EZ1 DNA Blood Handbook
Contents
1. Figure 4 Typical EZ1 worktoble First row Elution tubes 1 5 ml are loaded here 2 Second row Tip holders containing filter tips are loaded here 3 Third row Empty unless option of 80 ethanol wash step is selected If 80 ethanol wash step is selected 2 ml tubes containing 1800 pl 80 ethanol are loaded here 4 Fourth row Sample tubes 2 ml are loaded here 5 Reagent cartridges are loaded into the cartridge rack 6 Heating block this is empty for Blood Protocols Data tracking with EZ1 Advanced and EZ1 Advanced XL The EZ Advanced and EZ1 Advanced XL enable complete tracking of a variety of data for increased process control and reliability The EZ1 Kit lot number and expiration dates are entered at the start of the protocol using the Q Card bar code A user ID and the Q Card bar code can be entered manually via the keypad or by scanning bar codes using the handheld bar code reader Sample and assay information can also be optionally entered at the start of the protocol At the end of each protocol run a report file is automatically generated The EZ1 Advanced and EZ1 Advanced XL can store up to 10 result files and the data can be transferred to a PC or directly printed on a printer for ordering information see Equipment and Reagents to Be Supplied by User on page 9 EZ1 DNA Blood Handbook 04 2010 15 To receive report files on a PC the EZ Advanced Communicator softwar2. ae u 4 EZ1 DNA Blood Handbook 04 2010 13 Reagent cartridges Reagents for the purification of nucleic acids from a single sample are contained in a single reagent cartridge Figure 3 Each well of the cartridge contains a particular reagent such as magnetic particles lysis buffer wash buffer or elution buffer Since each well contains only the required amount of reagent generation of waste due to leftover reagent at the end of the purification procedure is avoided a m gt f Figure 3 Ease of setup using reagent cartridges EV A sealed prefilled reagent cartridge Fill levels vary depending on the type of reagent cartridge Loading reagent cartridges into the cartridge rack The cartridge rack itself is labeled with an arrow to indicate the direction in which reagent cartridges must be loaded Worktable The worktable of EZ1 instruments is where the user loads samples and the components of the EZ1 Kits Figure 4 Details on worktable setup are provided in the protocols in this handbook and are also displayed in the vacuum fluorescent display of the EZ1 Advanced and the EZ1 Advanced XL or the liquid crystal display LCD of the BioRobot EZ1 control panel when the user starts worktable setup The display also shows protocol status during the automated purification procedure 14 EZ1 DNA Blood Handbook 04 2010
3. EZ1 DNA Blood Handbook 04 2010 7 EZ1 DNA Blood Procedure Whole blood or buffy coot Magnet Magnet wmm Pure high quality DNA Lysis Magnetic particles added to samples DNA binds magnetic particles Magnetic separation Wash Magnetic separation Elute 8 EZ1 DNA Blood 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 All protocols Sterile RNase free pipet tips Soft paper tissue Water 70 ethanol Pipets Thermomixer heated orbital incubator heating block or water bath For BioRobot EZ1 users BioRobot EZ1 cat no 9000705 and disposables EZ Card see Table 2 page 13 For EZ1 Advanced users EZ Advanced cat no 9001410 or 900141 1 EZ Advanced Card see Table 2 page 13 For EZ1 Advanced XL users EZ Advanced XL cat 9001492 EZ Advanced XL Card see Table 2 page 13 For EZ1 Advanced and EZ1 Advanced XL users For documentation purposes one of the following is required EZ Advanced Communicator software supplied with the EZ Advanced and the EZ1 Advanced XI PC can be connected with up to 4 EZ1 Advanced and EZ1 Advanced XL instruments and monitor cat no for PC and monitor 9016643 EZ Ad
4. 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 giagen com 2009 2010 GIAGEN all rights reserved www qiagen com Australia Orders 1 800 243 800 Fox 03 9840 9888 Technicol 1 800 243 066 Austria Orders 0800 28 10 10 Fax 0800 28 10 19 Technicol 0800 28 10 11 Belgium Orders 0800 79612 Fax 0800 79611 Technical 0800 79556 Brazil Orders 0800 557779 Fox 55 11 5079 4001 Technicol 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 0
5. for 150 pl sample volume or press 3 for 300 pl sample volume Press any key to proceed through the text shown on the display The text summarizes the following steps which describe the loading of the worktable Open the instrument door Invert reagent cartridges 4 times to mix the magnetic particles Then tap the cartridges to deposit the reagents at the bottom of their wells Check that the magnetic particles are completely resuspended Load the reagent cartridges into the cartridge rack Note After sliding a reagent cartridge into the cartridge rack ensure that you press down on the cartridge until it clicks into place Load opened elution tubes into the first row Load tip holders containing filter tips into the second row Note The buffy coat samples should be 75 300 yl depending on the type of sample used see Table 5 page 21 Close the instrument door Press START to start the purification procedure The automated purification procedure takes approximately 20 min When the protocol ends the display shows Protocol finished A Press ENT to generate the report file The EZ1 Advanced and EZ1 Advanced XL can store up to 10 report files Report files can be printed directly on a connected printer or transferred to a computer Open the instrument door Remove the elution tubes containing the purified DNA Discard the sample preparation waste See Figure 3B on page 14 1 See Figure 4 on
6. into the second row 12 Load opened sample tubes containing 200 pl or 350 pl blood into the fourth row 13 Close the instrument door 14 Press START to start the purification procedure The automated purification procedure takes approximately 20 min 15 When the protocol ends the display shows Protocol finished A Press ENT to generate the report file The EZ1 Advanced and EZ1 Advanced XL can store up to 10 report files Report files can be printed directly on a connected printer or transferred to a computer 16 Open the instrument door 17 Remove the elution tubes containing the purified DNA Discard the sample preparation waste 18 A Optional Follow the onscreen instructions to perform UV decontamination of the worktable surfaces 19 To run another protocol press ESC prepare samples and follow the procedure from step 4 onward Otherwise press STOP twice to return to the first screen of the display close the instrument door and switch off the EZ1 instrument 20 Clean the EZ1 instrument Follow the maintenance instructions in the user manual See Figure 3B on page 14 1 See Figure 4 on page 15 t Sample waste contains guanidine salts and is therefore not compatible with bleach See page for safety information 20 EZ1 DNA Blood Handbook 04 2010 Protocol Purification of from Buffy Select the appropriate EZ1 Kit and EZ1 Card according to the sample type and the EZ1 instrument
7. slot of the EZ1 Advanced XL or the EZ1 DNA Blood Card completely into the EZ1 Card slot of the BioRobot EZ1 2 Switch on the EZ1 instrument 3 Press START to start protocol setup A Follow the onscreen instructions for data tracking Note When using the data tracking option ensure that the sample ID follows the same order as the samples on the worktable to avoid data mixup 4 Press 1 or 2 to start worktable setup for the 200 pl Protocol or 350 pl Protocol respectively Note When using the data tracking option with the EZ1 Advanced XL the display shows which kit has been selected according to the Q Card scanned 5 Choose the elution volume press 1 to elute in 50 pl 2 to elute in 100 pl or 3 to elute in 200 pl 6 Proceed through the text shown on the display The text summarizes the following steps which describe the loading of the worktable 7 Open the instrument door EZ1 DNA Blood Handbook 04 2010 19 8 Invert reagent cartridges 4 times to mix the magnetic particles Then tap the cartridges to deposit the reagents at the bottom of their wells Check that the magnetic particles are completely resuspended 9 Load the reagent cartridges into the cartridge Note After sliding a reagent cartridge into the cartridge rack ensure that you press down on the cartridge until it clicks into place 10 Load opened elution tubes into the first row 11 Load tip holders containing filter tips
8. 2 2626 5703 Technical 080 000 7145 Luxembourg Orders 8002 2076 Fax 8002 2073 Technical 8002 2067 Mexico Orders 01 800 7742 639 Fax 01 800 1 122 330 Technical 01 800 7742 639 The Netherlands Orders 0800 0229592 Fax 0800 0229593 Technical 0800 0229602 Norway Orders 800 18859 Fax 800 18817 Technical 800 18712 Singapore Orders 1800 742 4362 Fax 65 6854 8184 Technical 1800 742 4368 Spain Orders 91 630 7050 Fax 91 630 5145 Technical 91 630 7050 Sweden Orders 020 790282 Fax 020 790582 Technical 020 798328 Switzerland Orders 055 254 22 11 Fax 055 254 22 13 Technical 055 254 22 12 UK Orders 01293 422 911 Fax 01293 422 922 Technical 01293 422 999 USA Orders 800 426 8157 Fax 800 718 2056 Technical 800 DNA PREP 800 362 7737 QIAGEN Sample amp Assay Technologies
9. April 2010 EZ1 DNA Blood Handbook EZ1 DNA Blood 200 yl Kit EZ1 DNA Blood 350 yl Kit For automated purification of DNA from blood and buffy coat using EZ1 instruments QIAGEN Sample and Assoy Technologies GIAGEN is the leading provider of innovative sample and assay technologies enabling the isolation and detection of contents of any biological sample Our advanced high quality products and services ensure success from sample to result GIAGEN sets standards in 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 QIAGEN is a member of the Forest Stewardship Council FSC For the production of printed materials including handbooks QIAGEN has a policy to select suppliers that comply with FSC standards for printing processes and well managed forests Contents Contents 4 Storage 4 Product Use Limitations 4 Product Warranty and Satisfaction Guarantee 5 Technical Assistance 5 Safety Information Quality Control Introduction 7 Principle and procedure 7 Equipment and Reagents to Be Supplied by User 9 Important Notes 10 Starting material 10 Precipitate in reagent cartridge 11 Quantification of DNA 11 Working with EZ1 instruments 11 Yield of purified DNA 17 Protocols Purific
10. Z Advanced 9018293 Card DNA blood protocols EZ Advanced XL DNA Blood Preprogrammed card EZ Advanced 9018695 Card XL DNA blood protocols EZ1 Advanced DNA Buffy Preprogrammed card for EZ1 Advanced 9018294 Coat Card DNA buffy coat protocols EZ Advanced XL DNA Buffy Preprogrammed card for EZ Advanced 9018697 Coat Card XL DNA buffy coat protocols Warranty PLUS 2 cat no 9237720 recommended 3 year warranty 1 preventive maintenance visit per year 48 hour priority response all labor travel and repair parts 30 EZ1 DNA Blood Handbook 04 2010 Ordering Information Product Contents Cat no Related products Filter Tips and Holders 50 Disposable Filter Tips 50 Disposable 994900 EZ1 50 Tip Holders additional tips and holders for use with EZ1 Kits 12Tube Magnet Magnet for separating magnetic 36912 particles in 12 x 1 5 ml or 2 ml tubes PC and TFT Monitor 17 PC capable of connection with up to 9016643 4 EZ Advanced EZ Advanced XL instruments Monitor for use with PC Printer Printer for connection with EZ1 9018464 Advanced or EZ1 Advanced XL instrument Printer Accessory Package Accessories for printer connected to 9018465 EZ1 Advanced or EZ1 Advanced XL instrument For up to date licensing information and productspecific disclaimers see the respective QIAGEN kit handbook or user manual QIAGEN kit handbooks and user manuals are available at www giagen com or can be requested from QIAGEN Technical Servi
11. Z1 Card Volume of sample required required eluted DNA 200 pl EZ1 DNA EZ1 Advanced XL 50 yl 100 yl Blood 200 yl DNA Blood or 200 pl Kit EZ1 Advanced 350 yl EZ DNA EZ Advanced XL 50 yl 100 yl DNA Blood Card or EZ1 DNA Blood Blood 350 DNA Blood Card or 200 yl Kit EZ Advanced DNA Blood Card EZ1 DNA Blood Card EZ Advanced XL Cards are only for use with the EZ1 Advanced XL t EZ Advanced Cards are only for use with the EZ1 Advanced t EZ Cards are only for use with the BioRobot EZ1 Important points before starting If using the EZ1 DNA Blood 200 pl Kit or EZ1 DNA Blood 350 pl Kit for the first time read Important Notes page 10 After receiving the kit check the kit components for damage If any kit components are damaged contact QIAGEN Technical Services or your local distributor In the case of liquid spillage refer to Safety Information page 6 Do not use damaged kit components since their use may lead to poor kit performance The protocols include an option to perform 80 ethanol washes instead of washes using the buffer provided in the reagent cartridge This may be advantageous for some downstream applications If this option is selected 2 ml tubes containing 1800 pl 80 ethanol should be placed in row 3 of the worktable by the user see Figure 4 page 15 Follow the instructions given in the onscreen messages The reagent cartridges contain guanid
12. al Workflow of EZ1 operation Insert EZ1 Card into EZ1 Card slot Switch the EZ1 instrument v Follow onscreen messages for data tracking v Follow onscreen messages for worktable setup v Start the protocol v Collect purified nucleic acids v UV decontamination EZ Advanced and EZ1 Advanced XL only 16 EZ1 DNA Blood Handbook 04 2010 Yield of purified DNA DNA yields depend on the sample type number of nucleated cells in the sample and the protocol used for purification of DNA Table 3 shows typical yields obtained from different sample volumes and sample types Table 3 DNA yields obtained from different sample types using EZ1 DNA procedures Sample type Sample amount DNA yield Blood 200 pl 4 8 pg Blood 350 yl 5 12 yg Buffy coat enriched 9x 75 yl 6 5 14 5 yg Buffy coat enriched lt 9x 150 yl 8 14 yg Buffy coat with low 300 pl Up to 14 leukocyte concentration Whole blood with 4 7 x 10 white blood cells ml elution volume 200 yl t Prepared from blood bag 10x enrichment This type of buffy coat preparation tends to result in very efficient leukocyte enrichment EZ1 DNA Blood Handbook 04 2010 17 Protocol Purification of DNA from Whole Blood Select the appropriate EZ1 Kit and EZ1 Cord according to the volume of your whole blood samples and the EZ1 instrument you are using Table 4 Selection of EZ1 Kit and EZ1 Card Volume of blood EZ Kit E
13. ation of DNA from Whole Blood 18 Purification of DNA from Buffy Coat 21 Troubleshooting Guide 25 Appendix A Storage Quantification and Determination of Purity of DNA 27 Appendix B Example of an EZ1 Advanced Report File 28 Ordering Information 30 EZ1 DNA Blood Handbook 04 2010 3 Kit Contents EZ1 DNA Blood Kits Blood 200 pl Blood 350 pl Catalog no 951034 951054 Preps per kit 48 48 Reagent Cartridge Blood 200 pl A8 1023745 Reagent Cartridge Blood 350 yl 48 1023729 Disposable Tip Holders 50 50 Disposable Filter Tips 50 50 Sample Tubes 2 ml 50 50 Elution Tubes 1 5 ml 50 50 Q Card 1 1 Handbook 1 1 For details about the EZ1 Cards to be used with these kits see Table 2 page 13 and visit www qiqgen com The information encoded in the bor code on the Q Cord is needed for reagent data tracking using the EZ Advanced and EZ1 Advanced XL instruments Storage The EZ1 DNA Blood Kits are shipped at ambient temperature All buffers and reagents can be stored at room temperature 15 25 Do not freeze the reagent cartridges When stored properly the reagent cartridges are stable until the expiration date on the QCard Product Use Limitations The EZ1 DNA Blood 200 pl Kit and EZ1 DNA Blood 350 pl Kit 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 t
14. cal Service Departments are staffed by experienced scientists with extensive practical and theoretical expertise in sample and assay technologies and the use of QIAGEN products If you have any questions or experience any difficulties regarding EZ1 DNA Blood Kits 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 see our Technical Support Center at www giagen com Support or call one of the QIAGEN Technical Service Departments or local distributors see back cover or visit www giagen com EZ1 DNA Blood Handbook 04 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 qiagen com support MSDS aspx where you can find view and print the MSDS for each QIAGEN kit and kit component CAUTION DO NOT add bleach or acidic solutions directly to the sample preparation waste Buffers in the reagent cartridges contain
15. ces or your local distributor Visit www qiagen com goto EZ1Advanced to find out more about other EZ1 Kits EZ1 DNA Blood Handbook 04 2010 31 Notes 32 EZ1 DNA Blood Handbook 04 2010 Notes EZ1 DNA Blood Handbook 04 2010 33 Notes 34 EZ1 DNA Blood Handbook 04 2010 Trademarks QIAGEN BioRobot EZ1 QIAGEN Group Limited License Agreement Use of this product signifies the agreement of any purchaser or user of EZ1 DNA Blood Kits to the following terms 1 EZI DNA Blood Kits may be used solely in accordance with the EZ DNA Blood Handbook and for use with components contained in the Kit only GIAGEN grants no license under any of its intellectual property to use or incorporate the enclosed components of this Kit with any components not included within this Kit except as described in the EZ DNA Blood 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 GIAGEN 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 GIAGEN may enforce the prohibitions of this Limited License
16. e needs to be installed The software receives the report file and stores it in a folder that you define After the PC has received the report file you can use and process the file with a LIMS Laboratory Information Management System or other programs An example of the report file is shown in Appendix B page 28 In report files the 6 pipetting channels of the EZ Advanced are named from left to right channels A to F or the 14 pipetting channels of the EZ1 Advanced XL are named from left to right channels 1 14 When scanning a user ID Q Card bar code with the bar code reader confirms data input After the information is displayed for 2 seconds it is automatically stored and the next display message is shown When scanning sample ID assay kit ID or notes a beep confirms data input the information is displayed and a message prompts you to enter the next item of information After scanning sample ID assay kit ID and notes press ENT once to confirm that the information entered is correct If for example a wrong bar code was scanned for one of the samples press ESC and then rescan all sample bar codes according to the onscreen instructions For user ID and notes you can enter the numbers using the keypad or you can easily generate your own bar codes to encode these numbers For details about data tracking and using EZ1 Advanced Communicator software see the EZ Advanced User Manual or the EZ Advanced XL User Manu
17. ed leukocytes to a new tube not supplied First pipet as much of the gray white interface as possible followed by equal portions of the layers directly over and under the interface In some cases it may be helpful to carefully aspirate off part of the plasma layer before harvesting the leukocytes A 1 8 ml whole blood sample should yield approximately 200 yl buffy coat Scaling up the preparation e g to obtain 1 ml buffy from 9 ml whole blood may improve the efficiency of the leukocyte harvest Proceed with DNA purification immediately or store samples at 20 C for purification at a later date DNA purification 4 22 Insert the EZ1 Advanced DNA Buffy Coat Card completely into the EZ1 Advanced Card slot of the EZ1 Advanced or the EZ1 Advanced XL DNA Buffy Coat Card completely into the EZ1 Advanced XL Card slot of the EZ1 Advanced XL or 6 the EZ DNA Buffy Coat Card completely into the EZ1 Card slot of the BioRobot EZ1 EZ1 DNA Blood Handbook 04 2010 12 13 Load opened sample tubes containing buffy coat samples into the fourth row 15 16 18 19 Switch on the EZ1 instrument Press START to start protocol setup A Follow the onscreen instructions for data tracking Note When using the data tracking option ensure that the sample ID follows the same order as the samples on the worktable to avoid data mixup Depending on the sample volume used in step 1 press 1 for 75 pl sample volume press 2
18. ed or EZ1 Advanced XL via the PC Printer serial port Check whether the serial port is set for use with a printer Report file not sent Check whether the PC is connected to EZ to the PC Advanced or EZ1 Advanced XL via the PC Printer serial port Check whether the serial port is set for use with a PC d Wrong Q Card ID If the wrong ID was entered instead of the Q Card ID entered the EZ1 Advanced or EZ1 Advanced XL will not accept the ID and will prompt for the Q Card ID until the correct ID is entered Press STOP twice to go to the main menu Low DNA yield a Magnetic particles not Ensure that you invert the reagent cartridges several completely resuspended times to resuspend the magnetic particles b Insufficient reagent After inverting the reagent cartridges to resuspend aspirated the magnetic particles ensure that you tap the cartridges to deposit the reagents at the bottom of the wells EZ1 DNA Blood Handbook 04 2010 25 Comments and suggestions Varying pipetting To ensure pipetting accuracy it is important to have volumes correct buffer volumes in the reagent cartridges and optimal fit of the filter tips to the tip adapter Ensure that samples are thoroughly mixed and that reagent cartridges have not passed their expiry date Perform regular maintenance as described in the instrument user manual Check the fit of the filter tips regularly as described in the user manual d Frozen blood o
19. er ID is allowed a maximum of 9 characters and that Assay kit ID and Note are allowed a maximum of 14 characters The EZ Advanced XL generates a similar report file containing instrument and protocol information relevant to the EZ1 Advanced XL and information for channels 1 14 REPORT FILE EZ1 Advanced Serial No EZ Advanced ________ 0301F0172 4121 Firmware version _ 2 222 22 2 L V 1 0 0 Installation date of instr __ Jan 05 2008 Weekly maintenance done on Apr 15 2008 Yearly maintenance done _____ Mar 10 2008 Date of last UV run _ Apr 20 2008 Start of last UV run s 16 06 End of last UV run __ s 16 26 Status UV run o k Protocol name Date of run Apr 21 2008 Start of run 12 57 End of run 13 17 Status run o k ErorCode Sample input Vol ul __ 200 Elution volume ul 100 Channel A Sample ID 123456789 Reagent Kit number _ __ 9801601 Reagent Lot number 23456789 Reagent Expiry date _ __________ Jun 14 2009 Assay kit ID l l 848373922 Note 2000 28 EZ1 DNA Blood Handbook 04 2010 Chonnel B Sample ID _ 234567890 Reagent number _ 9801601 Reagent Lot number _ 23456789 Reagent Expiry date Jun 14 2009 Assay ID 836266738 Note l LLL LL LLL Channel C Sample ID 345678901 Reagent Kit number 9801601 Reagent Lot number _ 23456789 Reagent Expiry da
20. ested preparation efficiently Low leukocyte count in Increase whole blood amount and keep the volume of the whole blood sample leukocytes harvested constant 26 EZ1 DNA Blood Handbook 04 2010 Appendix A Storage Quantification and Determination of Purity of DNA Storage of DNA Purified DNA may be stored at 2 8 C for 24 hours or at 20 C for longer storage Quantification of DNA The concentration of DNA should be determined by measuring the absorbance at 260 nm A260 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 ml A260 1 50 pg ml Use buffer of neutral pH e g 10 mM Tris Cl pH 7 0 to dilute the samples and to calibrate the spectrophotometer Carryover of magnetic particles in the eluate may affect the A260 reading but should not affect the performance of the DNA in downstream applications If the purified DNA is to be analyzed 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 see below To quantify DNA isolated using the EZ1 system Apply the tube containing the DNA to a suitable magnetic separator e g QIAGEN 12 Tube Magnet cat no 36912 for 1 minute If a suitable magnetic separator is not available centrifuge the tube containing the DNA for 1 minute at full speed
21. f two choices can be made Choose A blue if using the EZ1 Advanced or the EZ1 Advanced XL choose red if using the BioRobot EZ1 EZ1 DNA Blood Handbook 04 2010 21 Things to do before storting The buffer in well 1 of the reagent cartridge i e well that is nearest to the front of the EZ1 instrument when the reagent cartridge is loaded may form a precipitate upon storage If necessary redissolve by warming at 37 C and then place at room temperature 15 25 B Equilibrote reagent cartridges to room temperature 15 25 before use E IF using fresh buffy coat mix the buffy coat samples thoroughly before loading them onto the EZ1 instrument to ensure homogeneity of the sample f using frozen buffy coat samples thaw the buffy coat samples and equilibrate to room temperature 15 25 Mix the buffy coat samples thoroughly before loading them onto the EZ1 instrument to ensure homogeneity of the sample Procedure Preparation of buffy coat 1 Centrifuge whole blood at 300 x g for 10 min at room temperature 15 25 C Whole blood samples containing a standard anticoagulant EDTA citrate or heparin should be used After centrifugation 3 different fractions are distinguishable the upper clear layer is plasma the intermediate layer is buffy coat containing concentrated leukocytes and the bottom layer contains concentrated erythrocytes Carefully transfer the middle layer containing the concentrat
22. guanidine hydrochloride guanidine thiocyanate which can form highly reactive compounds when combined with bleach IF liquid containing these buffers is spilt clean with suitable laboratory detergent and water If the spilt liquid contains potentially infectious agents clean the affected area first with laboratory detergent and water and then with 176 v v sodium hypochlorite If liquid containing potentially infectious agents is spilt on the EZ1 instrument clean the affected area first with laboratory detergent and water and then with 1 v v sodium hypochlorite followed by water The following risk and safety phrases apply to the components of the 21 DNA Blood Kits Reagent cartridge Contains ethanol guanidine hydrochloride and guanidine thiocyanate highly flammable harmful and irritant Risk and safety phrases R11 20 21 22 32 36 38 13 26 36 37 39 46 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 Quality Control In accordance with QIAGEN s ISO certified Quality Management System each lot of EZ1 DNA Blood Kits is tested against predetermined specifications to ensure consistent product quality R11 Highly flammable R20 21 22 Harmful by inhalation in contact with skin and if swallowed R32 Contact with acids liberates very toxic gas R36 38 Irritating to eyes and ski
23. he handling of the products We recommend all users of QIAGEN products to adhere to the NIH guidelines that have been developed for recombinant DNA experiments or to other applicable guidelines 4 EZ1 DNA Blood Handbook 04 2010 Product Warranty and Satisfaction Guarantee GIAGEN guarantees the performance of all products in the manner described in our product literature The purchaser must determine the suitability of the product for its particular use Should any product fail to perform satisfactorily due to any reason other than misuse QIAGEN will replace it free of charge or refund the purchase price We reserve the right to change alter or modify any product to enhance its performance and design If a QIAGEN product does not meet your expectations simply call your local Technical Service Department or distributor We will credit your account or exchange the product as you wish Separate conditions apply to QIAGEN scientific instruments service products and to products shipped on dry ice Please inquire for more information A copy of QIAGEN terms and conditions can be obtained on request and is also provided on the back of our invoices If you have questions about product specifications or performance please call QIAGEN Technical Services or your local distributor see back cover or visit www qiagen com Technical Assistance At QIAGEN we pride ourselves on the quality and availability of our technical support Our Techni
24. ification from opening of reagent cartridges to elution of nucleic acids with no manual centrifugation steps EZ1 DNA Blood Handbook 04 2010 11 Additional features of the EZ Advanced and the EZ1 Advanced XL include Bar code reading and sample tracking Kit data tracking with the Q Card provided in the kit B UV lamp to help eliminate sample carryover from run to run and to allow pathogen decontamination on the worktable surfaces Note UV decontamination helps to reduce possible pathogen contamination of the EZ Advanced and EZ1 Advanced XL worktable surfaces The efficiency of inactivation has to be determined for each specific organism and depends for example on layer thickness and sample type QIAGEN cannot guarantee complete eradication of specific pathogens EZ1 Cards Protocols for nucleic acid purification are stored on preprogrammed EZ Cards integrated circuit cards The user simply inserts an EZ1 Card into the BioRobot EZ1 or an EZ1 Advanced Card into the EZ1 Advanced or an EZ1 Advanced XL Card into the EZ1 Advanced XL and the instrument is then ready to run a protocol Figure 1 The availability of various protocols increases the flexibility of EZ1 instruments EZ1 Advanced 4 Figure 1 Ease of protocol setup using EZ1 Cards Inserting an EZ1 Card containing protocol into an EZ1 instrument The instrument should only be switched on after an EZ1 Card is inserted EZ1 Cards should not be exchanged while the in
25. in a microcentrifuge to pellet any remaining magnetic particles Once separation is complete carefully withdraw 10 50 yl of isolated DNA and dilute to a final volume of 100 pl in buffer of neutral pH Measure the absorbance at 320 nm and 260 nm Subtract the absorbance reading obtained at 320 nm from the reading obtained at 260 nm to correct for the presence of magnetic particles Concentration of DNA sample 50 pg ml x A260 A320 x dilution factor Total amount of DNA isolated concentration x volume of sample in ml Purity of DNA Purity is determined by calculating the ratio of corrected absorbance at 260 nm to corrected absorbance at 280 nm i e Aoso A320 Pure DNA has As o Ao o ratio of 1 7 1 9 Use buffer of slightly alkaline pH e g 10 mM Tris Cl pH 7 5 to dilute the samples and to calibrate the spectrophotometer 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 the samples are not diluted use water to calibrate the spectrophotometer EZ1 DNA Blood Handbook 04 2010 27 Appendix B Example of an EZ1 Advanced Report File This appendix shows typical report file generated on the EZ Advanced The values for each parameter will differ from the report file generated on your EZ1 Advanced Please note that Us
26. ine salts and are therefore not compatible with disinfecting reagents containing bleach Take appropriate safety measures and wear gloves when handling See page 6 for safety information EZ1 DNA Blood Handbook 04 2010 All steps of the protocol should be performed at room temperature 15 25 During the setup procedure work quickly In some steps of the procedure one of two choices can be made Choose A blue if using the EZ1 Advanced or the EZ1 Advanced XL choose red if using the BioRobot EZ1 Things to do before starting The buffer in well 1 of the reagent cartridge i e the well that is nearest to the front of the EZ1 instrument when the reagent cartridge is loaded may form a precipitate upon storage If necessary redissolve by warming at 37 C and then place at room temperature 15 25 C Equilibrote reagent cartridges to room temperature 15 25 before use E If using fresh blood mix the blood samples thoroughly before loading them onto the EZ instrument to ensure homogeneity of the sample E If using frozen blood samples thaw the blood samples and equilibrate to room temperature 15 25 Mix the blood samples thoroughly before loading them onto the EZ instrument to ensure homogeneity of the sample Procedure 1 Insert the EZ1 Advanced DNA Blood Card completely into the EZ1 Advanced Card slot of the EZ1 Advanced or the EZ1 Advanced XL DNA Blood Card completely into the EZ1 Advanced XL Card
27. n 513 Keep away from food drink and animal feedingstuffs 526 In case of contact with eyes rinse immediately with plenty of water and seek medical advice 536 37 39 Wear suitable protective clothing gloves and eye face protection 546 If swallowed seek medical advice immediately and show this container or label 6 EZ1 DNA Blood Handbook 04 2010 Introduction The EZ1 DNA Blood 200 pl Kit and EZ1 DNA Blood 350 yl Kit are for purification of genomic DNA from whole blood samples and buffy coat Magnetic particle technology provides high quality DNA that is suitable for direct use in downstream applications such as amplification or other enzymatic reactions The EZ1 instruments perform all steps of the sample preparation procedure and the procedure can be scaled up or down allowing purification from varying amounts of starting material Up to samples using BioRobot EZ and EZ1 Advanced instruments or up to 14 samples using the EZ1 Advanced XL instrument are processed in a single run Principle and procedure Magnetic particle technology combines the speed and efficiency of silica based DNA purification with the convenient handling of magnetic particles see flowchart page 8 DNA is isolated from lysates in one step through its binding to the silica surface of the particles in the presence of a chaotropic salt The particles are separated from the lysates using a magnet The DNA is then efficiently washed and eluted in elution buffer
28. page 15 Sample waste contains guanidine salts and is therefore not compatible with bleach See page 6 for safety information EZ1 DNA Blood Handbook 04 2010 23 20 A Optional Follow the onscreen instructions to perform UV decontamination of the worktable surfaces 21 To run another protocol press ESC prepare samples and follow the procedure from step 7 onward Otherwise press STOP twice to return to the first screen of the display close the instrument door and switch off the EZ1 instrument 22 Clean the EZ1 instrument Follow the maintenance instructions in the user manual i w o J H 24 EZ1 DNA Blood Handbook 04 2010 Troubleshooting Guide This troubleshooting guide may be helpful in solving any problems that may arise For more information see also the Frequently Asked Questions page at our Technical Support Center www qiagen com FAQ FAQList aspx The scientists in QIAGEN Technical Services are always happy to answer any questions you may have about either the information and protocols in this handbook or sample and assay technologies for contact information see back cover or visit www qiagen com Comments and suggestions General handling a Error message in Refer to the user manual supplied with your EZ1 instrument display instrument b Report file not printed Check whether the printer is connected to the EZ1 Advanc
29. r buffy Thow frozen blood or buffy coat samples at room coat samples not mixed temperature 15 25 with mild agitation to ensure properly after thawing thorough mixing DNA does not perform well in downstream applications a Insufficient DNA used Quantify the purified DNA spectrophotometric in downstream measurement of the absorbance at 260 nm see application Quantification of DNA page 27 b Excess DNA used in Excess DNA can inhibit some enzymatic reactions downstream application Quantify the purified DNA spectrophotometric measurement of the absorbance at 260 nm see Quantification of DNA page 27 c Inhibition of Some downstream applications may show superior downstream application performance if 80 ethanol wash is performed instead of washes using buffers in the reagent cartridges see page 18 Low A260 A280 ratio for purified nucleic acids Absorbance reading at To correct for the presence of magnetic particles in the 320 nm not subtracted eluate an absorbance reading at 320 nm should be from the absorbance taken and substracted from the absorbance readings readings obtained obtained at 260 nm and 280 nm see Quantification at 260 nm and 280 of DNA page 27 Low DNA yield from buffy coat a Clogging due to Reduce the amount of sample The maximum sample overload recommended amount of cells to use as starting material is 5 x 10 b Poor buffy coat Ensure that the leukocyte fraction is harv
30. strument is switched on 12 EZ1 DNA Blood Handbook 04 2010 EZ instruments should only be switched on after EZ1 Card is inserted Make sure that the EZ1 Card is completely inserted Figure 2 otherwise essential instrument data could be lost leading to a memory error EZ1 Cards should not be exchanged while the instrument is switched on Figure 2 Complete insertion of EZ1 Card The EZ1 Card must be completely inserted before the EZ1 instrument is switched on The EZ1 Kit and EZ1 Card required depend on the purification procedure to be carried out and the EZ1 instrument used Table 2 Table 2 Purification of DNA from various sample types Sample type EZ1 Card required EZ1 Kit required Blood 200 yl EZ1 Advanced XL DNA Blood Card EZ1 DNA Blood EZ1 Advanced DNA Blood Card or 200 yl Kit EZ1 DNA Blood Blood 350 yl EZ1 Advanced XL DNA Blood EZ1 DNA Blood EZ Advanced DNA Blood or 350 yl Kit EZ1 DNA Blood Card Buffy coat EZ1 Advanced XL DNA Buffy Coat EZ1 DNA Blood Card EZ1 Advanced DNA Buffy Coat 350 pl Kit Card or EZ1 DNA Buffy Coat EZ1 Advanced XL Cards are only for use with the EZ1 Advanced XL t EZ1 Advanced Cards are only for use with the EZ Advanced EZ Cards are only for use with the BioRobot EZ1 Visit www qiagen com literature for the latest supplementary protocols which describe how to use EZ1 DNA Kits and 21 DNA Cards for other applications
31. te Jun 14 2009 Assay kit ID 883727832 Notes l l LLL LLL LLL 1000 Channel D Sample ID 456789012 Reagent Kit number _ 5 9801601 Reagent Lot number _ 23456789 Reagent Expiry date _ Jun 14 2009 Assay 763684837 Channel Sample ID _ 567890123 Reagent Kit number gt 2 9801601 Reagent Lot number _ 23456789 Reagent Expiry date Jun 14 2009 Assay 4387728002 Channel Sample ID 678901234 Reagent Kit number _ 5 2 9801601 Reagent Lot number _ 23456789 Reagent Expiry date _ __________ Jun 14 2009 Assay 509389403 Note 50 EZ1 DNA Blood Handbook 04 2010 29 Ordering Information Product Contents Cat no EZ1 DNA Blood 200 yl Kit 48 48 Reagent Cartridges Blood 200 yl 951034 50 Disposable Tip Holders 50 Disposable Filter Tips 50 Sample Tubes 2 ml 50 Elution Tubes 1 5 ml EZ1 DNA Blood 350 yl Kit 48 48 Reagent Cartridges Blood 350 yl 951054 50 Disposable Tip Holders 50 Disposable Filter Tips 50 Sample Tubes 2 ml 50 Elution Tubes 1 5 ml EZ1 Advanced Robotic instrument for automated 9001410 purification of nucleic acids from up to 6 samples using EZ1 Kits 1 year warranty on parts and labor EZ1 Advanced XL Robotic instrument for automated 9001492 purification of nucleic acids from up to 14 samples using EZ1 Kits 1 year warranty on parts and labor Accessories EZ1 Advanced DNA Blood Preprogrammed card for E
32. ting material should be used see Table 1 page 10 for recommended starting volumes Efficiency of buffy coat enrichment depends on the sample preparation procedure used and on the accuracy used when extracting the buffy coat layer Three different protocols are available for purification of genomic DNA from buffy coat on EZ instruments The amounts of recommended starting material for the 3 different protocols are shown in Table 1 Precipitate in reagent cartridge The buffer in well 1 of the reagent cartridge the well that is nearest to the front of the EZ instrument when the reagent cartridge is loaded may form a precipitate upon storage If necessary redissolve by mild agitation at 37 C Quantification of DNA Carryover of magnetic particles may affect the absorbance reading at 260 nm of the purified DNA but should not affect downstream applications The measured absorbance at 320 nm A320 should be subtracted from all absorbance readings See Quantification of DNA page 27 for more information Working with EZ1 instruments The main features of EZ1 instruments include Purification of high quality nucleic acids from 1 6 or 1 14 samples per run Small footprint to save laboratory space Preprogrammed EZ Cards containing ready to use protocols for nucleic acid purification Prefilled sealed reagent cartridges for easy safe and fast setup of EZI instruments Complete automation of nucleic acid pur
33. vanced Communicator Software supplied with the EZ1 Advanced and the EZ1 Advanced XL and your own PC and monitor connection with up to 4 21 Advanced and EZ1 Advanced XL instruments not recommended MH Printer cat no 9018464 and accessory package for printer cat no 2018465 For Blood Protocols E Optional 80 ethanol 2 ml screw capped tubes if optional 80 ethanol wash step is performed see Important points before starting page 18 For Buffy Coat Protocol B 1 5 ml or 2 ml screwcapped tube EZ1 DNA Blood Handbook 04 2010 9 Important Notes Starting material The amounts of starting material for use in EZ1 DNA Blood procedures are shown in Table 1 Table 1 Amounts of starting material for EZ1 DNA Blood procedures Sample type Amount of starting Elution material volume Blood 200 yl or 350 yl 50 yl 100 pl or 200 yl Buffy coat Buffy coat enriched gt 9 50 75 yl 200 yl Buffy coat enriched lt 9x 100 150 pl 200 yl Buffy coat with low leukocyte concentration 200 300 pl 200 yl For each buffy coat protocol the maximum number of cells to use as starting material is 5 x 10 cells t For example 1 ml leukocyte containing fraction harvested from 10 ml centrifuged whole blood 10x enrichment This is recommended for preparation of buffy coat 3 For example from certain leukemia patients or other donors where leukocyte count is low Storage of blood Whole blood samples treated
34. with EDTA ACD or heparin can be used and may be either fresh or frozen Frozen samples should be thawed at room temperature 15 25 with mild agitation before beginning the procedure Yield and quality of the purified DNA depend on storage conditions of the blood Fresher blood samples may yield better results For short term storage up to 10 days collect blood in tubes containing EDTA as an anticoagulant and store the tubes at 2 8 C However for applications requiring maximum fragment size such as Southern blotting we recommend storage at 2 8 C for up to 3 days only as low levels of DNA degradation will occur after this time For longterm storage collect blood in tubes containing a standard anticoagulant preferably EDTA if high molecular weight DNA is required and store the tubes at 70 C 1 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 10 EZ1 DNA Blood Handbook 04 2010 Buffy coat Buffy coat may vary considerably in leukocyte concentration depending upon the number of nucleated cells in the original whole blood sample and the efficiency of leukocyte harvesting during the buffy coat preparation In order to avoid overloading the isolation procedure if using highly enriched buffy coat samples gt 9x enrichment smaller volumes of star
35. you are using Table 5 Selection of EZ1 Kit and EZ1 Card Volume of 21 Kit EZ1 Card Volume of sample required required eluted DNA 75 yl buffy EZ1 DNA EZ Advanced XL 200 pl coat enriched gt 9x Blood 350 yl DNA Buffy Coat or 150 yl buffy Kit Card EZ1 coat enriched lt 9x Advanced DNA or 300 pl buffy Buffy Coat coat with low or EZ DNA leukocyte Bufty Coat Card concentration EZ1 Advanced XL Cards are only for use with the EZ1 Advanced XL t EZ1 Advanced Cards are only for use with the EZ Advanced EZ Cards are only for use with the BioRobot EZ1 Important points before starting E If using the EZ1 DNA Blood Kit or working with buffy coat for the first time read Important Notes page 10 After receiving the kit check the kit components for damage If any kit components are damaged contact GIAGEN Technical Services or your local distributor In the case of liquid spillage refer to Safety Information page 6 Do not use damaged kit components since their use may lead to poor kit performance The reagent cartridges contain guanidine salts and are therefore not compatible with disinfecting reagents containing bleach Take appropriate safety measures and wear gloves when handling See page 6 for safety information All steps of the protocol should be performed at room temperature 15 25 During the setup procedure work quickly some steps of the procedure one o
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