that require a larger starting volume elution volume can be increased up to 150 ul However an increase in elution volume will decrease the concentration of nucleic acids in the eluat
4. 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 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 436 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
5. 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 01293 422 922 Technical 01293 422 999 o e o 00000 USA Orders 800 426 8157 Fax 800 718 2056 Technical 800 DNA PREP 800 362 7737 QIAGEN WWW QIAGEN COM diua
6. column into a VacConnector on the vacuum manifold Insert an extension tube into the open QlAamp MinElute column Note Keep the 2 ml collection tube for the dry spin in step 16 12 Carefully pipet all of the lysate from step 10 into the extension tube of the QlAamp MinElute column Switch on the vacuum pump After the lysate has been completely drawn through the column switch off the vacuum pump and release the pressure to O mbar If the lysate from an individual sample has not completely passed through the membrane despite the VacValves of all other QlAamp MinElute columns being closed place the QlAamp MinElute column into a clean 2 ml collection tube close the lid and centrifuge at full speed for 3 min or until the lysate has completely passed through Additional 2 ml collection tubes can be purchased separately see ordering information page 32 Note For fast and convenient release of vacuum pressure a vacuum regulator should be used see Equipment and Reagents to Be Supplied by User page 11 18 QlAamp MinElute Media Handbook 04 2010 13 Apply 750 ul of Buffer AW2 into the extension tube of the QlAamp MinElute column Switch on the vacuum pump After Buffer AW2 has been completely drawn through the QlAamp MinElute column switch off the vacuum pump and release the pressure to O mbar 14 Apply 750 ul of ethanol 96 100 into the extension tube of the QlAamp MinElute column Switch on the vacuum pump After the ethanol
7. has been completely drawn through the QlAamp MinElute column switch off the vacuum pump and release the pressure to 0 mbar 15 Remove and discard the extension tube Note To avoid cross contamination ensure that removed extension tubes do not pass over neighboring QlAamp MinElute columns 16 Close the lid of the QlAamp MinElute column Remove it from the vacuum manifold and discard the VacConnector Place the QlAamp MinElute column in the clean 2 ml collection tube saved from step 11 and centrifuge at full speed 20 000 x g 14 000 rpm for 3 min to dry the membrane completely 17 Place the QlAamp MinElute column in a clean 1 5 ml collection tube provided and discard the 2 ml collection tube with the filtrate Open the lid of the QlAamp MinElute column and incubate the column at room temperature 15 25 C for 15 min Note Alternatively for faster incubation heat the opened QlAamp MinElute column at 56 C for 3 min 18 Apply 120 ul of Buffer AVE to the center of the membrane in the QlAamp MinElute column Close the lid and incubate at room temperature 15 25 C for 1 min Centrifuge at full speed 20 000 x g 14 000 rpm for 1 min Important Ensure that Buffer AVE is already equilibrated to room temperature Incubating the QlAamp MinElute column loaded with Buffer AVE for 5 min at room temperature before centrifugation generally increases yield Elution volume is flexible 20 150 ul and can be adjusted depending on th
8. the blister pack and attach each column to a VacConnector The collection tubes can be saved for the dry spin in step 16 of the procedure page 17 6 Insert an extension tube into each QlAamp MinElute column 7 For nucleic acid purification follow the instructions for the QlAamp MinElute Media procedure page 17 Discard the VacConnectors appropriately after use Leave the lid of the QlAamp MinElute column open while applying vacuum VacValves can be closed individually when each sample is completely drawn through its column allowing parallel processing of samples of different volumes or viscosities 8 After processing of samples discard the liquid waste in the waste bottle appropriately Clean and decontaminate the vacuum system as described in Appendix C page 30 Note Buffer AL used in the QlAamp MinElute Media procedure is not compatible with disinfecting agents containing bleach see safety information page 6 QlAamp MinElute Media Handbook 04 2010 29 Appendix C Cleaning and Decontaminating the Vacuum Manifold The QlAvac 24 or QlAvac 24 Plus vacuum manifold must be decontaminated after QlAamp MinElute columns have been processed The vacuum manifold must also be decontaminated before removal from the laboratory Important points before starting E When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles E Do not use cleaning materials that contain abrasives W I
9. up to date online database of scientific publications utilizing 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 a complete list of references visit the QIAGEN Reference Database online at www qiagen com RefDB search asp or contact QIAGEN Technical Services or your local distributor D H P BONUM QlAamp MinElute Media Handbook 04 2010 31 Ordering Information Product QlAamp MinElute Media Kit 50 Accessories QIAGEN Proteinase K 2 ml Buffer ATL 200 ml Buffer AL 216 ml Buffer AW1 concentrate 242 ml Buffer AW2 concentrate 324 ml Collection Tubes 2 ml Extension Tubes 3 ml QlAvac 24 QlAamp Vac Accessory Set VacConnectors 500 Vacuum Regulator QlAvac 24 Plus QlAvac Connecting System 32 Contents For 50 minipreps 50 QlAamp MinElute Columns QIAGEN Proteinase K Carrier RNA Buffers Extension Tubes 3 ml Collection Tubes 1 5 ml 2 ml gt 600 mAU ml solution 200 ml Tissue Lysis Buffer 216 ml Lysis Buffer AL 242 ml Wash Buffer 1 Concentrate 324 ml Wash Buffer 2 Concentrate 1000 Collection Tubes 2 ml For use with QIAGEN Mini or MinElute Columns on vacuum manifolds 100 per pack Vacuum manifold for processing 1 24 sp
10. your amplification reaction Reduce or increase the volume of eluate added to the amplification reaction accordingly The elution volume can be adjusted proportionally d Performance of purified Salt and ethanol components of Buffer AW2 may nucleic acids in have separated out after being unused for a long downstream assays period Always mix buffers thoroughly before varies with the age of each preparation the reconstituted wash buffers e Anew combination of If enzymes are changed it may be necessary to reverse transcriptase readjust the amount of carrier RNA added to and Taq DNA Buffer AL and the amount of eluate used polymerase was used QlAamp MinElute Media Handbook 04 2010 21 Comments and suggestions f PCR is inhibited Depending on the sample type it may be necessary to include a wash step with Buffer AW not included with the kit see ordering information page 32 in the QlAamp MinElute Media procedure After completing step 12 of the procedure apply 600 ul ethanol reconstituted Buffer AW into the extension tube of the QlAamp MinElute column Switch on the vacuum pump After Buffer AW1 has been completely drawn through the QlAamp MinElute column switch off the vacuum pump and release the pressure to O mbar Continue with step 13 of the procedure General handling a Clogged QlAamp Remove the QlAamp MinElute column from the MinElute column vacuum manifold place it in a 2 ml collection tube and centrifuge at
11. April 2010 QlAamp MinElute Media Handbook For purification of nucleic acids from liquid media using vacuum systems 0000 00000 QIAGEN WWW QIAGEN COM Trademarks QIAGEN QlAamp MinElute QIAGEN Group PreservCyt Cytyc Corporation SurePath TriPath Imaging Inc Tween ICI Americas Inc The PCR process is covered by U S Patents 4 683 195 and 4 683 202 and foreign equivalents owned by Hoffmann La Roche AG 2004 QIAGEN all rights reserved Contents Kit Contents 4 Storage 4 Quality Control 5 Product Use Limitations 5 Product Warranty and Satisfaction Guarantee 5 Technical Assistance 6 Safety Information 6 Introduction 8 Principle and procedure 8 Equipment and Reagents to Be Supplied by User 11 Important Notes 12 Elution of pure nucleic acids 12 Yield and size of nucleic acids 12 Carrier RNA 12 Addition of internal controls 13 Handling of QlAamp MinElute columns 13 Preparation of buffers 15 Protocol H Purification of Nucleic Acids from Media 17 Troubleshooting Guide 20 Appendix A Handling Guidelines for the QlAvac 24 24 Appendix B Handling Guidelines for the QlAvac 24 Plus 28 Appendix C Cleaning and Decontaminating the Vacuum Manifold 30 References 31 Ordering Information 32 QlAamp MinElute Media Handbook 04 2010 3 Kit Contents QlAamp MinElute Media Kit 50 Catalog no 1025106 Number of preps 50 QlAamp MinElute Columns each packaged with 50 a 2 ml Collection Tube Exte
12. ants Nucleic acids remain bound to the QlAamp MinElute column membrane while contaminants are efficiently washed away during a sequence of wash steps using 2 different wash solutions In a single step highly pure nucleic acids are eluted in Buffer AVE equilibrated to room temperature 15 25 C QlAamp MinElute Media Handbook 04 2010 9 GlAamp MinElute Media Procedure Sample Bind Wash Buffer AW2 Wash ethanal Dry spin Bute at ox uae fe fad e feed f faba ot Pure nucleic acids 10 QlAamp MinElute Media 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 M Ethanol 96 100 M 2 ml microcentrifuge tubes Wi Pipettips pipet tips with aerosol barriers for preventing cross contamination are recommended M Disposable gloves E Heating block thermomixer or heated orbital incubator for lysis of samples one for 56 C incubation and another for 70 C incubation B Microcentrifuge B Vortexer E Vacuum manifold e g QlAvac 24 cat no 19403 or QlAvac 24 Plus cat no 19413 E Vacuum pump with a capacity of 34 liter min and capable of producing a vacuum of 800 to 900 mbar e g Vacuum Pump cat no 84000 84010 or 840208 If using the QlAvac 24
13. aranteed for every virus species and must be validated by the user Sample volumes Each QlAamp MinElute column can bind nucleic acids that are longer than 200 bases but yield depends on sample volume and nucleic acid content The procedure is optimized for use with a starting volume of 250 ul Lysis with QIAGEN proteinase K Samples are lysed under denaturing conditions at elevated temperatures Lysis is performed in the presence of QIAGEN proteinase K and Buffer ATL Addition of Buffer AL enhances lysis efficiency and ensures inactivation of RNases Adsorption to the QlAamp MinElute column membrane Binding conditions are adjusted by adding ethanol to the lysates to ensure optimal binding of nucleic acids to the QlAamp MinElute column membrane Lysate is applied to a QlAamp MinElute column and nucleic acids are adsorbed onto the silica gel membrane as the lysate is drawn through by vacuum pressure Salt and pH conditions ensure that protein and other contaminants which can inhibit PCR and other downstream enzymatic reactions are not retained on the membrane 8 QlAamp MinElute Media Handbook 04 2010 A vacuum manifold and a vacuum source or pump capable of producing a vacuum of 800 to 900 mbar are required for the procedure A vacuum regulator should be used for easy monitoring of vacuum pressures and convenient vacuum release For details see Equipment and Reagents to Be Supplied by User page 11 Removal of residual contamin
14. ase Carrier RNA Buffers Collection Tubes 2 ml QIAamp DNA Micro Kit for purification of genomic and mitochondrial DNA from small amounts of fresh or frozen blood tissue forensic samples and dried blood spots QlAamp DNA Micro Kit For 50 DNA preps 50 QlAamp 50 MinElute Columns QIAGEN Proteinase K Carrier RNA Buffers Collection Tubes 2 ml QlAamp DNA Mini Kit for isolation of genomic mitochondrial bacterial parasite or viral DNA QlAamp DNA Mini Kit For 50 DNA preps 50 QlAamp Mini 50 Spin Columns QIAGEN Proteinase K Buffers Collection Tubes 2 ml QlAamp MinElute Media Handbook 04 2010 Cat no 84000 84010 84020 57714 57704 56304 51304 33 Product Contents Cat no QlAamp DNA Mini Kit For 250 DNA preps 250 QlAamp Mini 51306 250 Spin Columns QIAGEN Proteinase K Buffers Collection Tubes 2 ml For up to date licensing information and product specific 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 E e S 34 QlAamp MinElute Media Handbook 04 2010 QIAGEN Companies Please see the back cover for contact information for your local QIAGEN office QlAamp MinElute Media Handbook 04 2010 35 www qiagen com Australia Orders 1 800 243 800 Fax 03 9840 9888 Technical 1 800 243 066 Austria Orders 0800
15. ation of nucleic acids from 250 ul of liquid media such as cervical swab transport media e g PreservCyt or SurePath solution Important points before starting W All centrifugation steps are carried out at room temperature 15 25 C E After applying lysate or wash solution to the QlAamp MinElute column in steps 12 13 and 14 wait for at least 1 minute before switching on the vacuum pump After all liquid has been drawn through the column wait for at least one minute before switching the vacuum pump off and releasing the vacuum Things to do before starting E Equilibrate samples to room temperature 15 25 C M Equilibrate Buffer AVE to room temperature for elution in step 18 E Ifa precipitate has formed in Buffer ATL or AL dissolve by heating to 70 C and gentle agitation M Prepare a 56 C heating block thermomixer or heated orbital incubator for use in step 4 and a 70 C heating block thermomixer or heated orbital incubator for use in step 7 E Ensure that Buffer AW2 has been prepared according to instructions on page 16 E Add carrier RNA dissolved in Buffer AVE to Buffer AL according to instructions on page 15 Procedure 1 Pipet 80 pl of Buffer ATL into a 2 ml microcentrifuge tube not provided 2 Add 250 pl of sample into the 2 ml microcentrifuge tube 3 Add 20 yl QIAGEN proteinase K Close the lid and mix by pulse vortexing for 10 s 4 Incubate at 56 C for 30 min Shake the samples to ensure high nucl
16. ations or performance please call QIAGEN Technical Services or your local distributor see back cover QlAamp MinElute Media Handbook 04 2010 5 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 QlAamp MinElute Media 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 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 qgiagen com ts msds asp where you can find view and print the MSDS for each QIAGEN kit and kit component
17. e The volume of eluate recovered can be up to 5 ul less than the volume of elution buffer applied to the column for example an elution buffer volume of 20 ul results in gt 15 ul final eluate The volume of eluate recovered also depends on the nature of the sample Eluted nucleic acids are collected in 1 5 ml collection tubes provided If the purified nucleic acids are to be stored for up to 24 hours storage at 2 8 C is recommended To store nucleic acids for longer than 24 hours storage at 20 C is recommended Yield and size of nucleic acids Yields of nucleic acids isolated from liquid media are normally below 1 ug and are therefore difficult to determine with a spectrophotometer Quantitative amplification methods are recommended for determination of yields When quantifying nucleic acids isolated using the QlAamp MinElute Media procedure remember that there will be much more carrier RNA in the sample than target nucleic acids The size distribution of nucleic acids purified using this procedure can be checked by agarose gel electrophoresis and hybridization to a target specific labeled probe followed by autoradiography Sambrook J Fritsch E F Maniatis T 1989 Molecular Cloning A Laboratory Manual 2nd ed Cold Spring Harbor NY Cold Spring Harbor Laboratory Press Carrier RNA Carrier RNA enhances binding of nucleic acids to the QlAamp MinElute column membrane especially if there are very few target molecules in th
18. e downstream application Reduction of elution volume yields more concentrated eluates which might increase assay sensitivity Remember that the recovered eluate volume will differ by 5 ul from the elution buffer volume applied onto the column QlAamp MinElute Media Handbook 04 2010 19 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 page for contact information Comments and suggestions Little or no nucleic acids in the eluate a Carrier RNA not added Reconstitute carrier RNA in Buffer AVE and mix to Buffer AL with Buffer AL as described on page 15 Repeat the purification procedure with new samples b Degraded carrier RNA Carrier RNA reconstituted in Buffer AVE was not stored at 20 C or underwent multiple freeze thaw cycles Alternatively Buffer AL carrier RNA mixture was stored for more than 48 hours at 2 8 C Prepare a new tube of carrier RNA dissolved in Buffer AVE and mix with Buffer AL Repeat the purification procedure with new samples c Low concentration of Samples were left at room temperature target nucleic acid 15 25 C for too long Repeat the purification obtained from the procedure with new samples samples d Insufficient sample lysis QIAGEN protei
19. e sample If carrier RNA is not added to Buffer AL this may lead to reduced nucleic acid recovery 12 QlAamp MinElute Media Handbook 04 2010 The amount of lyophilized carrier RNA provided is sufficient for the volume of Buffer AL supplied with the kit The concentration of carrier RNA has been adjusted so that the QlAamp MinElute Media procedure can be used as a universal purification system compatible with many different amplification systems and is suitable for a wide range of samples Different amplification systems vary in efficiency depending on the total amount of nucleic acids present in the reaction Eluates from this kit contain both target nucleic acids and carrier RNA and amounts of carrier RNA will greatly exceed amounts of target nucleic acids The amount of eluate to add to downstream amplifications should therefore be based on the amount of carrier RNA added To obtain the highest levels of sensitivity in amplification reactions it may be necessary to adjust the amount of carrier RNA added to Buffer AL Addition of internal controls Using the QlAamp MinElute Media procedure in combination with commercially available amplification systems may require the introduction of an internal control into the procedure An internal control should be added together with the carrier RNA to Buffer AL For optimal purification efficiency internal control molecules should be longer than 200 nucleotides as smaller molecules are not efficie
20. eic acid yields For optimal results use a thermomixer at 900 rpm If using a heating block vortex the samples occasionally throughout the incubation period 5 Briefly centrifuge the 2 ml tube to remove drops from the inside of the lid QlAamp MinElute Media Handbook 04 2010 17 6 Add 250 ul of Buffer AL containing 10 pg ml of carrier RNA Close the lid and mix by pulse vortexing for 10 s To ensure efficient lysis it is essential that the sample Buffer ATL QIAGEN proteinase K and Buffer AL are mixed thoroughly to yield a homogeneous solution A white precipitate may form when Buffer AL is added to Buffer ATL The precipitate does not interfere with the procedure and will dissolve during the incubation in step 7 7 Incubate at 70 C for 15 min Shake the samples to ensure high nucleic acid yields For optimal results use a thermomixer at 900 rpm If using a heating block vortex the samples occasionally throughout the incubation period 8 Briefly centrifuge the 2 ml tube to remove drops from the inside of the lid 9 Add 300 pl of ethanol 96 1009 to the sample Close the lid and mix thoroughly by pulse vortexing for 15 s Incubate the lysate with the ethanol for 5 min at room temperature 15 25 C Note If ambient temperature exceeds 25 C ethanol should be cooled on ice before adding to the lysate 10 Briefly centrifuge the 2 ml tube to remove drops from the inside of the lid 11 Insert a QlAamp MinElute
21. f the vacuum manifold is still not clean after following the procedure below soak the vacuum manifold in warm detergent solution for at least 4 hours Then repeat the procedure E Sample preparation waste may contain guanidine hydrochloride for details see the safety information on page 6 Mixing of this chemical with sodium hypochlorite inside a closed container can cause buildup of gas bubbles Follow the procedure below to avoid this hazard E If liquid containing guanidine salt 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 196 w v sodium hypochlorite Procedure 1 Thoroughly rinse the inside and outside of the vacuum manifold first using laboratory detergent solution and then using water 2 Soak the vacuum manifold in 196 w v sodium hypochlorite for 10 min Thoroughly rinse the inside and outside of the vacuum manifold using water 3 Wipe the outside of the vacuum manifold using 7096 v v ethanol Then wipe dry 4 Thoroughly soak the VacValves in laboratory detergent solution Rinse thoroughly using water 5 Soak the VacValves in 196 w v sodium hypochlorite for 10 min Rinse thoroughly using water 6 Rinse the VacValves using 7096 v v ethanol Wipe dry or allow to air dry 30 QlAamp MinElute Media Handbook 04 2010 References QIAGEN maintains a large
22. full speed until the sample has completely passed through the membrane b Variable elution If different types of sample are processed oat the volumes same time on the vacuum manifold elution volumes may vary between samples e E 22 QlAamp MinElute Media Handbook 04 2010 Comments and suggestions c Vacuum pressure of The QlAVac 24 vacuum manifold is not tightly 800 900 mbar not closed Press down the lid of the vacuum reached manifold after vacuum is switched on Check if vacuum pressure is reached The gasket of the QlAvac 24 lid has worn out Check the seal of the manifold visually and if necessary replace it The VacValves have worn out Remove all VacValves and insert VacConnectors directly into the luer extensions slots Insert QlAamp MinElute columns into the VacConnectors close the lids of the columns and switch on the vacuum Check if vacuum pressure is reached Replace the VacValves if necessary Connection to the vacuum pump is leaky Close all luer extensions slots by attaching luer caps if using the QlAvac 24 or closed VacValves if using the QlAvac 24 Plus and switch on the vacuum pump Check if vacuum pressure is stable after the pump is switched on and the vacuum regulator valve is closed Exchange the connections between pump and vacuum manifold if necessary If the problem is not resolved after all above checks have been made replace the vacuum pump with a stronger one QlAamp MinElute Med
23. g bleach see safety information page 6 Setting Up the QlAvac 24 Figure 1 Setting up the QlAvac 24 with QlAamp MinElute columns using VacValves and VacConnectors 1 GlAvac 24 base 5 VacValve 2 GlAvac 24 lid 6 VacConnector 3 Luer extension of QlAvac 24 7 QlAamp MinElute column 4 Luer extension closed with luer cap 8 Extension tubet Optional see step 3 page 25 Must be purchased separately as part of the QlAamp Vac Accessory Set see ordering information page 32 t Included with the QlAamp MinElute Media Kit 26 QlAamp MinElute Media Handbook 04 2010 Vacuum Regulator Regulator gauge Figure 2 Schematic diagram of the Vacuum Regulator The Vacuum Regulator measures the pressure difference between the inside and outside of a vacuum system in millibars Use of a vacuum source that does not generate a vacuum 800 to 900 mbar may reduce the yield and purity of the nucleic acid preparation Use of the Vacuum Regulator makes it easy to monitor the pressure generated by the vacuum source ensuring that it is sufficient for the appropriate QIAGEN purification chemistry In addition the Vacuum Regulator facilitates easy and fast release of vacuum pressure SSS SSS 1 XI QlAamp MinElute Media Handbook 04 2010 27 Appendix B Handling Guidelines for the QlAvac 24 Plus The QlAvac 24 Plus QlAvac Connecting System and Vacuum Pump see ordering information page 32 form a complete sys
24. ia Handbook 04 2010 23 Appendix A Handling Guidelines for the QlAvac 24 The following guidelines should be followed when working with the QlAvac 24 vacuum manifold E Always place the QlAvac 24 on a secure bench top or work area If dropped the QlAvac 24 may crack E Always store the QlAvac 24 clean and dry To clean simply rinse all components with distilled water and allow to air dry or dry with paper towels M The components of the QlAvac 24 are not resistant to certain solvents Table 2 below If these solvents are spilt on the manifold rinse it thoroughly with water M To ensure consistent performance do not apply silicone or vacuum grease to any part of the QlAvac 24 E Always use caution and wear safety glasses when working near a manifold under pressure M Contact QIAGEN Technical Services or your local distributor for information concerning spare or replacement parts M The vacuum pressure is the pressure differential between the inside of the manifold and the atmosphere standard atmospheric pressure is 1013 millibar or 760 mm Hg and can be measured using the Vacuum Regulator see Figure 2 page 27 The procedure requires a vacuum pump with a capacity of 34 liter min and capable of producing a vacuum of 800 to 900 mbar e g QIAGEN Vacuum Pump see ordering information on page 32 Higher vacuum pressures must be avoided Use of vacuum pressures lower than recommended may reduce nucleic acid yield and purity a
25. in columns includes QlAvac 24 Base Lid Luer Caps For processing QlAamp Mini spin columns on QlAvac 24 12 VacValves 500 VacConnectors 500 disposable connectors for use with QlAamp spin columns on luer connectors For use with QlAvac manifolds Vacuum manifold for processing 1 24 spin columns System to connect vacuum manifold with vacuum pump Cat no 1025106 19131 19076 19075 19081 19072 19201 19587 19403 19409 19407 19530 19413 19419 QlAamp MinElute Media Handbook 04 2010 Product Contents Vacuum Pump 110 V Universal vacuum pump capacity 34 liter min 8 mbar vacuum abs 110 V 60 Hz Vacuum Pump 115 V Universal vacuum pump capacity 34 liter min 8 mbar vacuum abs 115 V 60 Hz Vacuum Pump 230 V Universal vacuum pump capacity 34 liter min 8 mbar vacuum abs 230 V 50 Hz Related products QlAamp MinElute Virus Vacuum Kit for simultaneous purification of viral DNA and RNA from plasma serum and cell free body fluids using vacuum processing QlAamp MinElute Virus For 50 minipreps 50 QlAamp MinElute Vacuum Kit 50 Columns QIAGEN Protease Carrier RNA Buffers Extension Tubes 3 ml Collection Tubes 1 5 ml QlAamp MinElute Virus Spin Kit for simultaneous purification of viral DNA and RNA from plasma serum and cell free body fluids using spin processing QlAamp MinElute Virus For 50 minipreps 50 QlAamp MinElute Spin Kit 50 Columns QIAGEN Prote
26. liter min and capable of producing a vacuum of 800 to 900 mbar Higher vacuum pressures must be avoided Use of vacuum pressures lower than recommended may reduce nucleic acid yield and purity and increase the frequency of clogged membranes Note If using the VacValves supplied with the QlAamp Vac Accessory Set VacConnectors must first be inserted into the luer slots of the QlAvac 24 Plus before the VacValves can be inserted 28 QlAamp MinElute Media Handbook 04 2010 Setting up the QlAvac 24 Plus For processing QlAamp MinElute columns on the QlAvac 24 Plus using VacConnectors and VacValves set up the manifold as follows 1 Connect the QlAvac 24 Plus to a vacuum source placing a vacuum trap between the manifold and the source and a vacuum regulator between the trap and the source Alternatively use the QlAvac Connecting System to connect the QlAvac 24 Plus to a vacuum source 2 Close unused luer slots by attaching closed VacValves 3 Optional Insert a VacValve into each luer slot used on the QlAvac 24 Plus If flow rates of samples differ significantly VacValves should be used to ensure consistent vacuum 4 Insert a VacConnector into each luer slot or VacValve used Perform this step just before starting the purification procedure to avoid exposure of VacConnectors to potential contaminants in the air 5 Place a QlAamp MinElute column into each VacConnector on the manifold Remove the QlAamp MinElute columns from
27. nase K was subjected to elevated in Buffer AL temperatures for a prolonged time Repeat the purification procedure using new samples and fresh QIAGEN proteinase K e Buffer AL carrier RNA Mix Buffer AL with carrier RNA by gently inverting mixture mixed the tube of Buffer AL carrier RNA at least insufficiently 10 times f Low percentage Repeat the purification procedure with new ethanol used instead of samples and use 96 10096 ethanol 96 100 g Buffer AW2 prepared Check that Buffer AW2 concentrate was diluted incorrectly with the correct volume of ethanol Repeat the purification procedure with new samples 20 QlAamp MinElute Media Handbook 04 2010 Comments and suggestions h Buffer AW2 prepared Check that Buffer AW2 concentrate was diluted with 70 ethanol with 96 100 ethanol Repeat the purification procedure with new samples RNA or DNA does not perform well in downstream enzymatic reactions a Little or no nucleic See Little or no nucleic acids in the eluate acids in the eluate above for possible reasons Increase the amount of eluate added to the reaction if possible b Too much or too little Determine the maximum amount of carrier RNA carrier RNA in the suitable for your amplification reaction Adjust eluate the concentration of carrier RNA added to Buffer AL accordingly see Addition of carrier RNA to Buffer AL page 15 c Reduced sensitivity Determine the maximum volume of eluate suitable for
28. nd increase the frequency of clogged membranes Table 2 Chemical Resistance Properties of the QlAvac 24 Resistant to Acetic acid Chlorine bleach Concentrated alcohols SDS Acetone Chromic acid Hydrochloric acid Tween 20 Chaotropic salts Sodium chloride Sodium hydroxide Urea Not resistant to Benzene Ethers Toluene Chloroform Phenol 24 QlAamp MinElute Media Handbook 04 2010 Setting up the QlAvac 24 For processing QlAamp MinElute columns on the QlAvac 24 using VacConnectors and VacValves set up the manifold as follows see Figure 1 page 26 1 Place the QlAvac 24 lid on top of the QlAvac 24 base Make sure that the gasket fits tightly in the groove of the QlAvac 24 Connect the QlAvac 24 to a vacuum source placing a vacuum trap between the manifold and the source and a vacuum regulator between the trap and the source 2 Close unused luer extensions with luer caps 3 Optional Insert a VacValve into each luer extension used on the QlAvac 24 lid Sample processing is most convenient if only 12 samples are processed in parallel i e every second luer extension If more than 12 samples are processed in parallel the knobs on the VacValves should point toward the sides of the QlAvac 24 If flow rates of samples differ significantly VacValves should be used to ensure consistent vacuum 4 Insert a VacConnector into each luer extension or VacValve used Perform this step just before starting the purificati
29. nsion Tubes 3 ml 50 Collection Tubes 1 5 ml 50 VacConnectors 50 Buffer ATL 10 ml Buffer AL 33 ml Buffer AW2 concentrate 13 ml Buffer AVE tubes with purple caps 4x2ml Carrier RNA tube with red cap 310 ug QIAGEN Proteinase K 1 25 ml Handbook 1 Contains a guanidine salt Not compatible with disinfectants containing bleach See page 6 for safety information t Contains sodium azide as a preservative Storage QlAamp MinElute columns should be stored at 2 8 C upon arrival Short term storage of up to 4 weeks at room temperature 15 25 C does not affect performance QIAGEN proteinase K solution can be stored at room temperature 15 25 C for up to one year after delivery For longer storage or if ambient temperatures exceed 25 C we suggest storing QIAGEN proteinase K at 2 8 C All buffers can be stored at room temperature 15 259 C 4 QlAamp MinElute Media Handbook 04 2010 Lyophilized carrier RNA is stable for up to 1 year when stored at room temperature 15 259 C Carrier RNA can only be dissolved in Buffer AVE or an internal control if used dissolved carrier RNA should be immediately added to Buffer AL as described in Addition of carrier RNA to Buffer AL on page 15 This solution should be prepared fresh and is stable at 2 8 C for up to 48 hours Unused portions of Buffer AVE dissolved carrier RNA should be frozen in aliquots at 20 C Quality Control As part of the stringent QIAGEN quali
30. ntly recovered Refer to the manufacturer s instructions in order to determine the optimal concentration of internal control Using a concentration other than that recommended may reduce amplification efficiency Handling of QlAamp MinElute columns Because of the sensitivity of nucleic acid amplification technologies the following precautions are necessary when handling QlAamp MinElute columns in order to avoid cross contamination between sample preparations E Carefully pipet the sample or solution into the QlAamp MinElute column without wetting the rim of the column M Avoid touching the QlAamp MinElute column membrane with the pipet tip E Change pipet tips between all liquid transfers The use of aerosol barrier pipet tips is recommended E After all pulse vortexing steps briefly centrifuge tubes to remove drops from the inside of the lid E Wear gloves throughout the entire procedure In case of contact between gloves and sample change gloves immediately QlAamp MinElute Media Handbook 04 2010 13 Processing QlAamp MinElute columns on QlAvac 24 or QlAvac 24 Plus QlAamp MinElute columns are processed on the QlAvac 24 or QlAvac 24 Plus vacuum manifold using disposable VacConnectors and reusable VacValves use of VacValves is optional VacValves are inserted directly into the luer extensions slots of the vacuum manifold and ensure a steady flow rate facilitating parallel processing of samples of different nature volume
31. on procedure to avoid exposure of VacConnectors to potential contaminants in the air 5 Place a QlAamp MinElute column into each VacConnector on the manifold Remove the QlAamp MinElute columns from the blister pack and attach each column to a VacConnector The collection tubes can be saved for the dry spin in step 16 of the procedure page 17 6 Insert an extension tube into each QlAamp MinElute column 7 For nucleic acid purification follow the instructions for the QlAamp MinElute Media procedure page 17 Discard the VacConnectors appropriately after use Leave the lid of the QlAamp MinElute column open while applying vacuum After vacuum is switched on it may be necessary to press down on the QlAvac 24 lid in order to achieve a tight seal Switch off vacuum between steps to ensure that a consistent even vacuum is applied during manipulations For faster release of vacuum a Vacuum Regulator see Figure 2 page 27 should be used VacValves can be closed individually when each sample is completely drawn through its column allowing parallel processing of samples of different volumes or viscosities QlAamp MinElute Media Handbook 04 2010 25 8 After processing of samples discard the liquid waste in the QlAvac 24 base appropriately Clean and decontaminate the vacuum system as described in Appendix C page 30 Note Buffer AL used in the QlAamp MinElute Media procedure is not compatible with disinfecting agents containin
32. or viscosity If sample flow rates differ significantly VacValves must be used to ensure consistent vacuum VacConnectors are disposable connectors that fit between the QlAamp MinElute columns and the VacValves or between the QlAamp MinElute columns and the luer extensions slots of the vacuum manifold VacConnectors prevent direct contact between the QlAamp MinElute columns and the VacValves or the vacuum manifold during purification avoiding any cross contamination between samples VacConnectors are discarded after a single use Handling guidelines for vacuum manifold If using the QlAvac 24 or QlAvac 24 Plus refer to the handling guidelines in Appendix A page 24 or Appendix B page 28 respectively In addition refer to Appendix C page 30 for guidelines on cleaning and decontaminating the QlAvac 24 or QlAvac 24 Plus after processing QlAamp MinElute columns If using another vacuum manifold refer to the manufacturer s guidelines for handling cleaning and decontamination Centrifugation For the dry spin at the end of the wash steps of the procedure and for elution centrifugation should be carried out at full speed All centrifugation steps should be carried out at room temperature 15 25 C Processing QlAamp MinElute columns in a microcentrifuge M Close the QlAamp MinElute column before placing it in the microcentrifuge Centrifuge as described M Remove the QlAamp MinElute column and collection tube from the microcen
33. r suitable protective clothing S36 37 Wear suitable protective clothing and gloves S46 If swallowed seek medical advice immediately and show this container or label QlAamp MinElute Media Handbook 04 2010 7 Introduction The QlAamp MinElute Media Kit uses well established technology for purification of nucleic acids The kit combines the selective binding properties of a silica based membrane with flexible elution volumes of between 20 and 150 ul The kit is suitable for use with liquid media containing nucleic acids such as cervical swab transport media e g PreservCyt or SurePath solution Nucleic acids are eluted in Buffer AVE ready for use in amplification reactions or storage at 20 C Purified nucleic acids are free of proteins nucleases and other impurities Principle and procedure The QlAamp MinElute Media procedure has 4 steps lyse bind wash elute see flowchart page 10 and uses QlAamp MinElute columns on a vacuum manifold The procedure is designed to ensure that there is no sample to sample cross contamination and allow safe handling of potentially infectious samples The simple QlAamp MinElute Media procedure is highly suited for simultaneous processing of multiple samples and yields pure nucleic acids from 24 samples in less than 90 minutes The QlAamp MinElute Media procedure can be used for isolation of genomic DNA and viral nucleic acids from a broad range of viruses However performance cannot be gu
34. tem for the vacuum processing of QlAamp MinElute columns The QlAvac 24 Plus is a vacuum manifold which is connected to the Vacuum Pump via the QlAvac Connecting System which includes tubing waste bottle inline filter and vacuum regulator A valve between the vacuum manifold and the first waste bottle allows control of vacuum in the vacuum manifold Up to 24 QlAamp MinElute columns can be connected to the surface of the vacuum manifold Upon application of vacuum liquid is drawn through the membranes of the QlAamp MinElute columns into the vacuum manifold and then collected in the waste bottles The following guidelines should be followed when working with the QlAvac 24 Plus vacuum manifold E Always place the QlAvac 24 Plus on a secure bench top or work area If dropped the QlAvac 24 Plus may crack E Always store the QlAvac 24 Plus clean and dry M To ensure consistent performance do not apply silicone or vacuum grease to any part of the QlAvac 24 Plus E Always use caution and wear safety glasses when working near a manifold under pressure E Contact QIAGEN Technical Services or your local distributor for information concerning spare or replacement parts E The vacuum pressure is the pressure differential between the inside of the manifold and the atmosphere standard atmospheric pressure is 1013 millibar or 760 mm Hg and can be measured using the vacuum regulator The procedure requires a vacuum pump with a capacity of 34
35. trifuge Discard the filtrate and the used 2 ml collection tube Place the QlAamp MinElute column in the 1 5 ml collection tube M Open only one QlAamp MinElute column at a time and take care to avoid generating aerosols 14 QlAamp MinElute Media Handbook 04 2010 Preparation of buffers Addition of carrier RNA to Buffer AL Add 310 ul Buffer AVE to the tube containing 310 ug lyophilized carrier RNA to obtain a solution of 1 ug ul 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 3 times Note that carrier RNA does not dissolve in Buffer AL It must first be dissolved in Buffer AVE and then added to Buffer AL Calculate the volume of Buffer AL carrier RNA mix needed per batch of samples by selecting the number of samples to be simultaneously processed from Table 1 page 15 For larger numbers of samples volumes can be calculated using the following sample calculation nx 0 3 ml y ml y ml x 10 ul ml z ul where n number of samples to be processed simultaneously y calculated volume of Buffer AL z volume of carrier RNA Buffer AVE to add to Buffer AL Gently mix by inverting the tube 10 times To avoid foaming do not vortex Table 1 Volumes of Buffer AL and Carrier RNA Buffer AVE Mix Required for the QlAamp MinElute Media Procedure No samples Vol Buffer AL ml Vol Carrier RNA AVE ul 0 3 3 2 0 6 6
36. ty assurance program the performance of QlAamp MinElute Media Kits is monitored routinely on a lot to lot basis All components are tested separately to ensure highest performance and reliability Product Use Limitations The QlAamp MinElute Media Kit is intended for molecular biology applications This product is not intended for the diagnosis prevention or treatment of 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 QIAGEN 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 or distributor We will credit your account or exchange the product as you wish 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 specific
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