MagAttract 96 DNA Plant Handbook
Contents
1. In addition to the BioRobot RapidPlate Workstation this protocol requires a BioRobot 3000 Workstation with the following configuration M 0 9 mm steel probes Optional Tip change system High Speed Pipetting System external Shaker System 4 plate 2500 yl Dilutor Units MagAttract 96 DNA Plant Handbook 08 2003 15 o A fe om o ga o im pu Q r o gt 020 014 c 2 Ses D Ss le Q O cc 2 a Cooling and Heating System with heattransfer adapter flat bottom Robotic Handling System T grip Two Reagent Holders 3 trough 20 ml Magnet 96 well Type B cat no 9012916 Consumables Non porous tape sheets for sealing 96 well microplates e g Tape Pads cat no 19570 Flat bottom 96 well microplates e g 96 Well Microplates FB cat no 36985 Round bottom 96 well microplates e g 96 Well Microplates RB cat no 19581 Disposable Troughs 20 ml cat no 9232764 Two 96 well blocks with 2 2 ml wells e g S Blocks cat no 19585 Optional One rack of Disposable Tips 300 pl for the BioRobot 3000 One rack of Disposable Tips 200 yl for the BioRobot RapidPlate Workstation Things to do before starting E Add 125 ml isopropanol and 1 vial RNase A 1 x 220 pl to each bottle of Buffer RPW 125 ml before use Transfer the solution into a 500 ml bottle with a luer adapter provided with the BioRobot Workstation Shake the bottle containing MagAttract Suspens2. S Blocks 96 well blocks with 2 2 ml wells 19585 24 per case Magnet 96 Well Type B 9012916 TissueLyser 24 Universal laboratory mixer mill Inquire for 24 samples TissueLyser 96 Universal laboratory mixer mill Inquire for 96 samples 28 MagAttract 96 DNA Plant Handbook 08 2003 Notes MagAttract 96 DNA Plant Handbook 08 2003 29 Notes 30 MagAttract 96 DNA Plant Handbook 08 2003 QIAGEN Companies Please see the back cover for contact information for your local QIAGEN office QIAGEN Distributors Argentina Egypt New Zealand Taiwan Tecnolab S A Clinilab Biolab Ltd TAIGEN Bioscience Corporation Tel 011 4555 0010 Tel 52 57 212 Tel 09 980 6700 Tel 02 2880 2913 Fax 011 4553 3331 Fax 52 57 210 or 0800 933 966 Fax 02 2880 2916 E mail infoGtecnolab com ar Email Clinilab link net Fax 09 980 6788 E mail taigen ms10 hinet net Web site www tecnolab com ar E mail biosciences nzl biolabgroup com Finland Web site www biolabgroup com nzl Thailand wi Tet 09 8043 uo el el i xc o Fax 09 8045 5200 Norway Fax 02 412 3244 Fax 01 576 00 600 E mail infoGfi vwr com VWR International AS E mail theetrad samart co th E mail infoGat vwr com Web site www vwr com E 2 ds i p T ax eR ne eRe SAN E mail info no vwr com Turkey Greece Web site www vwr com Medek Medikal Ur nler BioAnalytica S A ve Saglik Hizmetleri A S Belgium Luxemburg Tel 210 640 03 18 Tel 216 302 15 80 Westbu
3. any difficulties regarding the MagAttract 96 DNA Plant Core Kit or QIAGEN products in general please do not hesitate to contact us QIAGEN customers are a major source of information regarding advanced or specialized uses of our products This information is helpful to other scientists as well as to the researchers at QIAGEN We therefore encourage you to contact us if you have any suggestions about product performance or new applications and techniques For technical assistance and more information please call one of the QIAGEN Technical Service Departments or local distributors see back cover Product Specifications MagAttract 96 DNA Plant Core Kit Plant starting material fresh frozen Plant starting material dried lyophilized DNA yield Final volume of eluate Depends on type of plant and preparation protocol used MagAttract 96 DNA Plant Handbook 08 2003 5 Recommended starting amounts and expected yields from different plants Yield ug DNA Total yield Concentration Sample Amount mg tissue pg ng pl Arabidopsis blossom and hull 3 pieces 0 46 4 6 57 Arabidopsis leaf 100mg 0 05 5 0 62 Barley leat 30 mg 0 15 4 5 56 Corn leaf 60 mg 0 20 120 Flax 40 mg 0 14 5 9 64 Rape Canola 40 mg 0 14 SA 67 Rye leaf 30 mg 0 17 5 1 63 Ryegrass Lolium spp 30 mg 0 13 4 0 47 Sunflower leaf 30 mg 0 16 4 8 60 Tobaco leaf 40 mg pes Tomato leaf 50 mg 0 15 7 9 93 Wheat leaf 30 mg 0 16 4 8 60 DNA has also been
4. 984 898 Tel 02 924 86 97 Tel 01 830 80 40 Fax 01 6520 266 Fax 02 924 86 96 Fax 01 830 8070 E mail E mail webmasterGlrslab co kr or 01 830 80 63 inel medicinska tehnika zg tel hr Web site www lrslab co kr E mail mediline siol net Cyprus Malaysia South Africa S enhirontes lid RESEARCH BIOLABS SDN BHD Southern Cross Biotechnology Tel 02 357 22 765416 Tel 603 8070 3101 Pty Ltd Fax 02 357 22 764614 Fax 603 8070 5101 Tel 021 671 5166 E mail a sarpetsas biotronics com cy Email biolabs tm net my Fax 021 671 7734 Web site www researchbiolabs com E mail info scb co za Czech Republic Mexico Spain BIO CONSULT spol s r o Quimica Valaner S A de C V IZASA S A Tel Fax 420 2 417 29 792 Tel 55 55 25 57 25 Tel 93 902 20 30 90 E mail bio cons login cz Fox 55 55 25 56 25 Fax 93 902 22 33 66 Web site www bio consult cz E mail qvalaner infosel net mx E mail suministros izasa es Denmark The Netherlands Sweden VWR International A S Westburg b v VWR International AB Tel 43 86 87 88 Tel 033 4950094 Tel 08 621 34 00 Fax 43 86 87 90 Fax 033 4951222 Fax 08 760 45 20 E mail info dk vwr com E mail info westburg nl E mail info se vwr com Web site www vwr com Web site www westburg nl Web site www vwr com MagAttract 96 DNA Plant Handbook 08 2003 31 Australia QIAGEN Pty Ltd ABN 75 072 382 944 PO Box 25 Clifton Hill Victoria 3068 Orders 03 9489 3666 Fax 03 9489 3888 Tec
5. AGEN Magnet 96 well Type B cat no 9012916 M Multichannel pipet with 300 pl maximum capacity for manual preparation M BioRobot Plant Science Workstation or other robotic workstation for automated processing Consumables M Reagent reservoirs for the multichannel pipet for manual preparation M Collection Microtubes with Collection Microtube Caps for disruption cat nos 19560 and 19566 or 120008 M Flat bottom 96 well microplates e g 96 Well Microplates FB cat no 36985 M Round bottom 96 well microplates e g 96 Well Microplates RB cat no 19581 Reagents M Isopropanol 99 100 M Ethanol 96 100 M Liquid nitrogen 10 MagAttract 96 DNA Plant Handbook 08 2003 Protocol Disruption of Plant Tissue and Preparation of Cleared Lysates This protocol can be used for disruption of 192 2 x 96 plant tissue samples using the Tissuelyser see Disruption of plant tissue page 9 After addition of lysis buffer cleared lysates are prepared by centrifugation IMPORTANT The optimal amount of starting material depends on the plant type and its state fresh or lyophilized We recommend using up to 100 mg fresh plant material or up to 30 mg lyophilized material and performing a preliminary experiment with different amounts of starting material Procedure l Place a plant tissue sample into each tube of two collection microtube racks Keep the clear covers from the collection microtube racks for use in ste
6. August 2003 MagAttraci 96 DNA Plant Handbook For efficient high throughput isolation of DNA from plant tissue Trademarks and disclaimers Patented or patent pending and or registered or registration pending trademarks of the QIAGEN Group QIAGEN QIAsoft BioRobot MagAttract RapidPlate is a registered trademark of Zymark Corporation Registered names trademarks etc used in this document even when not specifically marked as such are not to be considered unprotected by law The PCR process is covered by U S Patents 4 683 195 and 4 683 202 and foreign equivalents owned by Hoffmann La Roche AG 2003 QIAGEN all rights reserved 2 MagAttract 96 DNA Plant Handbook 08 2003 Contents Kit Contents 4 Storage Conditions 4 Product Warranty and Satisfaction Guarantee 4 Product Use Limitations 5 Technical Assistance 5 Product Specifications 5 Safety Information 6 Introduction 8 Principle and procedure 9 Disruption of plant tissues v Centrifugation 9 Handling guidelines for the Magnet 96 well Type B 9 Equipment and Reagents to Be Supplied by the User 10 M Protocol Disruption of Plant Tissue and Preparation of Cleared Lysates 11 M Protocol Manual DNA Purification 13 M Protocol DNA Purification Using the MagAttract 96 DNA Plant Core Kit and BioRobot Plant Science Workstation 15 Recommendations for Using MagAttract Technology with Robotic Systems 19 M Protocol Adaptation of MagAttract Technology to Robotic Syst
7. age 15 Dispensing MagAttract Suspension A MagAttract particles sediment if stored without agitation To ensure a uniform distribution of particles shake the magnetic particle suspension before dispensing To resuspend the MagAttract particles we recommend pipetting 4 ml of MagAttract Suspension into a 20 ml trough and shaking the trough for 90 seconds at 600 rpm on a plate shaker that is integrated in the worktable The trough holder should have an SBS Standard footprint for fitting into the plate shaker Resuspension of the pelleted MagAttract particles after magnetic separation Some robotic devices are not capable of resuspending MagAttract particles by pipetting repeatedly after magnetic separation If this is the case we recommend l Addition of 100 pl Buffer RPW wash buffer 2 Resuspension of magnetic particles on a plate shaker 2 minutes at 800 rpm 3 Adding an additional 100 pl buffer in each of the three wash steps to increase the wash efficiency MagAttract 96 DNA Plant Handbook 08 2003 17 When using repeated pipetting to resuspend magnetic particles the following guidelines may help 1 Apply low speed pipetting to loosen the pellet 2 Apply high speed pipetting for efficient homogenization of the particles once the pellet is loosened Removal of the supernatant after magnetic separation Before elution the MagAttract particles should be dried to remove the remaining ethanol This step can be done at
8. before resuspending in Buffer RLT Make sure that the correct amount of Buffer RB was added to the samples Note Some plants contain secondary metabolites which may influence the binding of DNA to the MagAttract particles see next point In future preparations try the protocol modifications for difficult plant materials suggested in Appendix A page 27 Before starting the procedure ensure that the MagAttract particles are fully resuspended Vortex for at least 5 min before first use and for 1 min before subsequent uses Isopropanol must be added to Buffer RPW before use Repeat procedure with correctly prepared Buffer RPW The robotic workstation could have problems with liquid detection if distilled water is used for elution Use Buffer AE for elution to prevent this problem MagAttract 96 DNA Plant Handbook 08 2003 21 Comments and suggestions Contamination of RNA in the eluate Buffer RPW did not contain RNase A must be added to Buffer RPW before use RNase Repeat procedure with correctly prepared Buffer RPW A low level of RNA contamination may not affect the results of quantitative PCR Magnetic particles in the eluate Magnetic particle carryover Remove supernatant from MagAttract particles carefully and from slightly off center of each well However magnetic particles do not interfere with enzymatic reactions such as PCR Overestimation of DNA yield upon spectrophotometric analysis a RNA c
9. can be repeated if particle carryover occurs We also recommend repeating it if the purified DNA is to be used in highly sensitive downstream applications MagAttract 96 DNA Plant Handbook 08 2003 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 Low or no DNA recovery a b c d e f g h Low DNA content of the plant tissue Insufficient sample disruption Insufficient sample lysis Incorrect binding conditions Oils or other secondary metabolites may interfere with the binding process MagAttract particles were not completely resuspended Buffer RPW did not contain isopropanol The robotic workstation could not detect the elution buffer Comments and suggestions Increase the amount of starting material but do not use more than 150 mg Ensure that the starting material is completely disrupted See Disruption of plant tissue page 9 Ensure that the starting material is resuspended thoroughly in Buffer RLT Frozen samples should be resuspended immediately after disruption Do not allow samples to thaw If multiple Collection Microtube racks are to be processed store the disrupted samples at 20 C
10. cient excess DNA used in downstream application Salt carryover Eluate is slightly green or yellow Aspirate as much of the ethanol in the final wash step as possible Dry the particles before elution to remove residual ethanol Optimize the amount of DNA used in the downstream application if necessary Downstream applications can be adversely affected by insufficient or excess DNA Change the pipetting speeds and their duration or increase the number of wash steps to improve the wash efficiency See Green red or yellow colored eluates MagAttract 96 DNA Plant Handbook 08 2003 Appendix A Protocol Modifications for Difficult Plant Materials Some plant material used for DNA purification may yield low amounts of DNA and or potential inhibitors of PCR may be co purified Carefully following the protocols in this handbook is important to optimize yields If low yields or co purification of inhibitors still occur we recommend the following strategy Note The automation protocols must be adapted to include the appropriate volume settings and worktable accessories Problem Suggestion Low yield of DNA Replace Buffer RB 65 pl with ethanol 225 pl For some plant material yields are higher if the binding conditions are modified by the addition of ethanol Check whether your magnetic separation principle device is suited for this option because addition of alcohol affects the physical behavior of the magnet
11. contains a manual protocol a general protocol for use of the MagAttract 96 DNA Plant procedure with robotic workstations magnetic separation devices and a specific protocol for use with the BioRobot Plant Science Workstation t QIAGEN robotic systems are not available in all countries please inquire MagAttract Procedure Plant leaf tissue Grind lyse and precipitate Microplate at Add cleared lysate to binding buffer and 1 magnetic particles mix and incubate Remove supernatant 96 Well Magnet Type B using the ipie and LU 1 MINN perform 4 wash cycles Elution Microplate RB Ready to use DNA 8 MagAttract 96 DNA Plant Handbook 08 2003 Principle and procedure Fresh frozen or lyophilized starting material 10 100 mg is mechanically disrupted to give a fine powder The powder is resuspended in lysis buffer carefully mixed and then sedimented by a short centrifugation step The lysates are transferred to 96 well flat bottom microplates before starting the MagAttract purification procedure Genomic DNA selectively binds to the surface of MagAttract particles and is further purified by washing the magnetic particles with alcohol containing buffers and ethanol Pure genomic DNA is eluted from the particles with low salt Buffer AE into a 96 Well Microplate and is ready for
12. duct does not meet your expectations simply call your local Technical Service Department or distributor We will credit your account or exchange the product as you wish A copy of QIAGEN terms and conditions can be obtained on request and is also provided on the back of our invoices If you have questions about product specifications or performance please call QIAGEN Technical Services or your local distributor see back cover Contains harmful chemicals Take appropriate laboratory safety measures and wear gloves when handling Not compatible with bleach See page 6 for detailed safety information 4 MagAttract 96 DNA Plant Handbook 08 2003 Product Use Limitations The MagAttract 96 DNA Plant Core Kit is developed designed and sold for research purposes only It is not to be used for human diagnostic or drug purposes or to be administered to humans unless expressly cleared for that purpose by the Food and Drug Administration in the USA or the appropriate regulatory authorities in the country of use All due care and attention should be exercised in the handling of many of the materials described in this text 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
13. e DNA eluates to a clean 96 well round bottom microplate or microcentrifuge tube 14 MagAttract 96 DNA Plant Handbook 08 2003 Protocol DNA Purification Using the MagAttract 96 DNA Plant Core Kit and the BioRobot Plant Science Workstation Plant genomic DNA minipreps can be prepared using the MagAttract 96 DNA Plant Core Kit on the BioRobot Plant Science Workstation The following information provides details of the preparation required for this procedure Full descriptions of the preparation procedure and protocol are provided with the BioRobot Plant Science System A MagAttract protocol using the BioRobot RapidPlate for automation of some steps is also available upon request from QIAGEN Technical Services The BioRobot Plant Science Workstation consists of a BioRobot 3000 Workstation and a RapidPlate 96 well pipetting device In the MagAttract Plant procedure the BioRobot 3000 Workstation distributes the MagAttract Plant suspension for binding and the buffers for washing and elution steps It also resuspends the beads by shaking and efficiently removes ethanol from the beads by drying on the integrated cooling and heating device Magnetic separation is performed on the RapidPlate 96 well pipetting device which also enables rapid removal of supernatants and wash buffers and transfers eluates into a microplate without cross contamination For further information refer to the BioRobot 3000 and BioRobot RapidPlate User Manual Equipment
14. e of removing the supernatant slightly off center of each well This minimizes the danger of particle carryover Add 200 pl of Buffer RPW and resuspend the pelleted MagAttract particles thoroughly by pipetting up and down or by shaking Place the flat bottom microplate onto the magnet allow the MagAttract particles to separate for 20 s and remove the supernatant Add 200 pl of ethanol 96 100 and resuspend the MagAttract particles thoroughly by pipetting up and down or by shaking MagAttract 96 DNA Plant Handbook 08 2003 19 0204014 Q PH o A e o o a on p 3 72 nn 5 LN o gt uv Vv o 0 8 O Ow t ulum Q 11 12 20 Place the flat bottom microplate onto the magnet allow the MagAttract particles to separate for 20 s and remove the supernatant Repeat steps 8 and 9 During the final removal of the supernatant try to aspirate as much of the ethanol as possible Dry the MagAttract particles for 5 10 min at room temperature Add 100 pl of Buffer AE to each well resuspend the MagAttract particles thoroughly by pipetting up and down and or vortexing and incubate for 5 min at room temperature Place the flat bottom microplate onto the magnet allow the MagAttract particles to separate for 1 min and transfer the supernatant to a clean 96 well round bottom microplate The DNA can be quantified directly or used for downstream reactions Note This step
15. ems 21 Troubleshooting Guide 23 Appendix A Protocol Modifications for Difficult Plant Material 27 Appendix B Using 1 ml Round Well Blocks for the MagAttract 96 DNA Plant Procedure 28 Appendix C Determination of DNA Concentration and Yield 29 Appendix D Recovery and Cleaning of Tungsten Carbide Beads 29 Appendix E Cleaning of S Blocks 29 Ordering Information 30 MagAttract 96 DNA Plant Handbook 08 2003 3 Kit Contents MagAttract 96 DNA Plant Core Kit 6 24 Catalog no 67161 67163 Number of preps 576 6 x 96 2304 24 x 96 MagAttract Suspension A 25 ml 100 ml Buffer RB 200 ml 2 x 200 ml Buffer RLT 220 ml x 220 ml Buffer RPW 125 ml 4 x 125 ml RNase A 100 mg ml 220 pl 4 x 220 yl Buffer AE 110 ml 3 x 128 ml Storage Conditions All kit components buffers and RNase A stock solution can be stored at room temperature 15 25 C After addition of RNase A and isopropanol Buffer RPW is stable for 6 months when stored at 2 8 C Product Warranty and Satisfaction Guarantee QIAGEN guarantees the performance of all products in the manner described in our product literature The purchaser must determine the suitability of the product for its particular use Should any product fail to perform satisfactorily due to any reason other than misuse 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 pro
16. he HCI Appendix E Cleaning of S Blocks To avoid cross contamination after each use rinse the S Blocks thoroughly in tap water incubate for 1 min at room temperature in 0 4 M HCI empty and wash thoroughly with distilled water Used S Blocks can also be autoclaved after washing Additional S Blocks can be ordered separately see Ordering Information page 30 MagAttract 96 DNA Plant Handbook 08 2003 27 Ordering Information Product Contents Cat No MagAttract 96 DNA Plant Core MagAtiract Suspension A and 67161 Kit 6 buffers for 6 x 96 minipreps MagAttract 96 DNA Plant Core MagAtiract Suspension A and 67163 Kit 24 buffers for 24 x 96 minipreps MagAttract 96 DNA Plant Core MagAttract Suspension A and 67165 Kit 10 x 24 buffers for 10 x 24 x 96 minipreps Accessories Collection Microtubes racked Nonsterile polypropylene tubes 19560 1 2 ml 960 in racks of 96 Collection Microtube Caps Nonsterile polypropylene caps for 19566 collection microtubes 1 2 ml 960 in strips of 8 Collection Microtubes 24 Nonsterile polypropylene tubes 120008 1 2 ml and caps 24 racks with 96 tubes 96 Well Microplates FB 24 96 well microplates with flat bottom 36985 wells 24 per case for use with the 96 Well Magnet 96 Well Microplates RB 24 96 well microplates with 19581 round bottom wells plus lids 24 per case Tape Pads 5 Adhesive tape sheets for sealing 19570 multiwell plates and blocks 25 sheets per pad 5 pads per pack
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18. ic particles Note that the ethanol substitution for Buffer RB is not suitable for the automated BioRobot Plant Science protocol Inhibition of PCR Replace Buffer RLT by Buffer AP1 not included in the kit contact QIAGEN Technical Services for details Note that in many cases the yield will be lower depending on the sample source but the performance in PCR may be better Increase the volume of Buffer RLT used for lysis to up to 400 600 ul but process only the recommended 200 jl of lysate for DNA purification These recommendations can also be followed when using Buffer APT Polyphenolic compounds may be copurified with some plant materials and these can inhibit downstream reactions Polyphenols can be removed by adding 33 mg ml insoluble polyvinyl polypyrrolidone PVPP to Buffer RLT before use PVPP forms complex hydrogen bonds with polyphenolic compounds which are then separated from the DNA during the lysate centrifugation step Prepare a small aliquot of Buffer RLT containing 33 mg ml PVPP and thoroughly vortex the modified lysis buffer before use MagAttract 96 DNA Plant Handbook 08 2003 25 Problem Suggestion Low yields and inhibition Replace Buffer RLT with Buffer APT Replace Buffer RB of PCR 65 pl with ethanol 225 yl Check whether your magnetic separation principle device is suited for this option because addition of alcohol affects the physical behavior of the magnetic particles Note that the ethanol substit
19. ing the MagAttract procedure on robotic systems Things to do before starting Add 125 ml isopropanol and 1 vial RNase A 1 x 220 yl to each bottle of Buffer RPW 125 ml before use Shake the bottle containing MagAttract Suspension A and vortex for 5 minutes before first use or 1 minute before subsequent uses to ensure that the magnetic particles are fully resuspended before filling the reagent reservoir Procedure m 2 Add 65 pl of Buffer RB to each well of a 96 well flat bottom microplate Add 20 pl of resuspended MagAttract Suspension A to each well of the 96 well flat bottom microplate containing Buffer RB Note Buffer RB and MagAttract Suspension A can be combined in appropriate proportions to make a master mix before starting the procedure Add 85 pl master mix to each well of the 96 well flat bottom microplate Ensure that the MagAttract particles are fully resuspended Carefully transfer 200 pl of each supernatant from the plant lysates to the 96 well flat bottom microplate containing MagAttract Suspension A and Buffer RB Mix the samples thoroughly by pipetting up and down several times Incubate the samples for 2 min at room temperature 15 25 C Mix thoroughly by pipetting up and down and incubate for an additional 2 min at room temperature Place the flat bottom microplate onto a suitable magnet allow the MagAttract particles to separate for 20 s and then remove the supernatant Some robotic systems are capabl
20. ion A and vortex for 5 minutes before first use or 1 minute before subsequent uses to ensure that the MagAttract particles are fully resuspended before filling the disposable trough Fill each well of a 96 well block 2 2 ml wells with 1 ml distilled water MagAttract 96 DNA Plant Handbook 08 2003 Recommendations for Using MagAttract Technology with Robotic Systems The following guidelines should be followed when using robotic liquid handling and processing platforms for the MagAttract 96 DNA Plant procedure and as a starting point for optimization All automated protocols for the MagAttract 96 DNA Plant Core Kit start with cleared plant lysates step 13 on page 12 Robotic platforms should be equipped with the following features M Atleast an 8 channel pipetting system ideally a 96 channel system in addition to a 4 or 8 channel system M Plate handling device robotic hand to move plates M Plate shaker with a shaking speed higher than 500 rpm for resuspension of magnetic particles M Magnet for separation e g QIAGEN Magnet 96 well Type B M Plate heating device to dry MagAttract particles optional Since different robotic platform have different features and programming options we can provide only general recommendations for the MagAttract 96 DNA Plant procedure The main steps of the protocol are summarized below A fully automated protocol on the QIAGEN BioRobot Plant Science Workstation is also available p
21. iven centrifuge centrifuge the plates at maximum speed Increase the time of centrifugation if necessary Handling guidelines for the Magnet 96 well Type B The QIAGEN Magnet 96 well Type B is designed for rapid efficient and convenient separation of MagAttract particles from solutions in 96 well microplates and round well blocks The magnet consists of an array of 24 magnetic NdFeB rods that fit between the wells of a microplate or round well block Each magnetic rod quickly attracts the particles in four adjacent wells to one side of each well and holds the particles in place while the buffer is removed When the microplate or round well block is removed from the magnet the MagAttract particles are easily resuspended in buffer allowing thorough washing and elution MagAttract 96 DNA Plant Handbook 08 2003 9 Equipment and Reagents to Be Supplied by the User 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 available from the supplier Equipment M Equipment for disrupting plant tissue We recommend the Tissuelyser with the Tissuelyser Adapter Set 2 x 96 and reusable 3 mm tungsten carbide or stainless steel beads for optimal disruption M Centrifuge 4 15C or 4K15C with Plate Rotor 2 x 96 M Magnet for separation compatible with 96 well flat bottom and round bottom microplates e g QI
22. loride harmful irritant Risk and safety phrases R36 38 S13 26 36 46 RNase A Contains ribonuclease sensitizer Risk and safety phrases R42 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 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 RA2 43 May cause sensitization by inhalation and skin contact 13 Keep away from food drink and animal feed 23 Do not breathe gas fumes vapor or spray S24 Avoid contact with skin S26 In case of contact with eyes rinse immediately with plenty of water and seek medical advice S36 Wear suitable protective clothing 536 37 Wear suitable protective clothing and gloves S46 If swallowed seek medical advice immediately and show this container or label MagAttract 96 DNA Plant Handbook 08 2003 7 Introduction The MagAttract 96 DNA Plant Core Kit combines the speed and efficiency of silica based DNA purification with the convenience of magnetic particles and is designed for fully automated high throughput minipreparation of genomic chloroplast and mitochondrial DNA from plant tissue MagAttract technology provides high purity DNA which is ready for use in downstream applications such as PCR This handbook
23. ocentrifuge tube on the magnet and remove the supernatant after magnetic separation Wash the pelleted MagAttract particles by adding 200 pl Buffer RPW resuspending the particles placing the plate or tube on the magnet and removing the supernatant Note Resuspension of the magnetic particles should be done very carefully since the efficiency of washing is directly related to how well the particles are resus pended Resuspension can be performed by pipetting see previous note or by vortexing If 96 well plates are vortexed the initial mixing should be done very carefully to avoid spilling the magnetic particle suspension out of the wells and contaminating others If this is too difficult with an initial volume of 200 yl add 100 pl of Buffer RPW vortex and then add an additional 100 yl Wash the pelleted MagAttract particles by adding 200 pl ethanol 96 100 resuspending the particles placing the plate or tube on the magnet and removing the supernatant MagAttract 96 DNA Plant Handbook 08 2003 13 Q gt cC a c le e D Q e im oO 020 044 Lum 5 o 9 EE e5 a 5 i e Repeat step 7 aspirating as much ethanol as possible 9 Dry the MagAttract particles for 5 10 min at room temperature 15 25 C 10 Resuspend the MagAttract particles in 100 pl Buffer AE 11 Incubate at room temperature for 5 min 12 Place the plate or microcentrifuge tube on a magnet and transfer th
24. ontamination in Check that RNase A was added to Buffer RPW the eluate Overestimation of yield can lead to failure of down stream applications b Insufficient washing of Change the pipetting speeds and their duration or the MagAttract particles increase the number of wash steps to improve the wash efficiency Salt peaks at 200 240 nm in spectrophotometric scans Insufficient washing of the Change the pipetting speeds and their duration or the MagAttract particles number of wash steps to improve the wash efficiency However salt carryover does not necessarily interfere with downstream applications Green red or yellow colored eluates Insufficient washing of the Chlorophyll or carotenoids can be efficiently removed MagAttract particles by adding one additional wash step using ethanol or by reducing the amount of starting material in future preparations Slight color in the eluate does not neces sarily interfere with downstream applications Pipetting problems a Lysate too viscous In future preparations reduce the amount of starting material and or increase the volume of Buffer RLT to 400 600 yl Use 200 yl of supernatant in the MagAttract 96 DNA Plant procedure 22 MagAttract 96 DNA Plant Handbook 08 2003 Comments and suggestions b Viscous and turbid lysates If seeds are used as starting material the crude lysate may be very viscous and turbid This may result in low yields of DNA problems with sample proce
25. p 4 Normally 30 mg of starting material is sufficient Do not use more than 50 mg wet weight unless preliminary experiments suggest that the optimal amount is higher Add one tungsten carbide or stainless steel bead to each collection microtube and seal the tubes with the caps supplied Note that Buffer RLT may corrode tungsten carbide beads if samples are stored for more than 6 hours Cool the collection microtubes in liquid nitrogen Ensure that the microtubes remain tightly closed Place a clear cover saved from step 1 over each rack of collection microtubes and knock the racks upside down against the bench 5 times to ensure that all tungsten carbide beads can move freely within the collection microtubes Ensure that no liquid nitrogen remains but do not allow the leaf material to thaw Remove the clear cover Sandwich each rack of collection microtubes between adapter plates and fix into the TissueLyser clamps as described in the TissueLyser instruction manual Ensure that the microtubes are properly sealed with caps IMPORTANT Two plate sandwiches must be clamped to the Tissuelyser to provide balance To process 96 or fewer samples assemble a second plate sandwich using a rack of collection microtubes containing tungsten carbide beads but no samples or buffers and fix it into the empty clamp Shake the samples for 1 min at 30 Hz Remove and dismantle the plate sandwiches Ensure that the collection microtubes are tightly clo
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27. room temperature 15 25 C but the time for drying can be reduced and the reproducibility improved if the robotic workstation has an integrated heating device for microplates We recommend drying the particles for 7 minutes at 50 C although the drying time is dependent on the efficiency of the removal of the supernatant after the last wash cycle Note The particles should not be overdried since this makes resuspension of the particles in elution buffer on an automated workstation very difficult An alternative to the drying step The particles can be rinsed with distilled water while the microplate is on the magnet The plate is kept on the magnet after removal of the supernatant from the last wash cycle to keep the particles fixed to the wall of the wells Distilled water is then dispensed into the wells and is immediately aspirated All pipetting is carried out at low speed Note Rinsing the MagAttract particles with water while they are attracted to the magnet may slightly decrease the yield of DNA 18 MagAttract 96 DNA Plant Handbook 08 2003 Protocol Adaptation of MagAttract Technology to Robotic Systems Some steps of the MagAttract 96 DNA Plant procedure can be automated using a standalone 96 channel pipetting device such as the BioRobot RapidPlate protocol available upon request from QIAGEN Technical Services Walkaway automation can be established on some workstations This section provides general guidelines for establish
28. sed Cool the collection microtube racks again in liquid nitrogen and then knock the racks against the bench 5 times to ensure that no tissue powder remains in the caps MagAttract 96 DNA Plant Handbook 08 2003 11 o un O g un O e 5 U ipe 2 Q e c ae o o 2 dE 6A gt N a _ 12 13 Reassemble the plate sandwiches so that the collection microtubes nearest the TissueLyser in steps 5 and 6 are now outermost Reinsert the plate sandwiches into the Tissuelyser Rotating the racks of collection microtubes in this way ensures that all samples are thoroughly disrupted IMPORTANT Merely rotating the entire plate sandwich so that the QIAGEN logos are upside down when reinserted into the Tissuelyser is not sufficient since the samples that were outermost during the initial disruption will remain outermost in the second disruption step Shake the samples for 1 min at 30 Hz Carefully remove the caps from the collection microtubes and immediately pipet 300 pl Buffer RLT into each collection microtube Reseal the tubes with the caps and shake the entire rack in an upright position 20 times back and forth Vortex the rack of collection microtubes upside down at full speed for 20 s Centrifuge the rack of collection microtubes for 5 min at 6000 x g Proceed with the relevant purification protocol MagAttract 96 DNA Plant Handbook 08 2003 Protocol Manual DNA Purifica
29. ssing and carryover of inhibitors Generally a turbid lysate indicates that too much plant material was used For optimal results reduce the amount of starting material If it is not possible to use less material we recommend increasing the volume of Buffer RLT to 400 600 yl If the ground material is highly absorbent larger buffer volumes may be used Use 200 yl of supernatant in the MagAttract 96 DNA Plant procedure c After binding of the In future preparations reduce the amount of starting DNA to the particles the material and or increase the volume of Buffer RLT to particles clump and form 400 600 pl Use 200 pl of supernatant in the MagAttract large complexes making 96 DNA Plant procedure liquid handling difficult Variation of yields across the plate a Varying amount of Adjust the amounts of starting materials accordingly starting material across the plate b Racks of collection It is essential to turn the racks of collection microtubes microtubes not turned during disruption in the TissueLyser to ensure that all during disruption when samples are evenly disrupted see page 12 step 8 using TissueLyser c Non uniform sample Ensure that all samples are uniformly disrupted disruption when using alternative disruption methods MagAttract 96 DNA Plant Handbook 08 2003 23 Comments and suggestions DNA does not perform well in downstream experiments a b cj d 24 Ethanol carryover Insuffi
30. successfully purified from many other plants including sugar beet leaves and seeds rose Lactuca serriola Amaranthus maple soy cotton rice peanut castor gherkin red gherkin spinach cauliflower onion white cabbage carrot red beet bleach celery and oat 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 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 RPW and RLT 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 The following risk and safety phrases apply to components of the MagAttract 96 DNA 6 MagAttract 96 DNA Plant Handbook 08 2003 Plant Core Kit Buffer RLT Contains guanidine thiocyanate harmful Risk and safety phrases R20 21 22 32 S13 26 36 46 Buffer RPW Contains guanidine hydroch
31. tion Things to do before starting Add 125 ml isopropanol and 1 vial RNase A 1 x 220 yl to each bottle of Buffer RPW 125 ml before use Shake the bottle containing MagAttract Suspension A and vortex for 5 minutes before first use or 1 minute before subsequent uses to ensure that the magnetic particles are fully resuspended before use Procedure l Add 65 yl of Buffer RB to each well of a flat bottom microplate or 1 5 ml micro centrifuge tube Add 20 yl of resuspended MagAttract Suspension A to each well of the 96 well flat bottom microplate or into the 1 5 ml microcentrifuge tube Note Buffer RB and MagAttract Suspension A can be combined in appropriate proportions to make a master mix before starting the procedure Add 85 ul of the master mix to each well of the 96 well flat bottom microplate Ensure that the MagAttract particles are fully resuspended Transfer 200 pl plant lysate supernatant into each well of the microplate or into the microcentrifuge tube and mix by pipetting up and down several times Incubate at room temperature 15 25 C for 5 min Mix once during incubation Note Mixing can be done by pipetting using an 8 channel pipet The pipet tips needed for this step can be reused if they are returned to the tip rack after use To avoid cross contamination ensure that the same tip is always used for the same well When using microcentrifuge tubes samples can be mixed by vortexing Place the plate or micr
32. use in downstream applications Disruption of plant tissue For disruption of plant tissue optimal results are obtained using the Tissuelyser together with the Tissuelyser Adapter Set 2 x 96 and Tungsten Carbide Beads stainless steel beads can also be used The Tissuelyser provides rapid and efficient disruption of 2 x 96 samples in 2 4 minutes Plant material and a 3 mm bead are added to each of 192 collection microtubes in two racks The racks are fixed into the clamps on the Tissuelyser using adapter plates and disrupted by two 1 minute high speed 30 Hz shaking steps We recommend the use of tungsten carbide beads as these provide better and more reproducible results than chrome steel beads For some samples such as small seeds 5 mm beads should be used for grinding to ensure production of a homogenous plant powder Either fresh or lyophilized plant tissue samples can be processed using the Tissuelyser Frozen material should be disrupted under freezing conditions using liquid nitrogen Fresh material can also be disrupted in lysis buffer but this may cause shearing of high molecular weight DNA Lyophilized material should be disrupted without lysis buffer Centrifugation The recommended speed for the centrifugation step in the protocol is 6000 x g using the Centrifuge 4 15C or the Centrifuge 4K15C equipped with the QIAGEN Plate Rotor 2 x 96 If these centrifuges are not available or the recommended speed cannot be applied on the g
33. ution for Buffer RB is not suitable for the automated BioRobot Plant Science protocol Appendix B Using 1 ml Round Well Blocks for the MagAttract 96 DNA Plant Procedure Round well blocks with 1 ml wells can be used on some robotic workstations allowing larger volumes of plant lysate to be processed The protocol can be adapted to these plates but note that standard 96 well microplates may provide better results on some workstations The use of 1 ml round well blocks may result in insufficient resuspension of the magnetic particles after magnetic separation The following recommendations for adaptation of the MagAttract 96 DNA Plant procedure to 1 ml round well blocks are provided as a guide M Resuspend the disrupted plant material in 500 pl Buffer RLT M Add 400 yl cleared plant lysate to 130 pl Buffer RB and 20 pl MagAttract Suspension M Use 200 400 pl of Buffer RPW for the first washing step followed by 200 400 pl ethanol for the second and third washing steps The volume of wash buffer required is dependent on the speed of the shaking platform used to resuspend the MagAttract particles 26 MagAttract 96 DNA Plant Handbook 08 2003 Appendix C Determination of DNA Concentration and Yield DNA concentration can be determined by measuring the absorbance at 260 nm A350 in a spectrophotometer For greatest accuracy absorbance readings at 260 nm should be between 0 1 and 1 0 Sample dilution should be adjusted accordingly for e
34. xample an eluate with an expected DNA concentration of 25 50 ng yl A0 0 5 1 should not be diluted more than fourfold for spectrophotometry An absorbance of 1 at 260 nm corresponds to 50 yg DNA per ml This relationship is only valid for measurements made in water therefore samples should be diluted in water Use water to zero the spectrophotometer We recommend measuring the absorbance at 260 nm of the purified MagAttract 96 DNA Plant samples using a fourfold dilution DNA yields from different plant species depend greatly on genome size and ploidy and also on the age and growth state of the plant Appendix D Recovery and Cleaning of Tungsten Carbide Beads or Stainless Steel Beads Tungsten carbide beads are designed for repeated use Used tungsten carbide beads can be recovered from cell debris pellets and cleaned using the procedure below 1 Seal the collection microtubes with caps Place a clear cover saved from step 1 of the MagAttract 96 DNA Plant procedure page 11 over each rack of collection microtubes and knock the racks upside down against the bench 5 times to free the tungsten carbide beads from the surrounding material 2 Empty the contents of the microtubes into a fine sieve and rinse the beads thoroughly with water 3 Incubate beads in 0 4 M HCI for 1 min at room temperature 15 25 C to degrade any DNA and avoid cross contamination in future preparations 4 Rinse beads thoroughly with distilled water to remove t
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