QIAsymphony® DNA Handbook
Contents
1. Co purified RNA may increase A o Asso ratios to values of up to 2 2 Treat samples with RNase A according to the protocol if RNA free DNA is required GlAsymphony DNA Handbook 09 2010 17 35 c 2 pP o o 2 o 2 Protocol General Purification Protocol The following is a general protocol for using GlAsymphony DNA Kits Detailed information for each protocol including volumes and tubes is provided in protocol sheets that can be downloaded at www giagen com QlAsymphonyDNAKits Click on the Resources tab Important points before starting Ensure that you are familiar with operating the QIAsymphony SP Refer to the user manuals supplied with your instrument for operating instructions Optional maintenance is not mandatory for instrument function but is highly rec ommended to reduce risk of contamination Before beginning the procedure read Important Notes starting on page 11 Ensure you are familiar with the protocol sheet corresponding to the procedure you want to use available by clicking on the Resources tab ot www giagen com GlAsymphonyDNAKits Before using a reagent cartridge for the first time check that Buffers QSL1 and QSB1 do not contain a precipitate If necessary remove the troughs containing Buffers QSL1 and QSB1 from the reagent cartridge and incubate for 30 minutes at 37 with occasional shaking to dissolve precipitate Make sure to replac2. For use with the QlAsymphony SP 200 ml Tissue Lysis Buffer for 1000 preps 500 ml Resuspension Buffer RNase A not included 2 5 ml 100 mg ml 7000 units ml solution Magnet for separating magnetic particles in 12 x 1 5 ml or 2 ml tubes Magnet for separating magnetic particles in wells of 96 well plates 2 x 96 Well Microplates FB 96 well blocks with 2 2 ml wells 24 per case Cat no 997002 997004 990332 997024 9013395 19076 19051 19101 36912 36915 19585 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 qiagen com or can be requested from QIAGEN Technical Services or your local distributor GlAsymphony DNA Handbook 09 2010 27 Notes 28 GlAsymphony DNA Handbook 09 2010 Notes GlAsymphony DNA Handbook 09 2010 29 Notes 30 GlAsymphony DNA Handbook 09 2010 Trademarks QIAGEN QlAsymphony QIAGEN Group Limited License Agreement Use of this product signifies the agreement of any purchaser or user of GlAsymphony DNA Kits to the following terms 1 QlAsymphony DNA Kits may be used solely in accordance with the QlAsymphony DNA 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 no
3. 36 38 513 26 36 37 39 46 QSW2 Contains ethanol highly flammable Risk and safety phrases R11 57 16 R11 Highly flammable R20 21 22 Harmful by inhalation in contact with skin and if swallowed R22 Harmful if swallowed R32 Contact with acids liberates very toxic gas R36 Irritating to eyes R36 38 Irritating to eyes and skin R67 Vapors may cause drowsiness and dizziness 57 Keep container tightly closed 513 Keep away from food drink and animal feedingstuffs 516 Keep away from sources of ignition No smoking S26 In case of contact with eyes rinse immediately with plenty of water and seek medical advice S36 Wear suitable protective clothing S36 37 39 Wear suitable protective clothing gloves and eye face protection S46 If swallowed seek medical advice immediately and show container or label 6 GlAsymphony DNA Handbook 09 2010 Proteinase K Contains proteinase 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 Product Use Limitations GlAsymphony DNA Kits are intended for molecular biology applications These products are 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 r
4. SP For use with the QlAsymphony SP Plate carrier for sample input For use with the QlAsymphony SP Primary tube adapter 11 mm for use with the QlAsymphony tube carrier Primary tube adapter 13 mm for use with the QlAsymphony tube carrier Secondary tube adapter for 2 ml screw cap tubes for use with the GlAsymphony tube carrier Cooling adapter for 2 ml screw cap tubes for use in the GlAsymphony Eluate drawer Cooling adapter for EMT racks for use in the GlAsymphony Eluate drawer Cooling adapter for round bottom microtiter plates MTP for use in the GlAsymphony Eluate drawer Cooling adapter for PCR plates for use in the GlAsymphony Eluate drawer Adapter for 2 ml screw cap tubes for use in the GlAsymphony Eluate drawer Cat no 931236 931255 997012 997008 9017660 9241033 9241034 9241032 9018088 9018086 9018085 9018087 9018577 QlAsymphony DNA Handbook 09 2010 Ordering Information Product Sample Prep Cartridges 8 well 336 8 Rod Covers 144 Filter Tips 200 pl 1024 Filter Tips 1500 pl 1024 Tip Disposal Bags 15 Buffer ATL 200 ml Buffer P1 500 ml RNase A 17 500 U 12 Tube Magnet 96 Well Magnet Type A S Blocks 24 Contents 8 well sample prep cartridges for use with the QlAsymphony SP 8 Rod Covers for use with the GlAsymphony SP Sterile Disposable Filter Tips racked 8 x 128 Sterile Disposable Filter Tips racked 8 x 128
5. initialization procedure has finished The power switch is located at the bottom left corner of the QlAsymphony SP Log on to the instrument Ensure the Waste drawer is loaded properly and perform an inventory scan of the Waste drawer including the tip chute and liquid waste Replace the tip disposal bag if necessary Load the required elution rack into the Eluate drawer Do not load a 96 well plate onto Elution slot 4 IF eluates should be cooled use Elution slot 1 with the corresponding cooling adapter When using a 96 well plate make sure that the A1 well of the plate is on the top left corner to avoid sample mixup in downstream analysis Load the required reagent cartridge s and consumables into the Reagents and Consumables drawer Perform an inventory scan of the Reagents and Consumables drawer Place the samples into the appropriate sample carrier and load them into the Sample drawer For Virus Blood applications The tube s containing the internal control Buffer ATE mixture should be placed in slot A of the Sample drawer For more information about preparing the mixture refer to the relevant protocol sheet and see Using an internal control for purification of viral DNA page 14 Using the touchscreen enter the required information for each batch of samples to be processed Enter the following information E Sample information depending on sample racks used E Protoco
6. transferred to sample prep cartridge DNA binds to magnetic particles Magnetic separation Pure high quality DNA GlAsymphony DNA Handbook 09 2010 Magnetic separation uonbapdeud jpnubw dS AuouduiAsyqo eui uo 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 All protocols 8 Sample Prep Cartridges 8 well cartridges cat no 997002 8 Rod Covers cat no 997004 Filter Tips 200 pl and 1500 yl cat nos 990332 and 997024 Sample tubes or racks e g 2 ml sample tubes with screw caps Sarstedt cat no 72 693 or without caps Sarstedt cat no 72 608 or S Blocks GIAGEN cat no 19585 Compatible primary and secondary tube and plate formats are listed at www qiagen com QlAsymphonyDNAKits Labware lists are available under the Resources tab in this page EM Elution tubes or racks Compatible elution tube and rack formats are listed at www qiagen com QlAsymphonyDNAKits Labware lists are available under the Resources fab in this page Phosphate buffered saline PBS may be required for diluting samples E Vortexer E Optional DNase free RNase A if RNA free DNA is required Tissues E Buffer ATL cat no 1
7. we strongly recommend using the cooling position Inventory scan Before starting a run the instrument checks that sufficient consumables for the queued batch es have been loaded into the corresponding drawers Using FIX labware Using liquid level detection LLD for sample transfer allows the use of primary and secondary tubes However this requires certain dead volumes in the respective tubes In order to minimize dead volumes secondary tubes should be used without liquid level detection Specific FIX labware is available e g SAR FIX 472 694 T2 0 ScrewSkirt which can also be selected on the touchscreen of the GlAsymphony SP This tube rack type imposes aspiration restrictions The sample is aspirated at a particular height in the tube that is defined by the volume of sample to be transferred Therefore it is essential to make sure that the volume listed in the labware list is used Sample tubes that can be used with or without liquid level detection and required sample volumes are listed at www giagen com QlAsymphonyDNAKits Labware lists are available under the Resources tab in this page Tubes that are using liquid level detection and tubes that are not using liquid level detection can be processed within one batch run GlAsymphony DNA Handbook 09 2010 13 Preparation of sample material GlAsymphony DNA Kits are suitable for use with a wide range of sample types including human whole blood and buffy coat tissues cult
8. yield a Magnetic particles were not Before starting the procedure ensure that the completely resuspended magnetic particles are fully resuspended Vortex for at least 3 min before use b Frozen blood or buffy Thaw frozen blood samples quickly in a 37 C coat samples were not water bath with mild agitation to ensure thorough mixed properly after mixing thawing GlAsymphony DNA Handbook 09 2010 21 Comments and suggestions d d 9 22 Incomplete sample lysis Incomplete digestion of tissue samples Clogging of pipet tip due to insoluble material Clogging of pipet tip due to sample overload Poor buffy coat preparation when using the buffy coat protocol Low leukocyte count in the whole blood sample Incomplete lysis of cultured cells or bacteria Before use check that Buffers QSL1 and QSB1 do not contain precipitates If necessary remove the troughs containing Buffers QSL1 and QSB1 from the reagent cartridge and incubate for 30 min at 37 C with occasional shaking to dissolve precipitate If the reagent cartridge is already pierced make sure that the trough is closed with a Reuse Seal Strip and incubate the complete reagent cartridge for 30 min at 37 C with occasional shaking in a water bath Ensure that the tissue is completely digested by extending the time of incubation with proteinase K Insoluble material such as undigested cartilage was not removed from the digested s
9. 65 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 Fax 91 630 5145 Technical 91 630 7050 Sweden Orders 020 790282 Fax 020 790582 Technical 020 798328 Switzerland Orders 055 254 22 11 Fax 055 254 22 13 Technical 055 254 22 12 UK Orders 01293 422 911 Fax 01293 422 922 Technical 01293 422 999 USA Orders 800 426 8157 Fax 800 718 2056 Technical 800 DN
10. 9076 B Thermomixer or shaker incubator Cultured cells Buffer P1 cat no 19051 Thermomixer or shaker incubator Bacterial cultures E For Gram negative bacteria Buffer ATL cat no 19076 EH For Gram positive bacteria m Buffer PI cat no 19051 Lysozyme B Thermomixer or shaker incubator Human whole blood viral DNA using internal controls Sample tubes 14 ml 17 x 100 mm polystyrene round bottom tubes from Becton Dickinson cat no 352051 www bd com or 2 ml Sarstedt cat no 72 693 or 72 608 www sarstedt com 10 GlAsymphony DNA Handbook 09 2010 Important Notes Automated purification on the QlAsymphony SP The GlAsymphony SP makes automated sample preparation easy and convenient Samples reagents and consumables and eluates are separated in different drawers Simply load samples reagents provided in special cartridges and preracked consumables in the appropriate drawer before a run Start the protocol and remove purified DNA from the Eluate drawer after processing Refer to the user manual supplied with your instrument for operating instructions We recommend following the maintenance instructions given in the user manual to reduce the risk of contamination The range of protocols available is continually expanding and additional QIAGEN protocols can be downloaded free of charge at www giagen com GIAsymphonyDNAKits Labware lists are available under the Resources tab in this pa
11. A PREP 800 362 7737 QIAGEN Sample amp Assay Technologies
12. IAGEN terms and conditions can be obtained on request and is also provided on the back of our invoices If you have questions about product specifications or performance please call QIAGEN Technical Services or your local distributor see back cover or visit www giagen com Technical Assistance At GIAGEN 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 GIAGEN products If you have any questions or experience any difficulties regarding GlAsymphony DNA Mini or Midi Kits or GIAGEN products in general please do not hesitate to contact us GIAGEN 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 see our Technical Support Center at www qiagen com Support or call one of the QIAGEN Technical Service Departments or local distributors see back cover or visit www giagen com Quality Control In accordance with GIAGEN s ISO certified Quality Management System each lot of GlAsymphony DNA Mini and Midi Kit is tested against predetermined
13. September 2010 QlAsymphony DNA Handbook QlAsymphony DNA Mini Kit GlAsymphony DNA Midi Kit For purification of genomic DNA from human whole blood buffy coat tissues cultured cells bacterial cultures and purification of viral DNA from human whole blood using the QlAsymphony SP 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 QIAGEN sets standards in Purification of DNA RNA and proteins Nucleic acid and protein assays 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 Contents Kit Contents 4 Storage 4 Product Warranty and Satisfaction Guarantee 5 Technical Assistance 5 Quality Control 5 Safety Information 6 Product Use Limitations 7 Introduction 8 Principle and procedure 8 Equipment and Reagents to Be Supplied by User 10 Important Notes 11 Automated purification on the QlAsymphony SP 11 Preparation of sample material 14 Using an internal control for purification of viral DNA 14 Lysis with proteinase K 15 Quantification of DNA Yield of purified DNA 16 Storage and quality of purified DNA 17 Protocol E General Purification Protoc
14. ample prior to starting the QlAsymphony DNA purification procedure To remove insoluble material centrifuge the sample at 300 x g for 1 min as indicated in the protocol and transfer the supernatant to a new sample tube Reduce the sample input volume Ensure that the leukocyte fraction is efficiently harvested If using the buffy coat protocol increase volume of whole blood used and keep the volume of leukocytes harvested constant If the lysate is viscous extend the proteinase K incubation time QlAsymphony DNA Handbook 09 2010 Comments and suggestions DNA does not perform well in downstream applications a Insufficient DNA used Quantify the purified DNA by spectrophotometric in downstream application measurement of the absorbance at 260 nm see the appendix page 24 b Excess DNA used in Excess DNA can inhibit some enzymatic reactions downstream application Quantify the purified DNA by spectrophotometric measurement of the absorbance at 260 nm see the appendix page 24 c Degraded DNA obtained much sample might have been used from tissue samples Overloading with too much sample may lead to insufficient lysis therefore insufficient inactivation of potential DNAses For recommended sample sizes refer to the protocol sheet at www qiagen com QlAsymphonyDNAKits Click on the Resources tab Azo Azgo ratio for purified DNA is low Absorbance reading at To correct for the presence of magnetic par
15. ccur magnetic particles in eluates will not affect most downstream applications If magnetic particles need to be removed before performing downstream applications tubes or racks containing eluates should first be placed in a suitable magnet and the eluates transferred to a clean tube see appendix page 24 IF the Eluate drawer is opened when a batch is running e g if elution racks that contain eluates are removed the run will be paused and an inventory scan of the Eluate drawer will be performed when the drawer is closed Result files are generated for each elution rack If a reagent cartridge is only partially used seal it with the provided Reuse Seal Strips and close tubes containing proteinase K with screw caps immediately after the end of the protocol run to avoid evaporation For more information about storage of partially used reagent cartridges see Storage page 4 Discard used sample tubes racks and waste according to your local safety regulations See page 6 for safety information Clean the GIAsymphony SP Follow the maintenance instructions in the user manuals supplied with your instrument Clean the tip guards regularly to minimize the risk of cross contamination Close the instrument drawers and switch off the QlAsymphony SP GlAsymphony DNA Handbook 09 2010 Troubleshooting Guide This troubleshooting guide may be helpful in solving any problems that may arise For more information see also th
16. e Frequently Asked Questions page at our Technical Support Center www qiagen com FAQ FAGlist 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 qiagen com Comments and suggestions General handling Error message displayed If an error message is displayed during a protocol in the touchscreen run refer to Troubleshooting in the user manual supplied with your instrument Precipitate in reagent trough of opened cartridge a Buffer evaporation Excessive evaporation can lead to increased salt concentration in buffers Discard reagent cartridge Make sure to seal buffer troughs of a partially used reagent cartridge with Reuse Seal Strips when not being used for purification b Storage of reagent Storage of reagent cartridges below 15 may cartridge lead to formation of precipitates If necessary remove the troughs containing Buffers QSL1 and QSB1 from the reagent cartridge and incubate for 30 min at 37 C with occasional shaking to dissolve precipitate Make sure to replace the trough in the correct position If the reagent cartridge is already pierced make sure that the trough is closed with a Reuse Seal Strip and incubate the complete reagent cartridge for 30 min at 37 C with occasional shaking in a water bath Low DNA
17. e the troughs in the correct positions If the reagent cartridge is already pierced make sure that the troughs are sealed with Reuse Seal Strips and incubate the complete reagent cartridge for 30 minutes at 37 C with occasional shaking in a water bath Try to avoid vigorous shaking of the reagent cartridge otherwise foam may be generated which can lead to liquid level detection problems Things to do before starting Before starting the procedure ensure that the magnetic particles are fully resuspended Vortex the trough containing the magnetic particles vigorously for at least 3 minutes before first use Before loading the reagent cartridge remove the cover from the trough containing the magnetic particles and open the enzyme tubes Make sure that the piercing lid is placed on the reagent cartridge or if using a partially used reagent cartridge make sure the Reuse Seal Strips have been removed If samples are bar coded orient samples in the tube carrier so that the bar codes face the bar code reader at the left side of the GlAsymphony SP For information about compatible sample tube and minimum sample volumes for samples in primary and secondary tubes see the corresponding protocol sheet at www qiagen com QlAsymphonyDNAKits Click on the Resources tab GlAsymphony DNA Handbook 09 2010 Procedure 10 Close all drawers and the hood Switch on the QlAsymphony SP and wait until the Sample Preparation screen appears and the
18. ecessary to create an additional Assay Control Set as described in the GlAsymphony Management Console User Manual Note When using the default Virus Blood 200 default IC Assay Control Set for protocols that do not use an internal control the use of Buffer ATE is still required Buffer ATE must be placed in slot A of the Sample drawer Lysis with proteinase K GlAsymphony DNA Kits contain proteinase K which possesses a high specific activity that remains stable over a wide range of temperatures and pH values Enzyme activity is substantially increased at higher temperatures Quantification of DNA Carryover of magnetic particles may affect the absorbance reading at 260 nm of the purified DNA The measured absorbance at 320 nm A320 should be subtracted from all absorbance readings See Quantification of DNA page 24 for more information Note For accurate quantification of DNA by absorbance at 260 nm we recommend diluting the sample in elution buffer Buffer ATE Dilution of the sample in water may lead to inaccurate values GlAsymphony DNA Handbook 09 2010 15 Yield of purified DNA DNA yields depend on the sample type number of nucleated cells in the sample the quality of the starting material and the protocol used for isolation of DNA Table 1 lists typical yields obtained from different sample volumes and types Elution in smaller volumes increases the final DNA concentration in the eluate but slightly reduce
19. ecommend all users of QIAGEN products to adhere to the NIH guidelines that have been developed for recombinant DNA experiments or to other applicable guidelines R36 37 38 Irritating to eyes respiratory system and skin RA2 43 May cause sensitization by inhalation and skin contact 23 Do not breathe vapor 24 Avoid contact with the skin S26 In case of contact with eyes rinse immediately with plenty of water and seek medical advice 536 37 Wear suitable protective clothing and gloves GlAsymphony DNA Handbook 09 2010 7 Introduction QlAsymphony DNA Kits are designed for automated purification of total DNA from human whole blood buffy coat human and animal tissues cultured cells and bacterial cultures as well as viral DNA from human whole blood Proven performance leading magnetic particle technology provides high quality DNA that is suitable for direct use in downstream applications such as amplification or other enzymatic reactions or storage for later use Purified DNA is free of proteins nucleases and other impurities Up to 96 samples are processed in a single run For tissues cultured cells and bacteria protocols manual sample pretreatment is required Principle and procedure GlAsymphony technology combines the speed and efficiency of silica based DNA purification with the convenient handling of magnetic particles Figure 1 The purification procedure is designed to ensure safe and reproducible handling of po
20. ent parameters within a single eluate Compatibility of different internal controls must be validated by the user Internal controls should be diluted in Buffer ATE A total volume of 60 yl internal control Buffer ATE mixture is added per sample When calculating the amount of internal control s and the titer of the processed sample it is necessary to take into consideration the actual volume of elution solution that is used for each sample Small volumes of liquid are lost during transfer and through contact with the magnetic particles Because of this dead volume the initial volume of elution buffers that is aspirated by the QlAsymphony is greater than the volume selected by the operator This ensures that the actual final volume matches the selected final volume The minimum volume of internal control Buffer ATE mixture must include sufficient additional volume to take into account liquid loss due to pipetting and evaporation We recommend preparing fresh mixtures for each run just before use 14 QlAsymphony DNA Handbook 09 2010 The Virus Blood protocol sheet provides detailed information compatible tube formats the initial elution volume and the minimum volume of internal control Buffer ATE mixture Assay Control Sets Assay Control Sets are used with protocols even when the protocol does not use an internal control A default Assay Control Set is preinstalled for each protocol When an internal control is used it might be n
21. ge Loading reagent cartridges into the Reagents and Consumables drawer Reagents for purification of DNA are contained in an innovative reagent cartridge see Figure 2 Each trough of the reagent cartridge contains a particular reagent such as magnetic particles lysis buffer wash buffer or elution buffer Partially used reagent cartridges can be closed with Reuse Seal Strips for later use which avoids generation of waste due to leftover reagents at the end of the purification procedure ee Reuse Seal Strip LA Magneticparticle Piercing lid E s trough Enzyme rack Frame with reagent troughs Slots for screw caps from enzyme tubes Reagent cartridge holder Figure 2 GlAsymphony reagent cartridge The reagent cartridge contains all reagents required for the protocol run Before starting the procedure ensure that the magnetic particles are fully resuspended Remove the magnetic particle trough from the reagent cartridge frame vortex it vigorously for at least 3 minutes and replace it in the reagent cartridge frame before the first use Place the reagent cartridge into the reagent cartridge holder Place the enzyme rack into the reagent cartridge holder Before using a reagent cartridge for the first time place the piercing lid on top of the reagent cartridge Figure 3 GlAsymphony DNA Handbook 09 2010 11 Important The piercing lid is sharp Take care when placing it onto the reagent cart
22. l to be run i e Assay Control Set E Elution volume and output position EH For Virus Blood applications Tube s containing internal control s After information about the batch has been entered the status changes from LOADED to QUEUED As soon as one batch is queued the Run button appears GlAsymphony DNA Handbook 09 2010 19 a x 9 a o 34 x o o 2 2 11 12 13 14 15 16 20 Press the Run button to start the purification procedure All processing steps are fully automated At the end of the protocol run the status of the batch changes from RUNNING to COMPLETED Retrieve the elution rack containing the purified nucleic acids from the Eluate drawer The DNA is ready to use or can be stored at 2 8 C 20 C or 80 C For Virus Blood applications For short term storage of up to 24 h we recommend storing purified nucleic acids at 2 8 C For long term storage of over 24 h we recommend storing purified nucleic acids at 20 We recommend removing the elution rack from the Eluate drawer immediately after the run has finished Depending on temperature and humidity elution racks left in the QlAsymphony SP after the run is completed may experience condensation or evaporation In general magnetic particles are not carried over into eluates If carryover does o
23. of DNA Purity is determined by calculating the ratio of corrected absorbance at 260 nm to corrected absorbance at 280 nm i e Aso A320 Pure DNA has A260 ratio of 1 7 1 9 References GIAGEN maintains a large up to date online database of scientific publications utilizing GIAGEN 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 GlAsymphony DNA Handbook 09 2010 25 Ordering Information Product GlAsymphony DNA Mini Kit 192 GlAsymphony DNA Midi Kit 96 Related products Accessory Trough 10 Reagent Cartridge Holder 2 Sample Carrier plate Qsym Tube Insert 11 mm sample carrier Tube Insert 13 mm sample carrier Qsym Tube Insert 2 ml sample carrier Qsym Cooling Adapter tubes 2 ml Qsym Cooling Adapter EMT Qsym Cooling Adapter MTP RB Qsym Cooling Adapter PCR Qsym Adapter tubes 2 ml Qsym 26 Contents For up to 192 preps of 200 pl each Includes 2 reagent cartridges and enzyme racks and accessories For 96 preps of 1000 pl each Includes 2 reagent cartridges and enzyme racks and accessories For use with the QlAsymphony
24. ol 18 Troubleshooting Guide 21 Appendix Handling Quantification and Determination of Purity of DNA 24 Storage of DNA 24 Quantification of DNA 24 Purity of DNA 25 References 25 Ordering Information 26 GlAsymphony DNA Handbook 09 2010 3 Kit Contents GlAsymphony DNA Kits Mini 192 Midi 96 Catalog no 931236 931255 Number of preps 192 96 Reagent Cartridge 2 2 Enzyme Rack 2 Piercing Lid 2 Buffer ATE 20 ml 20 ml Reuse Seal Set 2 2 Handbook 1 1 For 96 x 1000 pl preps or 144 x 400 pl preps t Contains guanidine salts Not compatible with disinfectants containing bleach See page 6 for safety information Contains sodium azide as a preservative A Reuse Seal Set contains 8 Reuse Seal Strips Storage GlAsymphony DNA Kits should be stored at room temperature 15 25 Do not store reagent cartridges at temperatures below 15 GlAsymphony DNA Kits contain ready to use proteinase K solution that can be stored at room temperature When stored properly the kit is stable until the expiration date on the kit box Partially used reagent cartridges can be stored for a maximum of 2 weeks enabling costefficient use of reagents and more flexible sample processing If a reagent cartridge is partially used replace the cover of the trough containing the magnetic particles seal the buffer troughs with the provided Reuse Seal Strips and close the enzyme tubes with screw caps immediately after the end of the pr
25. otocol run to avoid evaporation To avoid reagent evaporation the reagent cartridge should be open for a maximum of 15 hours including run times at a maximum environmental temperature of 30 Running batches with low sample numbers lt 24 will increase both the time that the reagent cartridge is open and the required buffer volumes potentially reducing the total number of sample preparations possible per cartridge Avoid exposure of the reagent cartridges to UV light e g used for decontamination as exposure may cause accelerated aging of the reagent cartridges and buffers 4 QlAsymphony DNA Handbook 09 2010 Product Warranty and Satisfaction Guarantee GIAGEN guarantees the performance of all products in the manner described in our product literature The purchaser must determine the suitability of the product for its particular use Should any product fail to perform satisfactorily due to any reason other than misuse 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 QIAGEN scientific instruments service products and to products shipped on dry ice Please inquire for more information A copy of Q
26. ridge Make sure to place the piercing lid onto the reagent cartridge in the correct orientation After the magnetic particle trough cover is removed and the enzyme rack tubes are opened screw caps can be stored in dedicated slots see Figure 2 the reagent cartridge is subsequently loaded into the Reagents and Consumables drawer Piercing lid A Figure 3 Easy worktable setup with reagent cartridges Partially used reagent cartridges can be stored until needed again see Storage page 4 Loading plasticware into the Reagents and Consumables drawer Sample prep cartridges 8 Rod Covers both preracked in unit boxes and disposable filtertips 200 pl tips provided in blue racks 1500 yl tips provided in gray racks are loaded into the Reagents and Consumables drawer Note Ensure that the covers of the unit boxes are removed before loading the unit boxes into the Reagents and Consumables drawer Note Both types of tips have filters to help prevent cross contamination Tip rack slots on the GlAsymphony worktable can be filled with either type of tip rack The GlAsymphony SP will identify the type of tips loaded during the inventory scan Note Do not refill tip racks or unit boxes with sample prep cartridges or 8 Rod Covers before starting another protocol run The GlAsymphony SP can use partially used tip racks and unit boxes For the consumables required see the relevant protocol sheet available at ww
27. s overall DNA yield We recommend using an elution volume appropriate for the intended downstream application GlAsymphony DNA Kits RNA and DNA if both are present in the sample If RNA free DNA is required add RNase A to the sample before starting the procedure The final RNase A concentration should be 2 mg ml e g add 4 ml of a 100 mg ml RNase A solution to a 200 ml sample Table 1 Typical genomic DNA yields obtained from a range of sample types Sample Elution volume Typical DNA yield Sample types size pl vg Whole blood 200 yl 200 4 12 400 yl 400 8 24 1000 yl 500 15 45 Buffy coat 200 yl 200 12 40 400 pl 400 24 72 Spleen 25 mg 200 40 80 Liver 25 mg 200 25 50 Muscle 50 mg 200 5 15 Lung 25 mg 200 10 25 Kidney 25 mg 200 15 30 Rat tail 50 mg 200 20 40 Jurkat cells 1x 107 cells 200 60 80 For donors with white blood cell counts of 4 11 x 10 cells ml 1 For buffy coat 5 6x enrichment from blood with a white blood cell count of 4 11 x 10 cells ml 16 QlAsymphony DNA Handbook 09 2010 Storage and quality of purified DNA Purified genomic DNA can be stored at 80 C 20 C or at 2 8 C Purified viral DNA can be stored at 2 8 C for up to 24 h before use in analysis and should be kept at 20 C or 80 C for long term storage GlAsymphony DNA procedures yield pure DNA with A A ratios of 1 7 1 9 Purified DNA is up to 50 kb in size and is suitable for use in all downstream applications
28. specifications to ensure consistent product quality GlAsymphony DNA Handbook 09 2010 5 Safety Information When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information please consult the appropriate material safety data sheets MSDSs These are available online in convenient and compact PDF format at www giagen com Support MSDS aspx where you can find view and print the MSDS for each GIAGEN kit and kit component CAUTION DO NOT add bleach or acidic solutions directly to the sample preparation waste Buffers in the reagent cartridge contain guanidine salts 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 The following risk and safety phrases apply to components of QlAsymphony DNA Kits 9511 Contains guanidine hydrochloride harmful and irritant Risk and safety phrases R22 36 38 13 26 36 46 QSB1 Contains isopropanol and guanidine thiocyanate highly flammable harmful irritant Risk and safety phrases R11 20 21 22 32 36 67 S13 26 36 37 39 46 QSWI Contains guanidine hydrochloride and ethanol highly flammable harmful irritant Risk and safety phrases R11 22
29. t included within this Kit except as described in the GIAsymphony DNA 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 2008 2010 QIAGEN all rights reserved www qiagen com Australia Orders 1 800 243 800 Fax 03 9840 9888 Technical 1 800 243 066 Austria Orders 0800 28 10 10 Fax 0800 28 10 19 Technical 0800 28 10 1 1 Belgium Orders 0800 79612 Fax 0800 7961 1 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 38
30. tentially infectious samples and comprises 4 steps lyse bind wash and elute see flowchart on next page The user can choose between different elution volumes depending on the protocol DNA yields depend on sample type and storage Magnetic ro Magnetic Fast up and down movement of rod cover to release magnetic particles Rod cover ses Reagent 1 and Reagent 2 and magnetic particles magnetic particles Slow up and down movement to collect magnetic particles Transfer gt gt Wel gt gt well 2 Figure 1 Schematic of the QIAsymphony SP principle The QIAsymphony SP processes a sample containing magnetic particles as follows A magnetic rod protected by a rod cover enters a well containing the sample and attracts the magnetic particles The magnetic rod cover is positioned above another well and the magnetic particles are released The QlAsymphony SP uses a magnetic head containing an array of 24 magnetic rods and can therefore process up to 24 samples simultaneously Steps 1 and 2 are repeated several times during sample processing 8 QlAsymphony DNA Handbook 09 2010 GlAsymphony DNA Procedures Blood and buffy coat Tissues and cells Sample plus lysis buffer and magnetic particles transferred to sample prep cartridge a 6 lysis d Transfer cleared lysate to fresh tube Cleared lysate and magnetic particles
31. the purified DNA is to be analyzed e g by fluorescent capillary sequencing the tube containing the eluate should first be applied to a suitable magnetic separator and the eluate transferred to a clean tube E Apply the tube containing the DNA to a suitable magnetic separator e g 12 Tube Magnet cat no 36912 until the magnetic particles are separated If DNA is in microplates apply the microplate to a suitable magnetic separator e g 96 Well Magnet Type A cat no 36915 until the magnetic particles are separated fa suitable magnetic separator is not available centrifuge the tube containing the DNA for 1 minute at full speed in a microcentrifuge to pellet any remaining magnetic particles Once separation is complete carefully withdraw the purified DNA and transfer to a new tube or rack 24 QlAsymphony DNA Handbook 09 2010 Note For accurate quantification of DNA by absorbance at 260 nm we recommend diluting the sample in the corresponding elution buffer Dilution of the sample in water may lead to inaccurate values Elution buffer has high absorbance at 220 nm which can lead to high background absorbance levels if the spectrophotometer is not properly zeroed Evaporation of eluates potentially increases the risk of impact on the measurement especially when low amounts of eluates are used undiluted Extra elution buffer to blank the spectrophotometer is provided in a separate bottle with GlAsymphony DNA Kits Purity
32. ticles 320 nm was not subtracted the eluate an absorbance reading at 320 nm from the absorbance should be taken and subtracted from the readings at 260 nm absorbance readings obtained at 260 nm and and 280 nm 280 nm see the appendix page 24 GlAsymphony DNA Handbook 09 2010 23 Appendix Handling Quantification and Determination of Purity of DNA Storage of DNA Purified genomic DNA can be stored at 80 C 20 C or at 2 8 C Purified viral DNA can be stored at 2 8 for up to 24 hours before use in analysis and should be kept at 20 C or 80 C for long term storage Quantification of DNA The concentration of DNA should be determined by measuring the absorbance at 260 nm A in a spectrophotometer Absorbance readings at 260 nm should fall between 0 1 and 1 0 to be accurate An absorbance of 1 unit at 260 nm corresponds to 50 pg of DNA per milliliter gt 1 50 pg ml The ratio between the absorbance values at 260 nm and 280 nm gives an estimate of purity see Purity of DNA on page 25 Measure the absorbance at 320 280 and 260 nm Subtract the absorbance reading obtained at 320 nm from the readings obtained at 260 and 280 nm to correct for effects of background absorbance Concentration of DNA sample 50 pg ml x Aso A320 x dilution factor Total amount of DNA purified concentration x volume of sample in millilitres Carryover of magnetic particles in the eluate may affect the reading If
33. ured cells and bacterial cultures Depending on the starting material sample pretreatment may be required Samples should be equilibrated to room temperature 15 25 C before starting the run Prevent formation of foam in or on the samples For more information about the automated procedure including information about sample tubes that can be used with specific protocols and specific sample pretreatments see the relevant protocol sheet available at w giagen com GlAsymphonyDNAKits Click on the Resources tab Using an internal control for purification of viral DNA Using a combination of the GlAsymphony DNA Mini Kit and protocol for purification of viral DNA from human whole blood with amplification systems that use an internal control may require the introduction of these internal controls into the purification procedure This allows the efficiency of both sample preparation and the downstream assay to be monitored The amount of internal control added depends on the assay system and the elution volume chosen in the QlAsymphony SP protocol Calculation and validation must be performed by the user Refer to the instructions of the manufacturer of the downstream assay to determine the optimal concentration of internal control Using a concentration other than that recommended lead to invalid or incorrect results if the internal control is used for calculation of titers A mix of several internal controls can be used to analyze differ
34. w qiagen com GlAsymphonyDNAKits Click on the Resources tab For ordering information see page 26 12 QlAsymphony DNA Handbook 09 2010 Loading the Waste drawer Used sample prep cartridges and 8 Rod Covers are placed in empty unit boxes in the Waste drawer Make sure that the Waste drawer contains sufficient empty unit boxes for plastic waste generated during the protocol run Note Ensure that the covers of the unit boxes are removed before loading the unit boxes into the Waste drawer If you are using 8 Rod Cover boxes for collecting used sample prep cartridges and 8 Rod Covers ensure that the box spacer has been removed A bag for discarding used filter tips must be attached to the front side of the Waste drawer Note The presence of a tip disposal bag is not checked by the system Make sure that the tip disposal bag is properly attached before starting a protocol run For more information see the user manual supplied with your instrument A waste container collects liquid waste generated during the purification procedure The Waste drawer can only be closed if the waste container is in place Loading the Eluate drawer Load the required elution rack into the Eluate drawer Do not load a 96 well plate onto Elution slot 4 If eluates should be cooled use Elution slot 1 with the corresponding cooling adapter As long term storage of eluates in the Eluate drawer may lead to evaporation of eluates
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