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EZ1® DNA Investigator Handbook - The Murder of Meredith Kercher

1. 9 a 2 224 we LL Protocol Pretreatment for Other Forensic Samples This protocol is designed as a generic protocol for isolation of total genomic and mitochondrial DNA from various forensic samples The protocol describes the preliminary lysis of samples using proteinase K Important point before starting Before beginning the procedure read Important Notes page 15 Things to do before starting As some sample types e g bloodstained fabrics tend to be very absorbent it may be necessary to add a greater volume of digestion buffer to the sample in step 2 To provide sufficient digestion buffer for absorbent samples Buffer G2 can be diluted with distilled water before use If necessary dilute Buffer C2 in distilled water using a ratio of 1 1 i e one volume of Buffer C2 to one volume of distilled water for n 1 samples where n is the number of samples to be digested Use of diluted Buffer G2 does not influence DNA yield or quality Heat a thermomixer heating block or water bath to 56 C for the proteinase digest in step 4 Procedure 1 Place the forensic sample in a 2 ml sample tube 2 Depending on the type of sample follow either step 2a for non absorbent samples or step 2b for absorbent samples 2a Non absorbent samples Add 190 yl Buffer G2 to the sample 2b Absorbent samples Add 190 pl diluted Buffer G2 to the sample Check if
2. 32 EZ1 DNA Investigator Handbook 04 2009 6 necessary flick the tube to remove drops from inside the lid 7 Continue with Protocol DNA Purification Tip Dance Protocol page 46 Using the tip dance protocol there is generally no need to remove solid material from the tube Alternatively to eliminate the risk of clogging the tips remove any solid material from the tube Using forceps press the solid material against the inside of the tube to obtain maximum sample volume The sample volume should be approximately 200 yl Continue with Protocol DNA Purification Trace Protocol page 44 D E a Q eo fe 3 5 EZ DNA Investigator Handbook 04 2009 33 Protocol Pretreatment for Stains on Fabric This protocol is designed for isolation of total genomic and mitochondrial DNA from stains on fabric e g blood or saliva stained fabrics or leather The protocol describes the preliminary lysis of stains on fabric using proteinase K Some samples may require larger volumes for lysis see Protocol DNA Purification Large Volume Protocol page 49 Important point before starting E Before beginning the procedure read Important Notes page 15 Things to do before starting fabrics tend to be very absorbent it is generally necessary to add a greater volume of digestion buffer to the sample in step 2 To provide sufficient digestion buffer for absorbent samples Buffer G2 should be diluted with
3. 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 L Figure 2 Complete insertion of EZ1 Card The EZ1 Card must be completely inserted before the EZ1 instrument is switched on 16 EZ1 DNA Investigator Handbook 04 2009 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 NIE Figure 3 Ease of setup using reagent cartridges EY A sealed prefilled reagent cartridge Fill levels vary depending on the type of reagent cartridge El 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 DNA Investigator Kit Figure 4 Details on worktable setup are provided in the protocols in this handbook and are also displayed in the vacuum fluorescent display VFD of the EZ1 Advanced and 21 Advanced XL or the liquid crystal disp
4. same order as the samples on the worktable to avoid data mixup 13 Close the instrument door 14 Press START to start the purification procedure The automated purification procedure takes 15 20 min 15 When the protocol ends the display shows Protocol finished A Press ENT to generate the report file The EZ1 Advanced and the 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 Retrieve the elution tubes containing the purified DNA The DNA is ready to use or can be stored at 2 8 C for 24 h or at 20 C for longer periods Discard the sample preparation waste IF the purified DNA is to be analyzed by realtime PCR tubes containing eluate should first be applied to a suitable magnetic separator and the eluate transferred to a clean tube in order to minimize the risk of magnetic particle carryover 18 A Optional Follow the onscreen instructions to perform UV decontamination of the worktable surfaces 19 Torun another protocol press ESC prepare samples as described in the relevant protocol 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 supplied with your EZ1 instrument Sample wa
5. Nail Scrapings 28 Pretreatment for Chewing Gum 29 Pretreatment for Cigarette Butts 30 Pretreatment for Postage Stamps 32 Pretreatment for Stains on Fabric 34 E Pretreatment for Human Tissues 36 E Pretreatment for Epithelial Cells Mixed with Sperm Cells 37 Pretreatment for Hair 39 Pretreatment for Bones or Teeth 40 EZ DNA Investigator Handbook 04 2009 3 E Pretreatment for Soil 41 B6 Pretreatment for Other Forensic Samples 42 Purification protocols DNA Purification Trace Protocol 44 E DNA Purification Tip Dance Protocol 46 DNA Purification Large Volume Protocol 49 Troubleshooting Guide 52 Appendix A Purification of Low Amounts of DNA 54 Appendix B Example of an EZ1 Advanced Report File 55 Ordering Information 57 4 71 DNA Investigator Handbook 04 2009 Kit Contents EZ1 DNA Investigator Kit 48 Catalog no 952034 Number of preps 48 Reagent Cartridge DNA Investigator 48 Disposable Tip Holders 50 Disposable Filter Tips 50 Sample Tubes 2 ml 50 Elution Tubes 1 5 ml 50 Buffer G2 1x11 ml Proteinase K 2 x 250 yl Carrier 1 x 310 pg QCard 1 Handbook 1 Contains a guanidine salt Not compatible with disinfectants containing bleach See page 8 for safety information t Use of carrier RNA is optional See Description of protocols page 12 and Appendix A for more information The information encoded in the bar code on the Q Card is needed for reagent data tracking using the EZ1 Adv
6. Reagent Cartridges Virus Mini v2 0 Disposable Tips Disposable Tip Holders Sample Tubes Elution Tubes Buffers For up to date licensing information and productspecific disclaimers see the respective QIAGEN kit handbook or user manual QIAGEN kit handbooks and user manuals are available at www giagen com or can be requested from QIAGEN Technical Services or your local distributor Visit www giagen com goto EZ1Advanced to find out more about other EZ1 Kits EZ1 DNA Investigator Handbook 04 2009 59 Notes 60 71 DNA Investigator Handbook 04 2009 Notes EZ DNA Investigator Handbook 04 2009 61 Notes 62 71 DNA Investigator Handbook 04 2009 Trademarks QIAGEN QlAcard BioRobot EZ1 InhibitEX QIAGEN Group 903 S amp S Biosciences Dacron E du Pont de Nemours and Company FTA Whatman PLC Puritan Hardwood Products Company Limited License Agreement Use of this product signifies the agreement of any purchaser or user of the 21 DNA Investigator Kit to the following terms 1 The EZ1 DNA Investigator Kit may be used solely in accordance with the EZ DNA Investigator Handbook and for use with components contained in the Kit only QIAGEN grants no license under any of its intellectual property to use or incorporate the enclosed components of this Kit with any components not included within this Kit except as described in the 21 DNA Investigator Handbook and additional protocols availa
7. distilled water before use Dilute Buffer G2 in distilled water using a ratio of 1 1 i e one volume of Buffer G2 to one volume of distilled water for n 1 samples where n is the number of samples to be digested Use of diluted Buffer G2 does not influence DNA yield or quality Heat a thermomixer heating block or water bath to 56 C for the proteinase digest in step 4 Y a 5 N 4 2 o Heat a thermomixer heating block or water bath to 95 C for use in step 5 Procedure 1 Place the fabric sample in a 2 ml sample tube 2 Add 190 pl diluted Buffer G2 to the sample Check if the sample has absorbed some or all of the buffer and if necessary add more diluted Buffer G2 to the sample tube until the sample volume is 190 pl Note Prepare diluted Buffer G2 as described above in Things to do before starting 3 10 pl proteinase and mix thoroughly by vortexing for 10 s 4 Incubate at 56 C for 15 min Vortex the tube once or twice during the incubation or place in a thermomixer 34 EZ1 DNA Investigator Handbook 04 2009 5 Recommended Incubate at 95 for 5 min Incubating the sample at 95 may increase the yield of DNA 6 necessary flick the tube to remove drops from inside the lid 7 Continue with Protocol DNA Purification Tip Dance Protocol page 46 Using the tip dance protocol there is generally no need to remove solid material from the tube Alternat
8. or you can easily generate your own bar codes to encode these numbers For details about data tracking and using 21 Advanced Communicator software see the EZ Advanced User Manual or the EZI Advanced XL User Manual Workflow of EZ1 operation Insert EZ1 Card into the EZ1 Card slot v Switch on 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 EZ1 Advanced and EZ1 Advanced XL only EZ DNA Investigator Handbook 04 2009 19 Yield of purified DNA DNA yields depend on the sample type number of nucleated cells in the sample and the protocol used for DNA purification Table 1 shows typical yields for some common reference sample types Table 1 DNA yields from common reference sample types using EZ1 DNA Investigator procedures Sample type Sample amount Protocol DNA yield Blood 10 200 yl Trace or Tip dance 150 ng 2 pg Dried blood 4 x 3 mm dics Tip dance 0 2 0 5 yg Buccal cells 1 swab Tip dance 100 ng 2 pg Whole blood with 3 7 x 10 white blood cells ml elution volume 200 yl 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 and then
9. products EZ1 DNA Blood 200 yl Kit 48 EZ1 DNA Blood 350 pl Contents Cat no 2 Grinding Jars 10 ml 2 Stainless 69985 Steel Grinding Balls 20 mm PC capable of connection with up 9016643 to 4 EZ1 Advanced or EZ Advanced XL instruments Monitor for use with PC Printer for connection with 9018464 EZ Advanced or EZ Advanced XL instrument Accessories for printer connected to 9018465 EZ1 Advanced or EZ Advanced XL instrument For collection and storage of 159201 100 samples 100 GlAcard FTA One Spots For collection and storage of 159203 100 x 2 samples 100 GlAcard FTA Two Spots For collection and storage of 159205 100 x 4 samples 100 GlAcard FTA Four Spots For collection and storage of 159214 25 x 4 samples 25 GlAcard FTA Indicator Four Spots For use with GlAcard FTA Spots 159300 500 ml QlAcard FTA Purification Reagent 48 Reagent Cartridges 951034 50 Disposable Tip Holders 50 Disposable Filter Tips 50 Sample Tubes 50 Elution Tubes 48 Reagent Cartridges 951054 Kit 48 50 Disposable Tip Holders 50 Disposable Filter Tips 50 Sample Tubes 50 Elution Tubes 58 EZ1 DNA Investigator Handbook 04 2009 Ordering Information Product Contents Cat no EZ1 DNA Tissue Kit 48 48 Reagent Cartridges 953034 50 Disposable Tip Holders 50 Disposable Filter Tips 50 Sample Tubes 50 Elution Tubes Buffer G2 Proteinase K EZ1 Virus Mini Kit v2 0 48 For 48 virus nucleic acid preps 955134
10. text shown on the display and start worktable setup The text summarizes the following steps which describe loading of the worktable Wear gloves when loading the required items on the worktable Open the instrument door Invert reagent cartridges twice 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 of the tip rack Load tip holders containing filter tips into the second row of the tip rack Load opened sample tubes containing digested samples into the back row of the tip rack Pretreat the samples following the individual protocols in this handbook 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 Close the instrument door Press START to start the purification procedure The automated purification procedure takes 15 20 min When the protocol ends the display shows Protocol finished A Press ENT to generate the report file The 71 Advanced and the EZ Advanced XL can store up to 10 report files Report files can be printed directly on a connected printer or tran
11. the sample has absorbed some or all of the buffer and if necessary add more diluted Buffer G2 to the sample tube until the sample volume is 190 yl Note Prepare diluted Buffer G2 as described above in Things to do before starting 3 Add 10 pl proteinase and mix thoroughly by vortexing for 10 s 42 EZ1 DNA Investigator Handbook 04 2009 4 Incubate at 56 C for 15 min Vortex the tube once or twice during the incubation or place in a thermomixer 5 If necessary flick the tube to remove drops from inside the lid 6 Continue with Protocol DNA Purification Tip Dance Protocol page 46 Using the tip dance protocol there is generally no need to remove solid material from the tube Alternatively to eliminate the risk of clogging the tips remove any solid material from the tube Using forceps press the solid material against the inside of the tube to obtain maximum sample volume The sample volume should be approximately 200 pl Continue with Protocol DNA Purification Trace Protocol page 44 Q n 1 EX 3 24 A 4 e 3 ek EZ DNA Investigator Handbook 04 2009 43 Protocol DNA Purification Trace Protocol This protocol is designed for isolation of total genomic and mitochondrial DNA from forensic samples that have been pretreated as described in the relevant protocols in this handbook pages 22 43 The protocol describes the simple procedure for setting up the EZ instrum
12. 3 10 pl proteinase K and mix thoroughly by vortexing for 10 s 4 Incubate at 56 C for 15 min Vortex the tube once or twice during the incubation or place in a thermomixer fn Centrifuge the tube briefly to remove drops from inside the lid 6 Remove any solid material from the tube Using forceps press the solid material against the inside of the tube to obtain maximum sample volume The sample volume should be approximately 200 yl 7 Centrifuge the tube at 15 000 x g for 5 min Carefully transfer the supernatant to a new tube without disturbing the sperm cell pellet DNA from epithelial cells can be purified from the tube containing the supernatant following Protocol DNA Purification Trace Protocol page 44 or if the epithelial cell fraction is very dilute Protocol DNA Purification Large Volume Protocol page 49 Note The cell pellet may not be visible EZ DNA Investigator Handbook 04 2009 37 s 95 weds y m pexiw si 5 8 Wash the sperm cell pellet by resuspending the pellet in 500 pl Buffer G2 Centrifuge the tube at 15 000 x g for 5 min and discard the supernatant 9 Repeat step 8 two or three times 10 Add 180 pl Buffer G2 to the pellet and resuspend the pellet 11 Add 10 pl proteinase and 10 pl 1 M DIT and mix thoroughly by vortexing for 10 s 12 Incubate at 56 overnight at 850 rpm in a shaker incubator or thermomixer 13 Centrifuge the tube briefly to remove drops fr
13. EX tablets contact GIAGEN Technical Services see back cover Starting material Up to 0 5 g of soil can be used depending on the type of soil With flocculent soil samples less starting material should be used Important points before starting E Before beginning the procedure read Important Notes page 15 E Proteinase is not required in this protocol This protocol requires InhibitEX tablets contact QIAGEN Technical Services see back cover Things to do before starting M Heat a thermomixer heating block or water bath to 95 C for use in step 2 Procedure 1 Place the soil sample in a 2 ml sample tube 2 Add 900 pl distilled water Resuspend the soil by vortexing and incubate at 95 for 10 min 3 Centrifuge the tube at 4000 x g for 10 min Transfer the supernatant to another 2 ml sample tube and add 190 yl Buffer G2 Mix by vortexing 4 Add 1 InhibitEX tablet and incubate at room temperature 15 25 for 1 min 5 by vortexing and centrifuge at 10 000 x g for 2 min Transfer 200 pl of the supernatant to an EZ1 sample tube if proceeding with Protocol DNA Purification Trace Protocol or transfer 500 pl of the supernatant to an EZ1 sample tube if proceeding with Protocol DNA Purification Large Volume Protocol 6 Continue with Protocol DNA Purification Trace Protocol page 44 or Protocol DNA Purification Large Volume Protocol page 49 EZ1 DNA Investigator Handbook 04 2009 Al
14. Fourth Edition 2009 EZ1 DNA Investigator Handbook For automated purification of DNA from forensic and biosecurity samples using EZ instruments QIAGEN GIAGEN Sample and Assay Technologies GIAGEN is the leading provider of innovative sample and assay technologies enabling the isolation and detection of contents of any biological sample Our advanced high quality products and services ensure success from sample to result 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 qgiagen com Contents Kit Contents 5 Storage 5 Quality Control 6 Product Use Limitations 6 Product Warranty and Satisfaction Guarantee 6 Technical Assistance 7 Safety Information 8 Introduction 9 Principle and procedure 9 Description of protocols 11 Equipment and Reagents to Be Supplied by User 13 Important Notes 15 Starting material 15 Working with EZ1 instruments 15 Yield of purified DNA 20 Precipitate in reagent cartridge 20 Equilibrating reagent cartridges 20 lysis with proteinase K 21 Quantification of DNA 21 Pretreatment protocols Pretreatment for Whole Blood 22 Pretreatment for Dried Blood 23 Pretreatment for Saliva 25 Pretreatment for Forensic Surface and Contact Swabs 26 E Pretreatment for
15. anced or EZI Advanced XL instrument Additional filter tips and tip holders are available separately Additional Buffer G2 and GIAGEN Proteinase K required for some protocols are available separately See page 57 for ordering information Storage The EZ1 DNA Investigator Kit is 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 Q Card Lyophilized carrier RNA is stable until the expiration date on the Q Card when stored at room temperature The ready to use proteinase solution is stable for up to one year after delivery when stored at room temperature EZ DNA Investigator Handbook 04 2009 5 Quality Control In accordance with QIAGEN s ISO certified Quality Management System each lot of 71 DNA Investigator Kits is tested against predetermined specifications to ensure consistent product quality Functional QC testing ensures that the EZ1 DNA Investigator Kit meets the high standards required by forensic scientists Product Use Limitations The EZ1 DNA Investigator Kit is intended for molecular biology applications This product is neither intended for the diagnosis prevention or treatment of a disease nor has it been validated for such use either alone or in combination with other products All due care and attention should be exercised in the handli
16. barriers Distilled water For BioRobot EZ1 users BioRobot EZ instrument cat no 9000705 and disposables B EZ DNA Investigator Card cat 9016387 For EZ1 Advanced users E EZ Advanced instrument cat no 9001410 B EZ Advanced DNA Investigator Card cat no 9018302 For EZ1 Advanced XL users B EZ Advanced XL instrument cat no 9001492 E EZ Advanced XL DNA Investigator Card cat no 9018699 For EZ1 Advanced and EZ1 Advanced XL users For documentation purposes one of the following is required E EZ Advanced Communicator Software supplied with the EZ1 Advanced and EZ1 Advanced XL instruments PC can be connected with up to 4 EZ1 Advanced and EZ1 Advanced XL instruments and monitor cat no for PC and monitor 9016643 B EZ Advanced Communicator Software supplied with the EZ1 Advanced and EZ1 Advanced XL instruments and your own PC and monitor connection with up to 4 EZ Advanced and EZ1 Advanced XL instruments not recommended Wb Printer cat no 9018464 and accessory package for printer cat no 9018465 For purification of DNA from dried blood Filter paper e g GlAcard FTA Spots see Ordering Information page 57 B Manual paper punch 3 e g Harris UNI CORE 3 00 mm Punch Kit 4 cat no 159331 or equivalent punch with cutting mat EZ DNA Investigator Handbook 04 2009 13 For purification of DNA from forensic surface and contact swabs Plastic swabs with c
17. ble at www giagen com 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 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 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 2009 QIAGEN all rights reserved 63 www qiagen com Australia Orders 03 9840 9800 Fax 03 9840 9888 Technical 1 800 243 066 Austria Orders 0800 28 10 10 Fax 0800 28 10 19 Technical 0800 28 10 11 Belgium Orders 0800 79612 Fax 0800 79611 Technical 0800 79556 Brazil Orders 0800 557779 Fax 55 11 5079 4001 Technical 0800 557779 Canada Orders 800 572 9613 Fax 800 713 5951 Technical 800 DNA PREP 800 362 7737 China Orders 0086 2 1 3865 3865 Fax 0086 21 3865 3965 Technical 800 988 0325 800 988 0327 Denmark Orders 80 885945 Fax 80 885944 Te
18. ce Protocol page 44 24 EZ1 DNA Investigator Handbook 04 2009 Protocol Pretreatment for Saliva This protocol is designed for isolation of total genomic and mitochondrial DNA from saliva samples The protocol describes the preliminary lysis of saliva samples using proteinase K Starting material The amount of saliva should not exceed 50 yl For larger volumes if the sample is very dilute see Protocol DNA Purification Large Volume Protocol page 49 Important point before starting E Before beginning the procedure read Important Notes page 15 Things to do before starting Heat a thermomixer heating block or water bath to 56 C for the proteinase digest in step 4 Procedure 1 Place up to 50 pl saliva in a 2 ml sample tube 2 Add 140 190 pl Buffer G2 to the sample to bring the total volume up to 190 yl 3 10 pl proteinase K and mix thoroughly by vortexing for 10 s 4 Incubate at 56 C for 15 min Vortex the tube once or twice during the incubation or place in a thermomixer fn If necessary flick the tube to remove drops from inside the lid 6 Continue with Protocol DNA Purification Trace Protocol page 44 EZ DNA Investigator Handbook 04 2009 25 Protocol Pretreatment for Forensic Surface and Contact Swabs This protocol is designed for isolation of total genomic and mitochondrial DNA from forensic surface and contact swabs The protocol describes the preliminary lysis of fore
19. ced 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 Retrieve the elution tubes containing the purified DNA The DNA is ready to use or can be stored at 2 8 C for 24 h or at 20 C for longer periods Discard the sample preparation waste IF the purified DNA is to be analyzed by realtime PCR tubes containing eluate should first be applied to a suitable magnetic separator and the eluate transferred to a clean tube in order to minimize the risk of magnetic particle carryover 18 A Optional Follow the onscreen instructions to perform UV decontamination of the worktable surfaces 19 Torun another protocol press ESC prepare samples as described in the relevant protocol 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 supplied with your EZ1 instrument Sample waste contains guanidine salts and is therefore not compatible with bleach See page 8 for safety information EZ DNA Investigator Handbook 04 2009 51 10204014 YNA Troubleshooting Guide This troubleshooting guide may be helpful in solving any problems that may arise For more information see also the F
20. chnical 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 02 33430 420 Fax 02 33430 426 Technical 800 787980 Japan Telephone 03 6890 7300 Fax 03 5547 0818 Technical 03 6890 7300 Korea South Orders 1544 7145 Fax 1544 7146 Technical 1544 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 65 67775366 Fax 65 67785177 Technical 65 67775366 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 e UK Orders 01293 422 911 Fax 01293 422 922 Technical 01293 422 999 e o o e e USA Orders 800 426 8157 Fax 800 718 2056 Technical 800 DNA PREP 800 362 7737 QIAGEN TE Sample amp Assay Technologies
21. dbook 04 2009 Important Notes Starting material The amount of starting material for use in EZ1 DNA Investigator procedures can vary greatly depending on the amount of DNA in the sample Specific guidance for starting amounts is given in the individual protocols EZ1 instruments can process 200 yl pretreated samples using the trace protocol page 44 or the tip dance protocol page 46 for DNA purification With the large volume protocol page 49 up to 500 yl pretreated samples can be processed Working with EZ1 instruments The main features of the EZ1 instruments include Purification of high quality nucleic acids from 1 6 or 1 14 samples per run H Small footprint to save laboratory space E Preprogrammed EZ Cards containing ready to use protocols for nucleic acid purification Prefilled sealed reagent cartridges for easy safe and fast setup of EZ1 instruments Complete automation of nucleic acid purification from opening of reagent cartridges to elution of nucleic acids with no manual centrifugation steps Additional features of the EZ1 Advanced and EZ1 Advanced XL include E Bar code reading and sample tracking Kitdata 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 EZ1 Advanced and EZ1 Advanced XL worktable s
22. e protocol describes the addition of carrier RNA to sample lysates For full details refer to Kishore R Hardy W R Anderson V J Sanchez N A and Buoncristiani M P H 2006 Optimization of DNA extraction from low yield and degraded samples using the BioRobot EZ1 and BioRobot M48 J Forensic Sci Vol 51 No 5 1055 The procedure has not been thoroughly tested and optimized by QIAGEN Important point before starting E Before beginning the procedure read Important Notes page 15 Things to do before starting Add 310 pl nuclease free water or TE buffer to the tube containing carrier RNA 310 pg to obtain a solution of 1 pg ypl Dissolve the carrier RNA thoroughly divide it into single use aliquots and store at 70 C Procedure 1 Pretreat samples according to the appropriate pretreatment protocol given on pages 22 43 of this handbook 2 Add 1 pl of thawed carrier RNA solution 1 pg to each lysate It is not necessary to incubate the carrier RNA and sample lysate 3 Continue immediately with Protocol DNA Purification Trace Protocol Protocol DNA Purification Tip Dance Protocol or Protocol DNA Purification Large Volume Protocol on pages 44 46 or 49 of this handbook 54 EZ1 DNA Investigator Handbook 04 2009 Appendix B Example of an EZ1 Advanced Report File This appendix shows a typical report file generated on the EZ1 Advanced The values for each parameter will differ from the report f
23. ead mill When using the Tissuelyser transfer the bone sample and the ball into the grinding jar Pour liquid nitrogen into the grinding jar over the ball and bone fragments Allow the temperature to equilibrate i e liquid nitrogen stops boiling Decant the excess liquid nitrogen close the grinding jar with the lid and transfer it to the Tissuelyser Grind the bone at 30 Hz for 1 min or until the bone is pulverized grinding times depend on type condition and size of bone Place 150 200 mg of powdered bone into a 2 ml microcentrifuge tube 3 Add 600 700 pl 0 5 M EDTA pH 8 3 and incubate at 37 C for 24 48 h After incubation set the temperature to 56 C for the next incubation step Add 20 pl QIAGEN Proteinase K and incubate at 56 C for 3 h 5 Centrifuge at 6000 rpm for 4 min Transfer 200 pl of the supernatant to an EZ1 sample tube if proceeding with Protocol DNA Purification Trace Protocol or transfer 500 pl of the supernatant to an EZ1 sample tube if proceeding with Protocol DNA Purification Large Volume Protocol 6 Continue with Protocol DNA Purification Trace Protocol page 44 or Protocol DNA Purification Large Volume Protocol page 49 40 EZ1 DNA Investigator Handbook 04 2009 Protocol Pretreatment for Soil This protocol is designed for isolation of total genomic and mitochondrial DNA from soil The protocol describes the preliminary lysis of soil samples and adsorption of inhibitors using Inhibit
24. eat a thermomixer heating block or water bath to 56 C for the proteinase digest in step 4 M Heat a thermomixer heating block or water bath to 95 C for use in step 5 26 EZ1 DNA Investigator Handbook 04 2009 Procedure 1 Carefully cut or break off the end part of the swab or brush into a 2 ml sample tube using an appropriate tool e g scissors Add 290 pl of diluted Buffer G2 to the sample Note Prepare diluted Buffer G2 as described in Things to do before starting Add 10 pl proteinase K and mix thoroughly by vortexing for 10 s If processing brush samples centrifuge the tube briefly at 10 000 x g for 30 s to force the brush to the bottom of the tube Incubate at 56 C for 15 min Vortex the tube 1 2 times during the incubation or place in a thermomixer Recommended Incubate at 95 for 5 min Incubating the sample at 95 may increase the yield of DNA If necessary flick the tube to remove drops from inside the lid Continue with Protocol DNA Purification Tip Dance Protocol page 46 Using the tip dance protocol there is generally no need to remove the swab or brush from the tube Alternatively to eliminate the risk of clogging the tips remove the swab or brush from the tube Using forceps press the swab or brush against the inside of the tube to obtain maximum sample volume The sample volume should be approximately 200 yl Continue with Protocol DNA Purification Trace Protocol pa
25. ent and starting a run Important points before starting Ifusing the EZ1 DNA Investigator Kit for the first time read Important Notes page 15 E The reagent cartridges contain guanidine salts and are therefore not compatible with disinfecting reagents containing bleach See page 8 for safety information MH Perform all steps of the protocol at room temperature 15 25 During the setup procedure work quickly E In some steps of the procedure one of 2 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 E f reagent cartridges have been stored at 2 8 C equilibrate to operating temperature before use See Equilibrating reagent cartridges page 20 Remove any solid material from the sample tube Using forceps press the solid material against the inside of the tube to obtain maximum sample volume The lysis buffer in the reagent cartridge may form a precipitate during storage If necessary redissolve by warming at 37 C and then place at room temperature 15 25 C Procedure l Insert A the EZ Advanced DNA Investigator Card completely into the EZ1 Advanced Card slot of the EZ1 Advanced or the EZ1 Advanced XL DNA Investigator Card completely into the EZ1 Advanced XL Card slot of the EZ1 Advanced XL or the EZ1 DNA Investigator Card completely into the EZ1 Card slot of the BioRobot EZ1 2 Switch on the EZ1 in
26. es not specifically included in this handbook the Protocol Pretreatment for Other Forensic Samples page 42 provides a generic protocol that can serve as a starting point for optimizing pretreatment for other sample types DNA purification protocols There are 3 DNA purification protocols which can be used in conjunction with the pre treatment protocols Within each protocol the user can specify elution in water or TE buffer with elution volumes of 40 yl EZ1 Advanced XL only 50 yl 100 pl or 200 pl The standard Protocol DNA Purification Trace Protocol page 44 can be used with all sample types In the Protocol DNA Purification Tip Dance Protocol page 46 the filter tip moves back and forth relative to the worktable platform while pipetting This enables processing of solid materials such as swabs fabrics blood discs or cigarette butts directly in the sample tube There is generally no need for prior centrifugation to remove solid materials that could clog the tip However when processing fluffy sample material such as cotton wool we recommend removing solid material if you cannot process a replicate sample or the sample material is precious The Protocol DNA Purification Large Volume Protocol page 49 enables fully automated processing of starting volumes up to 500 pl This not only allows efficient DNA purification from dilute samples with low concentrations of DNA such as diffuse stains but also enables purificati
27. esigned for isolation of total genomic and mitochondrial DNA from forensic cigarette butt samples The protocol describes the preliminary lysis of saliva and epithelial cells on paper from cigarette butts using proteinase Starting material Use of approximately 1 cm paper from the end of the cigarette or filter is recommended Important point before starting Before beginning the procedure read Important Notes page 15 Things to do before starting As cigarette butts tend to be absorbent it is generally necessary to add a greater volume of digestion buffer to the sample in step 2 To provide sufficient digestion buffer for absorbent samples Buffer G2 should be diluted with distilled water before use Dilute Buffer G2 in distilled water using a ratio of 1 1 i e one volume of Buffer G2 to one volume of distilled water for n 1 samples where n is the number of samples to be digested Use of diluted Buffer G2 does not influence DNA yield or quality Heat a thermomixer heating block or water bath to 56 C for the proteinase digest in step 4 M Heat a thermomixer heating block or water bath to 95 C for use in step 5 Procedure 1 Place the cigarette butt sample in a 2 ml sample tube 2 Add 190 pl diluted Buffer G2 to the sample Check if the sample has absorbed some or all of the buffer and if necessary add more diluted Buffer G2 to the sample tube until the sample volume is 190 yl Note Prepare di
28. et of 4 discs to a 2 ml sample tube 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 EZ DNA Investigator Handbook 04 2009 23 A Add 190 pl diluted Buffer G2 to the sample Check if the sample has absorbed some or all of the buffer and if necessary add more diluted Buffer G2 to the sample tube until the sample volume is 190 pl Note Prepare diluted Buffer G2 as described in Things to do before starting 5 Add 10 pl proteinase and mix thoroughly by vortexing for 10 s Incubate at 56 for 15 min Vortex the tube once or twice during the incubation or place in a thermomixer 7 Recommended Incubate at 95 for 5 min Incubating the sample at 95 may increase the yield of DNA 8 necessary flick the tube to remove drops from inside the lid TO 2 TO E i a Continue with Protocol DNA Purification Tip Dance Protocol page 46 Using the tip dance protocol there is generally no need to remove solid material from the tube Alternatively to eliminate the risk of clogging the tips remove any solid material from the tube Using forceps press the solid material against the inside of the tube to obtain maximum sample volume The sample volume should be approximately 200 yl Continue with Protocol DNA Purification Tra
29. fication Trace Protocol page 44 28 EZ1 DNA Investigator Handbook 04 2009 Protocol Pretreatment for Chewing Gum This protocol is designed for isolation of total genomic and mitochondrial DNA from forensic chewing gum samples The protocol describes the preliminary lysis of chewing gum samples using proteinase K Starting material Use of up to 40 mg of chewing gum cut into small pieces is recommended Important point before starting E Before beginning the procedure read Important Notes page 15 Things to do before starting Heat a thermomixer heating block or water bath to 56 C for the proteinase digest in step 4 Procedure l Place the chewing gum sample in a 2 ml sample tube 2 Add 190 pl Buffer G2 to the sample 3 10 pl proteinase K and mix thoroughly by vortexing for 10 s 4 Incubate at 56 C for 15 min Vortex the tube once or twice during the incubation or place in a thermomixer a If necessary flick the tube to remove drops from inside the lid 6 Remove any solid material from the tube Using forceps press the solid material against the inside of the tube to obtain maximum sample volume The sample volume should be approximately 200 pl Continue with Protocol DNA Purification Trace Protocol page 44 EZ1 DNA Investigator Handbook 04 2009 29 wing Buimay gt E 5 pS 89 9 Protocol Pretreatment for Cigarette Butts This protocol is d
30. g the BioRobot EZ1 Things to do before starting E f reagent cartridges have been stored at 2 8 C equilibrate to operating temperature before use See Equilibrating reagent cartridges page 20 Remove any solid material from the sample tube Using forceps press the solid material against the inside of the tube to obtain maximum sample volume BW The lysis buffer in the reagent cartridge may form a precipitate during storage If necessary redissolve by warming at 37 C and then place at room temperature 15 25 EZ DNA Investigator Handbook 04 2009 49 020104g uonp2ylund YNA DNA Purification Large Volume Protocol Procedure Insert A the EZ1 Advanced DNA Investigator Card completely into EZ1 Advanced Card slot of the EZ1 Advanced or the EZ1 Advanced XL DNA Investigator Card completely into the EZ1 Advanced XL Card slot of the EZ1 Advanced XL or amp the EZ1 DNA Investigator 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 4 Press 3 for Large Volume protocol 5 Choose the elution buffer and volume press 1 to elute in water or 2 to elute in TE buffer Then press 1 2 or 3 or 4 EZ1 Advanced XL only to select the elution volume 6 Press any key to proceed through the text shown on the display and start
31. ge 44 EZ DNA Investigator Handbook 04 2009 27 Protocol Pretreatment for Nail Scrapings This protocol is designed for isolation of total genomic and mitochondrial DNA from forensic nailscraping samples The protocol describes the preliminary lysis of nail scraping samples using proteinase Starting material The amount of biological sample material should not exceed 40 mg Important point before starting E Before beginning the procedure read Important Notes page 15 Things to do before starting Heat a thermomixer heating block or water bath to 56 C for the proteinase digest in step 4 Procedure 1 Place nail scraping sample in a 2 ml sample tube 2 Add 190 pl Buffer G2 to the sample 3 Add 10 pl proteinase and mix thoroughly by vortexing for 10 s 4 Incubate at 56 C for 15 min Vortex the tube once or twice during the incubation or place in a thermomixer If necessary flick the tube to remove drops from inside the lid 6 Continue with Protocol DNA Purification Tip Dance Protocol page 46 Using the tip dance protocol there is generally no need to remove solid material from the tube Alternatively to eliminate the risk of clogging the tips remove any solid material from the tube Using forceps press the solid material against the inside of the tube to obtain maximum sample volume The sample volume should be approximately 200 yl Continue with Protocol DNA Puri
32. hat you tap the cartridges to deposit the reagents at the bottom of the wells EZ1 DNA Investigator Handbook 04 2009 Comments and suggestions c Purified DNA stored in water Elute in TE buffer instead of water Elution in TE buffer gives comparable performance and provides increased stability for long term storage of small amounts of purified DNA d Varying pipetting volumes To ensure pipetting accuracy it is important that buffer volumes in the reagent cartridges are correct and that the filter tips fit optimally 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 DNA does not perform well in downstream applications a Insufficient DNA used in If possible repeat the downstream downstream applications application using more eluate b Excess DNA used in Excess DNA can inhibit some enzymatic downstream application reactions Dilute the eluate or use less in the downstream application Quantify the purified DNA by measurement of the absorbance using an appropriate method EZ DNA Investigator Handbook 04 2009 53 Appendix A Purification of Low Amounts of DNA This protocol is designed for purification of isolation of total genomic and mitochondrial DNA from forensic samples that contain 100 ng DNA Th
33. ifficult to quantify using a spectrophotometer In addition eluates prepared with carrier RNA may contain much more carrier RNA than target nucleic acids We recommend using quantitative amplification methods to determine yields Carryover of magnetic particles may affect the absorbance reading at 260 nm A of the purified DNA but should not affect downstream applications The measured absorbance at 320 nm should be subtracted from all absorbance readings To eliminate carried over magnetic particles the tube containing the eluate should first be applied to a suitable magnetic separator and the eluate transferred to a clean tube EZ1 DNA Investigator Handbook 04 2009 21 Protocol Pretreatment for Whole Blood This protocol is designed for isolation of total genomic and mitochondrial DNA from fresh or frozen blood Starting material This protocol is designed for processing up to 200 pl of human whole blood o E 7 E Storage of blood samples Whole blood samples treated 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 C 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
34. ile generated on your EZ1 Advanced Please note that User ID is allowed a maximum of 9 characters and that Assay kit ID and Note are allowed a maximum of 14 characters The EZ1 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 21 Advanced ________ 0301F0172 Users en m Ir sr ete a 4121 Firmware version _______________ V 1 0 0 Installation date of inst Jan 05 2008 Weekly maintenance done on ___ _ Apr 15 2008 Yearly maintenance done on __ Mar 10 2008 Date of last UV run 2 2 L Apr 20 2008 Start of last UV run _______________ 16 06 End of last UV run 16 26 Status Vr nz oe oe ee LL o k Protocol name ____ DNA Investigator ee ee ee Trace Date of run ______________ Aprl 21 2008 Start of run ___________________ 12 57 Endofrun 13 31 UTE ne cR s sh e cere o k Error Code _______________________ Sample input Vol ul _______________ 200 Elution volume ul 100 Channel A Sample ID ________________ 123456789 Reagen Kit number 2 ___ 9801301 Reagen Lot number ____________ 23456789 Reagent Expiry date 1208 Assay 848373922 Note 2000 EZ1 DNA Investigator Handbook 04 2009 55 Channel B Sample ID 234567890 Reagen Kit number 2 2 2 2 9801301 Reagen Lot number 2 2 23456789 Reagent Ex
35. inter for ordering information see Equipment and Reagents to Be Supplied by User on page 13 18 71 DNA Investigator Handbook 04 2009 To receive report files on a PC the 21 Advanced Communicator software 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 a report file is shown in Appendix B page 55 In report files the 6 pipetting channels of the EZ1 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 or Q Card bar code with the bar code reader a beep 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
36. ion 5 Homogenize the sample by pipetting up and down several times Transfer the supernatant to a new 2 ml sample tube 6 Continue with Protocol DNA Purification Tip Dance Protocol page 46 Using the tip dance protocol there is generally no need to remove solid material from the tube Alternatively to eliminate the risk of clogging the tips remove any solid material from the tube Remove large pieces of insoluble material and centrifuge at 300 x g for 1 min The sample volume should be approximately 200 yl Continue with Protocol DNA Purification Trace Protocol page 44 36 71 DNA Investigator Handbook 04 2009 Protocol Pretreatment for Epithelial Cells Mixed with Sperm Cells This protocol is designed for purification of total genomic and mitochondrial DNA from epithelial cells mixed with sperm cells The protocol describes the preliminary lysis of samples using proteinase and dithiothreitol Important points before starting E Before beginning the procedure read Important Notes page 15 As some sample types e g fabrics tend to be very absorbent it may be necessary to add a greater volume of digestion buffer to the sample in step 2 Things to do before starting Heat a thermomixer heating block or water bath to 56 C for the proteinase digest in steps 4 and 12 Procedure 1 Place the forensic sample in a 1 5 ml or 2 ml sample tube 2 Add 190 pl Buffer G2 to the sample
37. iratory system and skin R42 43 May cause sensitization by inhalation and skin contact 513 Keep away from food drink and animal feedingstuffs 523 Do not breathe spray 524 Avoid contact with 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 5336 37 39 Wear suitable protective clothing gloves and eye face protection S46 If swallowed seek medical advice immediately and show container or label 8 EZ1 DNA Investigator Handbook 04 2009 Introduction EZ1 instruments and the EZ1 DNA Investigator Kit reproducibly automate purification of genomic DNA from 1 6 samples EZ1 Advanced and BioRobot EZ1 or 1 14 samples EZ1 Advanced Xl encountered in forensic human identity and biosecurity applications Purification is efficient and purified DNA performs well in downstream analyses such as quantitative PCR and STR analysis with high signal to noise ratios Magnetic particle technology provides high quality DNA that is suitable for direct use in downstream applications such as STR analysis or other enzymatic reactions EZ1 instruments perform all steps of the sample preparation procedure and the user can choose sample input volumes of 200 yl or 500 yl allowing purification from varying amounts of starting material Up to 6 samples BioRobot EZ1 EZ1 Advanced or up to 14 samples EZ1 Advanced XL are processed in a single run Princip
38. ively to eliminate the risk of clogging the tips remove any solid material from the tube Using forceps press the solid material against the inside of the tube to obtain maximum sample volume The sample volume should be approximately 200 yl Continue with Protocol DNA Purification Trace Protocol page 44 2 5 o E ax a a A EZ1 DNA Investigator Handbook 04 2009 35 Protocol Pretreatment for Human Tissues This protocol is designed for isolation of total genomic and mitochondrial DNA from human tissues The protocol describes the preliminary lysis of tissues using proteinase K Important point before starting E Before beginning the procedure read Important Notes page 15 Things to do before starting Heat a thermomixer heating block or water bath to 56 C for the proteinase digest in step 4 Procedure 1 Transfer the tissue sample into a 1 5 ml screw capped tube not supplied 2 Add 190 yl Buffer G2 Ensure that tissue pieces are fully submerged in Buffer G2 3 Add 10 pl proteinase K solution and mix by tapping the tube gently 4 Incubate at 56 C until the tissue is completely lysed Vortex 2 3 times per hour during incubation to disperse the sample or place in a thermomixer shaking water bath or on a rocking platform lysis time varies depending on the type of tissue processed lysis is usually complete in 3 h lysis overnight is possible and does not influence the preparat
39. lay LCD of the BioRobot EZ1 control panel when the user starts worktable setup The display also shows protocol status during the automated purification procedure EZ1 DNA Investigator Handbook 04 2009 17 Figure 4 Typical EZ1 worktable 1 First row Elution tubes 1 5 ml are loaded here 2 Second row Tip holders containing filter tips are loaded here 3 Third row Tip holders containing filter tips are loaded here In some protocols this row is empty or loaded with 2 ml Sarstedt tubes 4 Fourth row Sample tubes 2 ml are loaded here 5 Reagent cartridges are loaded into the cartridge rack 6 Heating block with 2 ml tubes in the reagent cartridges for lysis Data tracking with the EZ1 Advanced and EZ1 Advanced XL The EZ1 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 date 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 the 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 pr
40. le 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 10 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 the user s choice of either water or TE buffer The user can choose elution volumes of 40 pl EZ1 Advanced XL only 50 yl 100 pl or 200 yl EZ DNA Investigator Handbook 04 2009 9 EZ1 Investigator Procedure Blood or pretreated sample Magnet Magnet Pure high quality DNA Lysis Magnetic particles added to samples DNA binds to magnetic particles Magnetic separation Wash Magnetic separation Elute 10 71 DNA Investigator Handbook 04 2009 Description of protocols This handbook contains two types of protocols E Pretreatment protocols detail the preliminary steps such as proteinase digestion prior to processing on the EZ1 instrument DNA purification protocols describe setting up the EZ1 instrument and starting a fully automated run Pretreatment protocols Since the type of samples that can be processed using the EZ1 DNA Investigator Kit can vary greatly there is also a variety of different pretreatments optimized for specific sample types For sample typ
41. luted Buffer G2 as described above in Things to do before starting 3 10 pl proteinase and mix thoroughly by vortexing for 10 s Incubate at 56 for 15 min Vortex the tube once or twice during the incubation or place in a thermomixer 5 Recommended Incubate at 95 for 5 min Incubating the sample at 95 may increase the yield of DNA 30 EZ1 DNA Investigator Handbook 04 2009 6 necessary flick the tube to remove drops from inside the lid 7 Continue with Protocol DNA Purification Tip Dance Protocol page 46 Using the tip dance protocol there is generally no need to remove solid material from the tube Alternatively to eliminate the risk of clogging the tips remove any solid material from the tube Using forceps press the solid material against the inside of the tube to obtain maximum sample volume The sample volume should be approximately 200 yl Continue with Protocol DNA Purification Trace Protocol page 44 Q Co Q 7 wo a EZ1 DNA Investigator Handbook 04 2009 31 5 5 o D fe a Protocol Pretreatment for Postage Stamps This protocol is designed for isolation of total genomic and mitochondrial DNA from postage stamps The protocol describes the preliminary lysis of postage stamp samples using proteinase K Starting material Use of a 0 5 2 5 cm piece of postage stamp is recommended Important point before
42. mples prepared in this manner are cheaper and safer to transport A disc 3 mm diameter punched out from filter paper stained with dried blood contains white blood cells from approximately 5 pl whole blood we recommend using 4 punched out discs as starting material Jg D Em o Important point before starting E Before beginning the procedure read Important Notes page 15 Things to do before starting E As filter paper tends to be absorbent it is generally necessary to add a greater volume of digestion buffer to the sample in step 4 To provide sufficient digestion buffer for absorbent samples Buffer G2 should be diluted with distilled water before use Dilute Buffer C2 in distilled water using a ratio of 1 1 i e one volume of Buffer G2 to one volume of distilled water for n 1 samples where n is the number of samples to be digested Use of diluted Buffer G2 does not influence DNA yield or quality Heat a thermomixer heating block or water bath to 56 C for the proteinase K digest in step Heat a thermomixer heating block or water bath to 95 C for use in step 7 Procedure 1 Collect 70 pl of each blood sample onto a ring marked on filter paper Allow the blood to air dry Either untreated blood or blood containing an anticoagulant EDTA ACD or heparin can be used 2 For each dried blood sample use the manual paper punch to cut out four 3 mm diameter discs 3 Transfer each s
43. nformation MH Perform all steps of the protocol at room temperature 15 25 During the setup procedure work quickly n some steps of the procedure one of 2 choices 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 E f reagent cartridges have been stored at 2 8 C equilibrate to operating temperature before use See Equilibrating reagent cartridges page 20 The lysis buffer in the reagent cartridge may form a precipitate during storage If necessary redissolve by warming at 37 C and then place at room temperature 15 25 Procedure l Insert A the EZ Advanced DNA Investigator Card completely into the EZ1 Advanced Card slot of the EZ1 Advanced or the EZ1 Advanced XL DNA Investigator Card completely into the EZ1 Advanced XL Card slot of the EZ1 Advanced XL or the EZ1 DNA Investigator Card completely into the EZ1 Card slot of the BioRobot EZ 1 46 71 DNA Investigator Handbook 04 2009 10 11 12 Switch on the EZ1 instrument Press START to start protocol setup A Follow the onscreen instructions for data tracking Press 2 for Trace TD protocol aye Choose the elution buffer and volume press 1 to elute in water or 2 to elute in TE Then press 1 2 or 3 or 4 EZ1 Advanced XL only to select the elution volume Press any key to proceed through the
44. ng of the products We recommend all users of QIAGEN products to adhere to the NIH guidelines that have been developed for recombinant DNA experiments or to other applicable guidelines Product Warranty and Satisfaction Guarantee GIAGEN guarantees the performance of all products in the manner described in our product literature The purchaser must determine the suitability of the product for its particular use Should any product fail to perform satisfactorily due to any reason other than misuse 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 GIAGEN terms and conditions can be obtained on request and is also provided on the back of our invoices If you have questions about product specifications or performance please call QIAGEN Technical Services or your local distributor see back cover or visit www qiagen com 6 71 DNA Investigator Handbook 04 2009 Technical Assistance At GIAGEN we pride ourselves on the quality and availability of our technical support Our Technical Ser
45. nsic surface and contact swabs using proteinase K Starting material Swabs may be processed on the same day as collection or stored for future processing While storage at 20 C is recommended DNA of suitable quality for single copy gene amplification has been documented from swabs stored at room temperature for 24 months Important points before starting This protocol has been tested using the following swab types plastic swabs with cotton or Dacron tips Puritan applicators with plastic shafts and cotton or Dacron tips are available from Hardwood Products Company www hwppuritan com item nos 25 806 1PC and 25 806 1PD and from Daigger www daigger com cat nos EF22008D and EF22008DA Nylon cytology brushes and other swab types may also be used E Before beginning the procedure read Important Notes page 15 Things to do before starting BW Allow the swab or brush to air dry for at least 2 h after sample collection E As swabs tend to be absorbent it is generally necessary to add a greater volume of digestion buffer to the sample in step 2 To provide sufficient digestion buffer for absorbent samples Buffer G2 should be diluted with distilled water before use Dilute Buffer G2 in distilled water using a ratio of 1 1 i e one volume of Buffer G2 to one volume of distilled water for 1 samples where n is the number of samples to be digested Use of diluted Buffer G2 does not influence DNA yield or quality H
46. om inside the lid DNA from sperm cells can now be purified from this tube 14 Continue with Protocol DNA Purification Trace Protocol page 44 The two tubes in which the epithelial and sperm cells have been separated are now ready for DNA purification 38 EZ1 DNA Investigator Handbook 04 2009 Protocol Pretreatment for Hair This protocol is designed for isolation of total genomic and mitochondrial DNA from the root ends of plucked hair samples The protocol describes the preliminary lysis of hair samples using proteinase K and dithiothreitol DTT Starting material We recommend using 0 5 1 cm from the root ends of plucked hair samples Important point before starting Before beginning the procedure read Important Notes page 15 Things to do before starting Heat a thermomixer heating block or water bath to 56 C for the proteinase digest in steps 4 and 6 Procedure l Place the hair sample in a 2 ml sample tube 2 Add 180 pl Buffer G2 to the sample 3 10 pl proteinase and 10 pl DTT solution and mix thoroughly by vortexing for 10 s 4 Incubate at 56 C for at least 6 h Vortex the tube once or twice during the incubation or place in a thermomixer 5 Add another 10 pl proteinase and 10 pl DTT solution and mix thoroughly by vortexing for 10 s 6 Incubate at 56 C for at least 2 h or until the hair samples are completely dissolved 7 If necessary flick the tube to remove dr
47. on from samples that require larger volumes for thorough lysis The ability to process larger sample volumes with the same elution volume as the standard trace protocol enables higher yields of more concentrated DNA for greater sensitivity in downstream applications EZ1 DNA Investigator Handbook 04 2009 11 The protocol for purification of low amounts of DNA in Appendix A describes the optional use of carrier RNA in the purification procedure Carrier RNA enhances binding of DNA to the silica surface of the magnetic particles especially if the sample contains low amounts of DNA 100 ng Recently published data suggest that addition of carrier RNA enables more efficient isolation of low amounts of DNA from forensic samples and may for some sample types provide improved DNA yields Addition of carrier RNA to sample lysates did not interfere with downstream STR analyses This protocol has not been thoroughly tested and optimized by QIAGEN 12 EZ1 DNA Investigator Handbook 04 2009 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 Bb Thermomixer heating block or water bath Vortexer E Pipets and pipet tips to prevent cross contamination we strongly recommend the use of pipet tips with aerosol
48. ops from inside the lid 8 Continue with Protocol DNA Purification Tip Dance Protocol page 46 Using the tip dance protocol there is generally no need to remove solid material from the tube Alternatively to eliminate the risk of clogging the tips remove any solid material from the tube Using forceps press the solid material against the inside of the tube to obtain maximum sample volume Continue with Protocol DNA Purification Trace Protocol page 44 EZ DNA Investigator Handbook 04 2009 39 E o E wn a Protocol Pretreatment for Bones or Teeth This protocol is designed for isolation of total genomic and mitochondrial DNA from bones or teeth The protocol describes the preliminary grinding decalcification using EDTA and lysis of bone or teeth samples using proteinase K Starting material The amount of biological sample material should not exceed 200 mg Important points before starting E Before beginning the procedure read Important Notes page 15 E time to familiarize yourself with the Tissuelyser before starting this protocol See the Tissuelyser Handbook Things to do before starting Heat a thermomixer heating block or water bath to 37 C for the decalcification in step 3 Procedure 1 Remove and discard the bone or teeth surfaces Grind the remaining bone or tooth root to a fine powder using the TissueLyser system or an equivalent b
49. otton or Dacron tips Puritan applicators with plastic shafts and cotton or Dacron tips are available from Hardwood Products Company www hwppuritan com item nos 25 806 1 and 25 806 1PD and from Daigger www daigger com cat nos EF22008D and EF22008DA Nylon cytology brushes and other swab types may also be used For purification of DNA from chewing gum B Forceps For purification of DNA from human tissues 1 5 ml screwcapped tubes For purification of DNA from epithelial cells mixed with sperm cells Buffer G2 cat 1014636 1M dithiothreitol DTT H Microcentrifuge Forceps For purification of DNA from hair QIAGEN Proteinase cat 19131 or 19133 DIT solution 1 M dithiothreitol 10 mM sodium acetate pH 5 2 For purification of DNA from bones or teeth QIAGEN Proteinase cat no 19131 or 19133 0 5 M EDTA pH 8 3 Liquid nitrogen 2 ml microcentrifuge tubes Microcentrifuge Tissuelyser cat no 85300 with the Grinding Jar Set S Steel cat no 69985 or an equivalent bead mill For purification of DNA from soil 8 InhibitEX tablets contact QIAGEN Technical Services see back cover E Microcentrifuge For DNA purification large volume protocol Buffer MTL contact QIAGEN Technical Services see back cover This is not a complete list of suppliers and does not include many important vendors of biological supplies P y Imp g pP 14 71 DNA Investigator Han
50. piry date 1208 Assay kit ID 836266738 Channel Sample ID 345678901 Reagen Kit number 2 2 2 9801301 Reagen Lot number ____________ 23456789 Reagent Expiry date _______________ 1208 Assay 883727832 Noles ___________ ___ __ ____ _ 1000 Channel D Sample ID nnn 456789012 Reagen Kit number _____________ 9801301 Reagen Lot number ____________ 23456789 Reagent Expiry date 1208 Assay kit ID _______________ 763684837 Bol o Channel E Sample ID 567890123 Reagen Kit number 2 2 2 9801301 Reagen Lot number ____________ 23456789 Reagent Expiry date _______________ 1208 Assay kit ID _______________ 4387728002 Mole DI Lum Channel F Sample ID 678901234 Reagen Kit number 2 2 2 9801301 Reagen Lot number ____________ 23456789 Reagent Expiry date _______________ 1208 Assay kit ID 509389403 Note 50 56 71 DNA Investigator Handbook 04 2009 Ordering Information Product Contents Cat no EZ1 DNA Investigator Kit 48 48 preps Reagent Cartridges 952034 Disposable Tip Holders Disposable Filter Tips Sample Tubes Elution Tubes Buffers and Reagents includes Certificate of Analysis 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 EZ1 Advanced Robotic instrument for automated 9001410
51. place at room temperature 15 25 Equilibrating reagent cartridges If reagent cartridges have been stored at 2 8 C they must be equilibrated to operating temperature before use Place the reagent cartridge into a shaker incubator and incubate at 30 40 with mild agitation for at least 2 hours before use If precipitates are visible at the bottom of the wells redissolve by incubating at 30 40 C with mild agitation for a further 2 hours Do not use the reagent cartridges if the precipitates do not redissolve 20 EZ1 DNA Investigator Handbook 04 2009 Lysis with proteinase K The EZ1 DNA Investigator Kit contains proteinase K which is the enzyme of choice for lysis buffers used in EZ1 DNA Investigator protocols Proteinase K is a recombinant protein expressed in Pichia pastoris and is particularly suitable for short digestion times It possesses a high specific activity and remains stable over a wide range of temperatures and pH values with substantially increased activity at higher temperatures The activity of the proteinase K solution is 600 mAU ml solution or 40 mAU mg protein This activity provides optimal results in EZ1 DNA Investigator protocols Additional QIAGEN Proteinase is required for purification of DNA from hair bones or teeth see page 57 for ordering information Quantification of DNA Depending on the sample type the yields of DNA obtained in the purification proce dure may be below 1 pg and therefore d
52. purification of nucleic acids using EZ Kits 1 year warranty on parts and labor EZ1 Advanced XL DNA Preprogrammed card for EZ1 9018699 Investigator Card Advanced XL DNA Investigator protocols on the EZ1 Advanced XL EZ1 Advanced DNA Preprogrammed card for EZ1 9018302 Investigator Card Advanced DNA Investigator protocols EZ1 DNA Investigator Card Preprogrammed card for BioRobot 9016387 EZ1 DNA Investigator protocols Accessories Filter Tips and Holders 50 Disposable Filter Tips 994900 21 50 50 Disposable Tip Holders additional tips and holders for use with EZ1 Kits 12 Tube Magnet Magnet for separating magnetic 36912 particles in 12 x 1 5 ml or 2 ml tubes Buffer G2 260 ml Lysis buffer for EZ1 DNA procedures 1014636 QIAGEN Proteinase K 2 ml 2 ml 600 mAU nml solution 19131 QIAGEN Proteinase 10 ml 10 ml 3600 mAU ml solution 19133 Tissuelyser II Universal laboratory mixer mill 85300 disruptor 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 EZ1 DNA Investigator Handbook 04 2009 57 Ordering Information Product Grinding Jar Set S Steel 2 x 10 ml PC and TFT Monitor 17 Printer Printer Accessory Package QlAcard One Spot 100 QlAcard FTA Two Spots 100 QlAcard FTA Four Spots 100 QlAcard FTA Indicator Four Spots 25 QlAcard FTA Purification Reagent 500 ml Related
53. relevant protocols in this handbook pages 22 43 This protocol describes the simple procedure for setting up the EZ instrument and starting a run Starting material Using this protocol up to 500 pl of pretreated sample can be processed This not only allows efficient DNA purification from dilute samples with low concentrations of DNA such as diffuse stains but also enables purification from samples that require larger volumes for thorough lysis For these samples increase the amount of Buffer C2 as required The amount of proteinase K generally does not need to be increased The ability to process larger sample volumes with the same elution volume as the standard trace protocol enables higher yields of more concentrated DNA for greater sensitivity in downstream applications Important points before starting f using the 71 DNA Investigator Kit for the first time read Important Notes page 15 The reagent cartridges contain guanidine salts and are therefore not compatible with disinfecting reagents containing bleach See page 8 for safety information E Perform all steps of the protocol at room temperature 15 25 During the setup procedure work quickly This protocol requires extra Buffer MTL contact QIAGEN Technical Services see back cover n some steps of the procedure one of 2 choices be made Choose A blue if using the EZ1 Advanced or the EZ1 Advanced XL choose red if usin
54. requently Asked Questions page at our Technical Support Center www giagen com FAQ FAQList aspx The scientists in QIAGEN Technical Services are always happy to answer any questions you may have about either the information and protocols in this handbook or sample and assay technologies for contact information see back cover or visit www giagen com Comments and suggestions General handling a Error message in instrument display b Report file not printed c Report file not sent to the PC d Wrong QCard ID entered Low DNA yield a Magnetic particles not completely resuspended b Insufficient reagent aspirated 52 Refer to the user manual supplied with your EZ instrument Check whether the printer is connected to the EZ1 Advanced or EZ1 Advanced XL via the PC Printer serial port Check whether the serial port is set for use with a printer Check whether the PC is connected to the EZ1 Advanced or EZ1 Advanced XL via the PC Printer serial port Check whether the serial port is set for use with a PC IF the wrong ID was entered instead of the QCard ID the EZI Advanced EZl 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 Ensure that you invert the reagent cartridges several times to resuspend the magnetic particles After inverting the reagent cartridges to resuspend the magnetic particles ensure t
55. sferred to a com puter Open the instrument door EZ1 DNA Investigator Handbook 04 2009 47 02010ug 22UuDq dij 17 Retrieve the elution tubes containing the purified DNA The DNA is ready to use or can be stored at 2 8 C for 24 h or at 20 C for longer periods Discard the sample preparation waste If the purified DNA is to be analyzed by real time PCR tubes containing eluate should first be applied to a suitable magnetic separator and the eluate transferred to a clean tube in order to minimize the risk of magnetic particle carryover 18 A Optional Follow the onscreen instructions to perform UV decontamination of the worktable surfaces 19 Torun another protocol press ESC prepare samples as described in the relevant protocol 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 supplied with your EZ1 instrument Sample waste contains guanidine salts and is therefore not compatible with bleach See page 8 for safety information 48 EZ1 DNA Investigator Handbook 04 2009 Protocol DNA Purification Large Volume Protocol This protocol is designed for isolation of total genomic and mitochondrial DNA from forensic samples that have been pretreated as described in the
56. starting E Before beginning the procedure read Important Notes page 15 Things to do before starting E postage stamps tend to be absorbent it is generally necessary to add a greater volume of digestion buffer to the sample in step 2 To provide sufficient digestion buffer for absorbent samples Buffer G2 should be diluted with distilled water before use Dilute Buffer G2 in distilled water using a ratio of 1 1 i e one volume of Buffer G2 to one volume of distilled water for n 1 samples where n is the number of samples to be digested Use of diluted Buffer G2 does not influence DNA yield or quality Heat a thermomixer heating block or water bath to 56 C for the proteinase digest in step 4 E Heat a thermomixer heating block or water bath to 95 C for use in step 5 Procedure 1 Place the piece of postage stamp in a 2 ml sample tube 2 Add 190 pl diluted Buffer G2 to the sample Check if the sample has absorbed some or all of the buffer and if necessary add more diluted Buffer G2 to the sample tube until the sample volume is 190 yl Note Prepare diluted Buffer G2 as described above in Things to do before starting 3 Add 10 pl proteinase and mix thoroughly by vortexing for 10 s Incubate at 56 for 15 min Vortex the tube once or twice during the incubation or place in a thermomixer 5 Recommended Incubate at 95 for 5 min Incubating the sample at 95 may increase the yield of DNA
57. ste contains guanidine salts and is therefore not compatible with bleach See page 8 for safety information EZ DNA Investigator Handbook 04 2009 45 DNA Purification Tip Dance Protocol Protocol DNA Purification Tip Dance Protocol This protocol is designed for isolation of total genomic and mitochondrial DNA from forensic samples that have been pretreated as described in the relevant protocols in this handbook pages 22 43 This protocol describes the simple procedure for setting up the EZ instrument and starting a run In the tip dance protocol the filter tip moves back and forth relative to the worktable platform while pipetting This enables processing of solid materials such as swabs fabrics blood discs or cigarette butts directly in the sample tube There is generally no need for prior centrifugation to remove solid materials that could clog the tip However when processing fluffy sample material such as cotton wool we recommend removing solid material if you cannot process a replicate sample or the sample material is precious Using forceps press the solid material against the inside of the tube to obtain maximum sample volume Important points before starting E f using the EZ1 DNA Investigator Kit for the first time read Important Notes page 15 The reagent cartridges contain guanidine salts and are therefore not compatible with disinfecting reagents containing bleach See page 8 for safety i
58. strument 3 Press START to start protocol setup A Follow the onscreen instructions for data tracking 4 Press 1 for Trace protocol 5 Choose the elution buffer and volume press 1 to elute in water or 2 to elute in TE buffer Then press 1 2 or 3 or 4 EZ1 Advanced XL only to select the elution volume 6 Press any key to proceed through the text shown on the display and start worktable setup The text summarizes the following steps which describe loading of the worktable Wear gloves when loading the required items on the worktable 44 EZ1 DNA Investigator Handbook 04 2009 7 Open the instrument door 8 Invert reagent cartridges twice 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 rack 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 of the tip rack 11 Load tip holders containing filter tips into the second row of the tip rack 12 Load opened sample tubes containing digested samples into the back row of the tip rack Pretreat the samples following the individual protocols in this handbook Note When using the data tracking option ensure that the sample ID follows the
59. the reagent cartridges 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 If liquid containing potentially infectious agents is spilt on the EZ1 Advanced XL EZ1 Advanced or BioRobot EZ1 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 components of the EZ1 DNA Investigator Kit Reagent cartridge Contains ethanol guanidine hydrochloride and guanidine thiocyanate highly flammable harmful and irritant Risk and safety phrases R11 20 21 22 32 36 38 513 26 36 37 39 46 QIAGEN proteinase 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 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 skin R36 37 38 Irritating to eyes resp
60. 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 long term 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 Important points before starting Before beginning the procedure read Important Notes page 15 Proteinase is not required in this protocol Procedure 1 Thaw and equilibrate up to 6 whole blood samples at room temperature 15 25 2 Transfer 200 yl of each sample into EZ1 sample tubes 2 ml For samples 200 yl bring the volume up to 200 yl with Buffer G2 3 Continue with Protocol DNA Purification Trace Protocol page 44 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 22 EZ1 DNA Investigator Handbook 04 2009 Protocol Pretreatment for Dried Blood This protocol is designed for isolation of total genomic and mitochondrial DNA from dried blood The protocol describes sample collection and the preliminary lysis of dried blood samples using proteinase K Starting material Drying blood on filter paper is an effective form of storage and sa
61. urfaces The efficiency of inactivation has to be determined for each specific organism and depends for example on layer thick ness and sample type QIAGEN cannot guarantee complete eradication of specific pathogens EZ1 Cards EZ1 Advanced Cards and EZ1 Advanced XL Cards Protocols for nucleic acid purification are stored on preprogrammed EZ1 Cards integrated circuit cards The user simply inserts an EZ1 Advanced XL Card into the EZ1 Advanced XL an EZ1 Advanced Card into the EZ1 Advanced or an EZ Card into the BioRobot EZ1 and the instrument is then ready to run a protocol Figure 1 The availability of various protocols increases the flexibility of EZ1 instruments EZ DNA Investigator Handbook 04 2009 15 Figure 1 Ease of protocol setup using EZ1 Cards Inserting an EZ1 Card containing a 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 instrument is switched on The EZ1 DNA Investigator Kit requires use of the EZ1 Advanced XL DNA Investigator Card with the EZ1 Advanced XL or use of the EZ1 Advanced DNA Investigator Card with the EZ1 Advanced or use of the EZ1 DNA Investigator Card with the BioRobot EZ1 These EZ1 Cards contain protocols for purification of DNA from forensic and human identity samples EZ1 instruments should only be switched on after an EZ1 Card is inserted Make sure that the EZ1 Card is completely inserted
62. vice 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 the EZ1 DNA Investigator Kit or QIAGEN products in general please do not hesitate 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 GIAGEN 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 goto TechSupportCenter or call one of the QIAGEN Technical Service Departments or local distributors see back cover or visit www giagen com EZ1 DNA Investigator Handbook 04 2009 7 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 GIAGEN kit and kit component CAUTION DO NOT add bleach or acidic solutions directly to the sample preparation waste Buffers in
63. worktable setup The text summarizes the following steps which describe loading of the worktable Wear gloves when loading the required items on the worktable 7 Open the instrument door 8 Invert reagent cartridges twice 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 rack 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 of the tip rack 11 Load tip holders containing filter tips into the second row of the tip rack 12 Add 400 pl Buffer MTL to each sample tube containing digested samples Load opened sample tubes containing Buffer MTL and digested samples into the back row of the tip rack Pretreat the samples following the individual protocols in this handbook 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 13 Close the instrument door 14 Press START to start the purification procedure The automated purification procedure takes 15 20 min 50 EZ1 DNA Investigator Handbook 04 2009 15 When the protocol ends the display shows Protocol finished A Press ENT to generate the report file The EZ1 Advanced and the EZ1 Advan

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