EZ1 DNA Handbook - Projects at NFSTC.org
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1. Syngen Biotech Sp z o o Tel 071 351 41 06 or 0601 70 60 07 Fax 071 351 04 88 Email info syngen pl Web site www syngen pl Portugal IZASA PORTUGAL LDA Tel 21 424 7312 Fac 21 417 2674 Email consultasbiotec izasa es Singapore Research Biolabs Pte Ltd Tel 67775366 Fax 67785177 Email salesGresearchbiolabs com Web site www researchbiolabs com Slovak Republic BIO CONSUIT Slovakia spol s r o Tel Fax 02 5022 1336 Email bio cons cdicon sk Web site www bioconsult cz Slovenia MEDILINE d o o Tel 01 830 8040 Fac 01 8308070 or 01 830 8043 E mail medilineGsiol net South Africa Southern Cross Biotechnology Pty Ltd Tel 021 671 5166 Fax 021 6717734 E mail infoGscb co za Spain IZASA S A Tel 93 902 20 30 90 Fax 93 902 22 33 66 Email consultasbiotec izasa es Sweden WR International AB Tel 08 621 34 00 Fax 08 760 45 20 Email info se vwr com Web site www vwr com Taiwan TAIGEN Bioscience Corporation Tel 02 2880 2913 Fax 02 2880 2916 Email taigen ms10 hinet net Thailand Theera Trading Co Ltd Tel 02 412 5672 Fax 02 412 3244 Email theetrad samart co th Turkey Medek Medikal Ur nler ve Saglik Hizmetleri A S Tel 216 302 15 80 Fax 216 302 15 88 E mail makialp med ek com All other countries GIAGEN GmbH Germany 23 Australia QIAGEN Pty Ltd ABN 75 072 382. The following risk and safety phrases apply to the components of the EZ1 DNA Kits Reagent cartridge Contains ethanol guanidine hydrochloride and guanidine thiocyanate highly flammable harmful and irritant Risk and safety phrases R1 1 20 21 22 32 36 38 13 26 36 37 39 46 Proteinase K Contains proteinase K sensitizer irritant Risk and safety phrases R36 37 38 42 43 S23 24 26 36 37 24 hour emergency information Emergency medical information in English French and German can be obtained 24 hours a day from Poison Information Center Mainz Germany Tel 49 6131 19240 R11 Highly flammable R20 21 22 Harmful by inhalation in contact with skin and if swallowed R36 38 Irritating to eyes and skin R32 Contact with acids liberates very toxic gas R36 37 38 Irritating to eyes respiratory system and skin RA2 43 May cause sensitization by inhalation and skin contact 13 Keep away from food drink and animal feed 523 Do not breathe spray S24 Avoid contact with skin S26 In case of contact with eyes rinse immediately with plenty of water and seek medical advice 36 37 Wear suitable protective clothing and gloves S36 37 39 Wear suitable protective clothing gloves and eye face protection S46 If swallowed seek medical advice immediately and show this container or label 6 EZ1 DNA Handbook 02 2004 Introduction The EZ1 DNA Blood 200 yl Kit and EZ1 DNA Blood 350 yl Kit are for purification of genomic
3. Contact QIAGEN Technical Services for more information about the TissueLyser and supplementary protocols Procedure 1 Transfer tissue into a 1 5 ml screw capped tube not supplied For most tissue types a sample size of 10 mg is recommended however for heart up to 20 mg and for muscle up to 40 mg may be used 2 Add 190 pl Buffer G2 Ensure tissue pieces are fully submerged in Buffer G2 3 Add 10 pl proteinase K solution and mix by tapping on 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 an Eppendorf 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 preparation 5 Homogenize the sample by pipetting up and down several times Large pieces of insoluble material which could clog pipet tips should be removed by a quick centrifugation 300 x g 1 min Transfer the supernatant to a new 2 ml sample tube See Table 3 page 14 for more information EZ1 DNA Handbook 02 2004 17 Insert the EZ1 Card completely into the EZ1 Card slot of the BioRobot EZ1 Switch on the BioRobot EZ1 Press START to display the Protocols menu aye Press 1 to start worktable setup for the Tissue Protocol So 0O Mo 0 Press any key to proceed through the
4. 09 8045 5200 Email info fi vwr com Web site www vwr com Greece BioAnalytica S A Tel 210 640 03 18 Fax 210 646 27 48 E mail bioanalyt hol gr Web site www bioanalytica gr Hungary KasztelMed Co Ltd Tel 01 385 3887 Fac 01 381 0695 Email info kasztel hu Web site www kasztel hu India Genetix Tel 011 2542 1714 or 011 2515 9346 Fax 01 1 2546 7637 E mail genetix nda ysnl net in Israel Westburg Israel Ltd Tel 08 6900655 or 1 800 20 22 20 toll free Fax 08 6900650 E mail info westburg co il Web site www westburg co il Korea LRS Laboratories Inc Tel 02 924 86 97 Fax 02 924 86 96 E mail webmasterGlrslab co kr Web site www Irslab co kr Malaysia RESEARCH BIOLABS SDN BHD Tel 603 8070 3101 Fax 603 8070 5101 Email biolabs tm net my Web site www researchbiolabs com Mexico Quimica Valaner S A de C V Tel 55 55 25 57 25 Fax 55 55 25 56 25 E mail ventas valaner com Web site www valaner com The Netherlands Westburg b v Tel 033 4950094 Fax 033 4951222 E mail info westburg nl Web site www westburg nl EZ1 DNA Handbook 02 2004 New Zealand Biolab Ltd Tel 09 980 6700 or 0800 933 966 Fax 09 980 6788 E mail biosciences nzl biolabgroup com Web site www biolabgroup com nzl Norway WR International AS Tel 02 290 Fax 22 90 00 40 Email info no ywr com Web site www vwr com Poland
5. performance of all products in the manner described in our product literature The purchaser must determine the suitability of the product for its particular use Should any product fail to perform satisfactorily due to any reason other than misuse GIAGEN will replace it free of charge or refund the purchase price We reserve the right to change alter or modify any product to enhance its performance and design If a QIAGEN product does not meet your expectations simply call your local Technical Service Department or distributor We will credit your account or exchange the product as you wish A copy of GIAGEN terms and conditions can be obtained on request and is also provided on the back of our invoices If you have questions about product specifications or performance please call GIAGEN Technical Services or your local distributor see back cover Technical Assistance At QIAGEN we pride ourselves on the quality and availability of our technical support Our Technical Service Departments are staffed by experienced scientists with extensive practical and theoretical expertise in molecular biology and the use of QIAGEN products If you have any questions or experience any difficulties regarding EZ1 DNA 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
6. see Table 3 page 14 200 pl Visit www qiagen com goto clinicalDNA to find out about supplementary protocols for other sample types 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 E 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 E 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 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 page 14 shows typical yields obtained from different sample volumes and sample types Precipitate in reagent cartridge The buffer in well 1 of the reagent cartridge the well that is nearest to the front of the BioRobot EZ1 when the reagent cartridge is loaded may form a
7. to one year after delivery when stored at room temperature To prolong the lifetime of proteinase K storage at 2 8 C is recommended Quality Control As part of the stringent QIAGEN quality assurance program the performance of EZ1 DNA Kits is monitored routinely on a lotto lot basis All components are tested separately to ensure highest performance and reliability 4 EZ1 DNA Handbook 02 2004 Product Use Limitations The BioRobot EZ1 and EZ1 DNA Kits are intended as general purpose devices No claim or representation is intended for their use to identify any specific organism or for a specific clinical use diagnostic prognostic therapeutic or blood banking It is the user s responsibility to validate the performance of the BioRobot EZ1 and EZ1 DNA Kits for any particular use since their performance characteristics have not been validated for any specific organism The BioRobot EZ1 and EZ1 DNA Kits may be used in clinical diagnostic laboratory systems after the laboratory has validated their complete system as required by CLIA 88 regulations in the U S or equivalents in other countries All due care and attention should be exercised in the handling of many of the materials described in this text We recommend all users of GIAGEN 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
8. to the researchers at QIAGEN We therefore encourage you to contact us if you have any suggestions about product performance or new applications and techniques For technical assistance and more information please call one of the QIAGEN Technical Service Departments or local distributors see back cover EZ1 DNA Handbook 02 2004 5 Safety Information When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information please consult the appropriate material safety data sheets MSDSs These are available online in convenient and compact PDF format at www giagen com ts msds asp 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 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 1 v v sodium hypochlorite If liquid containing potentially infectious agents is spilt on the BioRobot EZ1 clean the affected area first with laboratory detergent and water and then with 1 v v sodium hypochlorite followed by water
9. 2 944 PO Box 25 Clifton Hill Victoria 3068 Orders 03 9489 3666 Fax 03 9489 3888 Technical 1 800 243 066 Canada QIAGEN Inc 2800 Argentia Road Unit 7 Mississauga Ontario L5N 8L2 Orders 800 572 9613 Fax 800 713 5951 Technical 800 DNA PREP 800 362 7737 France QIAGEN S A 3 avenue du Canada LP 809 91974 COURTABOEUF CEDEX Orders 01 60 920 920 Fax 01 60 920 925 Technical 01 60 920 930 Germany QIAGEN GmbH QIAGEN Strasse 1 40724 Hilden Orders 02103 29 12000 Fax 02103 29 22000 Technical 02103 29 12400 Italy QIAGEN S p A Via Grosio 10 10 20151 Milano Orders 02 3343041 1 Fax 02 33430426 Technical 02 33430414 Japan QIAGEN K K Forefront Tower Il 13 1 Kachidoki 3 Chome Chuo ku Tokyo 104 0054 Telephone 03 5547 081 1 Fax 03 5547 0818 Technical 03 5547 081 1 Switzerland QIAGEN AG Auf dem Wolf 39 4052 Basel Orders 061 3 19 30 30 Fax 061 3 19 30 33 Technical 061 3 19 30 31 UK and Ireland QIAGEN Ltd QIAGEN House Fleming Way Crawley West Sussex RH10 9NQ Orders 01293 422 911 Fax 01293 422 922 Technical 01293 422 999 USA QIAGEN Inc 27220 Turnberry Lane Valencia CA 91355 Orders 800 426 8157 Fax 800 718 2056 Technical 800 DNA PREP 800 362 7737 QIAGEN WWW QIAGEN COM
10. DNA from whole blood samples and blood products The EZ1 DNA Tissue Kit is for purification of genomic DNA from tissue and other samples Magnetic particle technology provides high quality DNA that is suitable for direct use in downstream applications such as amplification or other enzymatic reactions The BioRobot EZ1 workstation performs 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 6 samples are processed in a single run Principle and procedure Magnetic particle technology combines the speed and efficiency of silicatbased 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 Working with the BioRobot EZ1 The unique design of the BioRobot EZ1 makes operation of the workstation convenient and easy The main features of the BioRobot EZ1 include E Purification of high quality nucleic acids from 1 6 samples per run H Small footprint with no external computer to save laboratory space MH Pre programmed EZ1 Cards containing ready to use protocols for nucleic acid purification E Prefiled sealed reagent cartridges for eas
11. Robot EZ1 DNA paraffin section protocols Pre programmed card for BioRobot EZ1 DNA protocols for dried blood samples Cat no 951034 951054 953034 9000705 9015585 9015588 9015587 9015589 9015862 9015863 21 Ordering Information Product Contents Cat no EZ1 DNA Forensic Card Pre programmed card for BioRobot EZ1 9015864 DNA protocols for forensic applications Filter Tips and Holders EZ1 50 50 Disposable Filter Tips 50 Disposable 994900 Tip Holders additional tips and holders for use with BioRobot EZ1 Kits 12 Tube Magnet Magnet for separating magnetic particles 36912 in 12 x 1 5 ml or 2 ml tubes Related products EZ1 RNA Cell Mini Kit for purification of total RNA from cells EZ RNA Cell Mini Kit 48 48 Reagent Cartridges RNA Cell 958134 100 Disposable Tip Holders 100 Disposable Filter Tips 50 Sample Tubes 2 ml 50 Elution Tubes 1 5 ml Buffer RLT RNase Free DNase EZ1 RNA Tissue Mini Kit for purification of total RNA from tissue EZ RNA Tissue Mini Kit 48 48 Reagent Cartridges RNA Tissue 959134 100 Disposable Tip Holders 100 Disposable Filter Tips 50 Sample Tubes 2 ml 50 Elution Tubes 1 5 ml Buffer RLT RNase Free DNase EZ1 RNA Card for running RNA purification protocols on the BioRobot EZ1 EZ1 RNA Card Pre programmed card for EZ1 RNA 9015590 purification protocols 22 EZ1 DNA Handbook 02 2004 QIAGEN Companies Please see the back cover for c
12. Second Edition February 2004 EZ1 DNA Handbook EZ1 DNA Blood 200 yl Kit EZ1 DNA Blood 350 yl Kit EZ1 DNA Tissue Kit For automated purification of DNA from blood tissue and other samples using the BioRobot EZ1 workstation 00000 00000 QIAGEN Trademarks GIAGEN BioRobot QIAGEN Group Eppendorf EppendorfNetheler Hinz GmbH QIAGEN Robotic Systems are not available in all countries please inquire The BioRobot EZ1 and EZ1 Kits are intended as general purpose devices No claim or representation is intended for their use to identify any specific organism or for a specific clinical use diagnostic prognostic therapeutic or blood banking It is the user s responsibility to validate the performance of the BioRobot EZ1 and EZ Kits for any particular use since their performance characteristics have not been validated for any specific organism The BioRobot EZ1 and EZ1 Kits may be used in clinical diagnostic laboratory systems after the laboratory has validated their complete system as required by CLIA 88 regulations in the U S or equivalents in other countries 2004 QIAGEN all rights reserved 2 EZ1 DNA Handbook 02 2004 Contents Kit Contents 4 Storage 4 Quality Control 4 Product Use Limitations 5 Product Warranty and Satisfaction Guarantee 5 Technical Assistance 5 Safety Information 6 Introduction 7 Principle and procedure 7 Working with the BioRobot EZ1 7 Important Notes 13 Starting material 13 Stor
13. Tissue Kit Paraffin section EZ1 DNA Paraffin Section Card EZ1 DNA Tissue Kit Dried blood EZ DNA Dried Blood Card EZ1 DNA Tissue Kit Forensic samples EZ1 DNA Forensic Card EZ1 DNA Tissue Kit Cultured cells EZ1 DNA Tissue Card EZ1 DNA Tissue Kit See the protocols on page 15 and 17 of this handbook for details on the purification procedure See the supplementary protocols at www giagen com goto clinicalDNA for details on the purification procedure Visit www qiagen com goto clinicalDNA to find out the latest supplementary protocols which describe how to use EZ1 DNA Kits and EZ1 DNA Cards for other applications EZ1 DNA Handbook 02 2004 9 Reagent cartridges Reagents for the purification of nucleic acids from a single sample are contained in a single reagent cartridge Figure 2 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 Ease of Workstation Setup Using Reagent Cartridges Figure 2 EY A sealed prefilled reagent cartridge Well 1 is indicated with an arrow 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 the BioRob
14. age of blood samples 13 Yield of purified DNA 13 Precipitate in reagent cartridge 13 Lysis with proteinase K 14 Quantification of DNA 14 Protocols Bi Purification of Genomic DNA from Whole Blood 15 E Purification of Genomic DNA from Tissue 17 Troubleshooting Guide 19 Appendix Storage Quantification and Determination of Purity of DNA 20 Ordering Information 21 GIAGEN Distributors 23 EZ1 DNA Handbook 02 2004 3 Kit Contents EZ1 DNA Kits Blood 200 pl Blood 350 pl Tissue Catalog no 951034 951054 953034 Preps per kit 48 48 48 Reagent Cartridge Blood 200 yl 48 1023745 Reagent Cartridge Blood 350 yl 48 1023729 Reagent Cartridge Tissue 48 1023869 Disposable Tip Holders 50 50 50 Disposable Filter Tips 50 50 50 Sample Tubes 2 ml 50 50 50 Elution Tubes 1 5 ml 50 50 50 Buffer G2 1x11 ml Proteinase K 2 x 250 yl Handbook 1 1 1 For details about the EZ1 Cards to be used with these kits see Table 1 page 9 and visit www giagen com goto clinicalDNA Storage Store the reagent cartridges at room temperature 15 25 C The magnetic particles in the reagent cartridges remain active when stored at this temperature For prolonged storage store the reagent cartridges at 2 8 C Do not freeze the reagent cartridges If the reagent cartridges contain precipitates see Precipitate in reagent cartridge page 13 The ready to use proteinase K solution is stable for up
15. be applied to a suitable magnetic separator and the eluate transferred to a clean tube see below To quantify DNA isolated using the BioRobot EZ1 system B Apply the tube containing the DNA to a suitable magnetic separator e g GIAGEN 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 in a microcentrifuge to pellet any remaining magnetic particles E 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 E 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 A260 A320 A280 A320 Pure DNA has an Aoso A280 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 a
16. e enzymatic reactions downstream application Quantify the purified DNA by spectrophotometric measurement of the absorbance at 260 nm see Quantification of DNA page 20 Low A 60 A2go ratio for purified nucleic acids Absorbance reading at 320 nm To correct for the presence of magnetic particles in the not subtracted from the eluate an absorbance reading at 320 nm should be absorbance readings obtained taken and substracted from the absorbance readings at 260 nm and 280 nm obtained at 260 nm and 280 nm see Quantification of DNA page 20 EZ1 DNA Handbook 02 2004 19 Appendix 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 Asso 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 Aggg 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
17. fy coat Up to 14 pg Dried blood 4 x 3 5 mm diameter discs 0 2 0 5 yg Buccal cells 1 swab 1 4 5 pg Cultured cells 2 x 106 cells 6 7 5 yg Skeletal muscle 200 yl 40 mg tissue digested Upto 9 pg Heart 200 pl 20 mg tissue digested Up to 12 pg Spleen 200 yl 10 mg tissue digested Up to 28 pg Lung 200 yl 10 mg tissue digested Up to 17 pg Kidney 200 pl 10 mg tissue digested Up to 18 pg Visit www giagen com goto clinicalDNA to find out about supplementary protocols for sample types other than blood and tissue t Whole blood with 3 0 7 0 x 106 white blood cells ml elution volume 200 yl 14 EZ1 DNA Handbook 02 2004 Protocol Purification of Genomic DNA from Whole Blood Select the appropriate EZ1 Kit and EZ1 Card according to the volume of your whole blood samples Table 4 Selection of EZ1 Kit and EZ1 Card Volume of blood EZ1 Kit EZ1 Card Protocol Volume of sample required required required eluted DNA 200 pl EZ1 DNA EZ DNA 200 yl 200 yl Blood 200 yl Blood Card Protocol Kit 350 yl EZ1 DNA EZ1 DNA 350 yl 200 yl Blood 350 yl Blood Card Protocol Kit Supplementary protocols for automated purification of genomic DNA from blood products using the BioRobot EZ system are available at www qiagen com goto clinicalDNA Important points before starting E Before beginning the procedure read Important Notes on page 13 E The yield of genomic DNA depends on the sample volume and on the number of white blood cell
18. ontact information for your local QIAGEN office QIAGEN Distributors Argentina Tecnolab S A Tel 011 4555 0010 Fax 011 4553 3331 Email info tecnolab com ar Web site www tecnolab com ar Austria VWR International GmbH Tel 01 576 000 Fax 01 576 00 600 Email info at vwr com Web site www vwr com Belgium Luxemburg Westburg b v Tel 0800 1 9815 Fax 31 33 4951222 Email infoGwestburg nl Web site www westburg nl Brazil Uniscience do Brasil Tel 011 3622 2320 Fax 011 3622 2323 Email infoGuniscience com Web site www uniscience com China Gene Company Limited Tel 852 2896 6283 Fax 852 25 15 9371 E mail Hong Kong info genehk com Beijing info_bj genecompany com Shanghai info_sh genecompany com Chengdu gene public cd sc cn Guangzhou info_gz genecompany com Croatia INEL Medicinska Tehnika d o o Tel 01 2984 898 Fax 01 6520 966 Email inel medicinskatehnika zg hinet hr Cyprus Scientronics Ltd Tel 02 357 22 765416 Fax 02 357 22 764614 E mail a sarpetsas biotronics com cy Czech Republic BIO CONSULT spol s r o Tel Fax 420 2 417 29 792 Email info bioconsult cz Web site www bioconsult cz Denmark VWR International A S Tel 43 86 87 88 Fax 43 86 87 90 Email info dk vwr com Web site www vwr com Egypt Clinilab Tel 5257212 Fax 5257210 Email Clinilab link net Finland VWR International Oy Tel 09 804 551 Fax
19. ot EZ1 is where the user loads samples and the components of the EZ1 DNA Kit Figure 3 next page Details on worktable setup are provided in the protocols in this handbook and in the supplementary protocols at www qgiagen com goto clinicalDNA and are also displayed in the liquid crystal display LCD of the control panel when the user starts worktable setup 10 EZ1 DNA Handbook 02 2004 Worktable of the BioRobot EZ1 Figure 3 The BioRobot EZ1 worktable 1 Elution tubes loaded into the first row 2 Tip holders containing filter tips loaded into the second row 3 Third row is empty 4 Sample tubes loaded into the fourth row 5 Reagent cartridges loaded into the cartridge rack The LCD also displays updates during the automated purification procedure EZ1 DNA Handbook 02 2004 11 Workflow of BioRobot EZ1 Operation Insert EZ1 Card completely into the EZ1 Card slot Switch on the BioRobot EZ1 Start worktable setup Load the sealed reagent cartridges elution tubes tip holders containing filter tips and sample tubes Start the protocol Collect purified DNA 12 EZ1 DNA Handbook 02 2004 Important Notes Starting material The amounts of starting material for use in EZI DNA procedures are shown in Table 2 Table 2 Amounts of Starting Material for EZ1 DNA Procedures Sample type Amount of starting material Elution volume Blood 200 pl or 350 pl 200 pl Tissue 10 40 mg
20. precipitate upon storage If necessary redissolve by mild agitation at room temperature 15 25 C t 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 EZ1 DNA Handbook 02 2004 13 Lysis with proteinase K The EZ1 DNA Tissue Kit contains proteinase K which is the enzyme of choice for lysis buffers used in the tissue protocol Proteinase K is isolated from the saprophytic fungus Tritirachium album and is particularly suitable for short digestion times It possesses a high specific activity which 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 Tissue protocols Quantification of DNA Carryover of magnetic particles may affect the absorbance reading at 260 nm A260 of the purified DNA but should not affect downstream applications The measured absorbance at 320 nm A390 should be subtracted from all absorbance readings See Quantification of DNA page 20 for more information Table 3 DNA Yields Obtained from Different Sample Types Using EZ1 DNA Procedures Sample type Sample volume DNA yield Blood 200 pl 4 8 yg Blood 350 yl 5 12 yg Buf
21. s in the sample Procedure 1 Thaw and equilibrate up to 6 whole blood samples at room temperature 15 25 C Insert the EZ1 Card completely into the EZ1 Card slot of the BioRobot EZ1 Switch on the BioRobot EZ1 Press START to display the Protocols menu Press 1 or 2 to start worktable setup for the 200 pl Protocol or 350 pl Protocol respectively a ROM 6 Press any key to proceed through the text displayed in the LCD The text summarizes the following steps which describe the loading of the worktable 7 Open the workstation door 8 Invert 1 6 reagent cartridges twice to mix the magnetic particles Then tap the cartridges to deposit the reagents at the bottom of their wells EZ1 DNA Handbook 02 2004 15 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 If there are fewer than 6 reagent cartridges you can load them in any order on the rack However when loading the other labware in steps 10 12 ensure that they also follow the same order 10 Load 1 6 opened elution tubes into the first row 11 Load 1 6 tip holders containing filter tips into the second row 12 Load 1 6 opened sample tubes containing 200 pl or 350 pl blood into the fourth row 13 Close the workstation door 14 Press START to start the protocol The automated purifica
22. tch off the BioRobot EZ1 22 Clean the BioRobot EZ1 Follow the maintenance instructions in the BioRobot EZ1 User Manual See Figure 2B on page 10 See Figure 3 on page 11 18 EZ1 DNA Handbook 02 2004 Troubleshooting Guide This troubleshooting guide may be helpful in solving any problems that may arise The scientists in QIAGEN Technical Services are always happy to answer any questions you may have about either the information and protocols in this handbook or molecular biology applications see back cover for contact information Comments and suggestions General handling Error message in LCD Refer to the BioRobot EZ1 User Manual Low DNA yield a Magnetic particles Ensure that you invert the reagent cartridges not completely several times to resuspend the magnetic particles resuspended b Insufficient reagent After inverting the reagent cartridges to resuspend the aspirated magnetic particles ensure that you tap the cartridges to deposit the reagents at the bottom of the wells c Frozen blood samples Thaw frozen blood samples at room temperature not mixed properly 15 25 C with mild agitation to ensure thorough after thawing mixing DNA does not perform well in downstream applications a Insufficient DNA used in Quantify the purified DNA by spectrophotometric downstream application measurement of the absorbance at 260 nm see Quantification of DNA page 20 b Excess DNA used in Excess DNA can inhibit som
23. text displayed in the LCD The text summarizes the following steps which describe the loading of the worktable 11 Open the workstation door 12 Invert 1 6 reagent cartridges twice to mix the magnetic particles Then tap the cartridges to deposit the reagents at the bottom of their wells 13 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 If there are fewer than 6 reagent cartridges you can load them in any order on the rack However when loading the other labware in steps 14 16 ensure that they also follow the same order 14 Load 1 6 opened elution tubes into the first row 15 Load 1 6 tip holders containing filter tips into the second row 16 Load 1 6 opened sample tubes containing tissue samples into the fourth row 17 Close the workstation door 18 Press START to start the protocol The automated purification procedure takes approximately 20 min 19 When the protocol ends the LCD displays Protocol finished Open the workstation door 20 Remove the elution tubes containing the purified DNA Discard the sample preparation waste 21 To run another protocol press ESC prepare samples as described in steps 1 5 and follow the procedure from step 9 onward Otherwise press STOP twice to return to the first screen of the LCD close the workstation door and swi
24. tion procedure takes approximately 20 min 15 When the protocol ends the LCD displays Protocol finished Open the workstation door 16 Remove the elution tubes containing the purified DNA Discard the sample preparation waste 17 Torun another protocol press ESC prepare samples as described in step 1 and follow the procedure from step 5 onward Otherwise press STOP twice to return to the first screen of the LCD close the workstation door and switch off the BioRobot EZ1 18 Clean the BioRobot EZ1 Follow the maintenance instructions in the BioRobot EZ1 User Manual See Figure 2B on page 10 See Figure 3 on page 11 16 EZ1 DNA Handbook 02 2004 Protocol Purification of Genomic DNA from Tissue Select the EZ1 Kit and EZ1 Card below Table 5 Selection of EZ1 Kit and EZ1 Card Volume of tissue EZ1 Kit EZ1 Card Protocol Volume of sample required required required eluted DNA 200 pl EZ1 DNA EZ1 DNA Tissue 200 pl pre digested Tissue Kit Tissue Card Protocol pa sample Supplementary protocols for automated purification of genomic DNA from other tissue types and cells using the BioRobot EZ1 system are available at www giagen com goto clinicalDNA Important points before starting E Before beginning the procedure read Important Notes on page 13 E The incubation time with proteinase K can be shortened by using the Tissuelyser see www giagen com products accessories
25. vailable from the product supplier t If the samples are not diluted use water to calibrate the spectrophotometer 20 EZ1 DNA Handbook 02 2004 Ordering Information Product EZ1 DNA Blood 200 yl Kit 48 EZ1 DNA Blood 350 pl Kit 48 EZ1 DNA Tissue Kit 48 Contents 48 Reagent Cartridges Blood 200 pl 50 Disposable Tip Holders 50 Disposable Filter Tips 50 Sample Tubes 2 ml 50 Elution Tubes 1 5 ml 48 Reagent Cartridges Blood 350 pl 50 Disposable Tip Holders 50 Disposable Filter Tips 50 Sample Tubes 2 ml 50 Elution Tubes 1 5 ml 48 Reagent Cartridges Tissue 50 Disposable Tip Holders 50 Disposable Filter Tips 50 Sample Tubes 2 ml 50 Elution Tubes 1 5 ml Buffer G2 Proteinase K BioRobot EZ1 for easy automated purification of nucleic acids from 1 6 samples BioRobot EZ1 Accessories EZ1 DNA Blood Card EZ1 DNA Tissue Card EZ1 DNA Buffy Coat Card EZ1 DNA Buccal Swab Card EZ1 DNA Paraffin Section Card EZ1 DNA Dried Blood Card EZ1 DNA Handbook 02 2004 Robotic workstation for automated purification of nucleic acids using EZ1 Kits installation 1 year warranty on parts and labor Pre programmed card for BioRobot EZ1 DNA blood protocols Pre programmed card for BioRobot EZ1 DNA tissue protocols Pre programmed card for BioRobot EZ1 DNA protocols for buffy coat samples Pre programmed card for BioRobot EZ1 DNA protocols for buccal swab samples Pre programmed card for Bio
26. y safe and fast setup of the BioRobot EZ1 worktable H Complete automation of nucleic acid purification from opening of reagent cartridges to elution of nucleic acids with no manual centrifugation steps EZ1 Cards Protocols for nucleic acid purification are stored on pre programmed EZ1 Cards integrated circuit cards The user simply inserts an EZ1 Card into the BioRobot EZ1 and the workstation is then ready to run a protocol Figure 1 page 9 The availability of various protocols increases the flexibility of the BioRobot EZ1 system EZ1 DNA Handbook 02 2004 7 EZ1 DNA Procedure Whole blood blood products or digested tissue Magnetic particles added to samples DNA binds to magnetic particles Magnet Magnetic separation Wash Magnetic separation tute WI Pure high quality DNA Magnet 8 EZ1 DNA Handbook 02 2004 Ease of Protocol Setup Using EZ1 Cards Figure 1 Inserting an EZ Card containing protocols into the BioRobot EZ1 The EZ1 Kit and EZ1 Card required depend on the purification procedure to be carried out Table 1 Table 1 Purification of DNA from Various Sample Types Sample type EZ1 Card required EZ1 Kit required Blood EZ1 DNA Blood Card EZ DNA Blood 200 yl Kit 200 pl or 350 pl or EZ1 DNA Blood 350 yl Kit Tissue EZ DNA Tissue Card EZ1 DNA Tissue Kit Buffy coat EZ1 DNA Buffy Coat Card EZ1 DNA Blood 350 pl Kit Buccal swab EZ1 DNA Buccal Swab Card EZ1 DNA
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