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1. 2 ml DNeasy Plant Mini Kit 250 250 DNeasy Mini Spin Columns 69106 250 QlAshredder Mini Spin Columns RNase A Buffers Collection Tubes 2 ml DNeasy Plant Maxi Kit 6 6 DNeasy Maxi Spin Columns 68161 6 QlAshredder Maxi Spin Columns RNase A Buffers Collection Tubes 50 ml DNeasy Plant Maxi Kit 24 24 DNeasy Maxi Spin Columns 68163 24 QlAshredder Maxi Spin Columns RNase A Buffers Collection Tubes 50 ml DNeasy 96 Plant Kit 6 6 DNeasy 96 Plates Buffers 69181 Reagents RNase A S Blocks Collection Microtubes 1 2 ml Caps AirPore Tape Sheets TissueRuptor TissueRuptor Handheld rotor stator homogenizer Inquire TissueRuptor Disposable 25 nonsterile plastic disposable 990890 Probes 25 probes for use with the TissueRuptor TissueLyser System Tissuelyser II Universal laboratory mixer mill 85300 disruptor 100 120 220 240 V 50 60 Hz Tissuelyser LT Bead mill for low to medium 85600 throughput sample disruption Tissuelyser LT Adapter Adapter for disruption of up to 69980 12 Tube 12 samples in 2 ml microcentrifuge tubes on the Tissuelyser LT The Tissuelyser Il must be used in combination with the Tissuelyser Adapter Set 2 x 24 or Tissuelyser Adapter Set 2 x 96 DNeasy Plant Handbook 10 2012 49 Ordering Information Product Contents Cat no Tissuelyser Adapter Set 2 sets of Adapter Plates and 2 racks 69982 2x 24 for use with 2 0 ml microcentrifuge tubes on the TissueLyser Tissuelyser Adapter Set 2 s
2. 22 34 Remove and discard the caps from the collection microtubes Carefully transfer 1 ml of each sample to each well of the DNeasy 96 plates Take care not to wet the rims of the wells to avoid aerosols during centrifugation Do not transfer more than 1 ml per well Note Lowering pipet tips to the bottoms of the wells may cause sample overflow and cross contamination Therefore remove one set of caps at a time and begin drawing up the samples as soon as the pipet tips contact the liquid Repeat until all the samples have been transferred to the DNeasy 96 plates Seal each DNeasy 96 plate with an AirPore Tape Sheet provided Centrifuge for 4 min at 6000 rpm AirPore Tape prevents cross contamination between samples during centrifugation After centrifugation check that all of the lysate has passed through the membrane in each well of the DNeasy 96 plates If lysate remains in any of the wells centrifuge for a further 4 min Remove the tape Carefully add 800 pl Buffer AW2 to each sample Note Ensure that ethanol has been added to Buffer AW2 prior to use See Things to do before starting page 30 Centrifuge for 15 min at 6000 rpm to dry the DNeasy membranes For efficient drying do not reseal the DNeasy 96 plate with AirPore Tape IMPORTANT Residual ethanol in the DNeasy membranes derived from Buffer AW2 may inhibit PCR and must be removed by centrifugation before elution of the DNA Note DNeasy membranes are
3. For example to 450 pl lysate add 675 pl Buffer AW1 Reduce the amount of Buffer AW 1 accordingly if the volume of lysate is smaller A precipitate may form after the addition of Buffer AW 1 but this will not affect the DNeasy procedure Note Ensure that ethanol has been added to Buffer AW 1 See Things to do before starting page 22 Note It is important to pipet Buffer AW1 directly onto the cleared lysate and to mix immediately 14 Pipet 650 pl of the mixture from step 13 including any precipitate that may have formed into the DNeasy Mini spin column placed in a 2 ml collection tube supplied Centrifuge for 1 min at gt 6000 x g corresponds to gt 8000 rpm for most microcentrifuges and discard the flow through Reuse the collection tube in step 15 15 Repeat step 14 with remaining sample Discard flow through and collection tube Flow through fractions contain Buffer AW1 and are therefore not compatible with bleach See page 6 for safety information ee ee 24 DNeasy Plant Handbook 10 2012 16 Place the DNeasy Mini spin column into a new 2 ml collection tube supplied add 500 pl Buffer AW2 and centrifuge for 1 min at 26000 x g 28000 rpm Discard the flow through and reuse the collection tube in step 17 Note Ensure that ethanol is added to Buffer AW2 See Things to do before starting page 22 17 Add 500 pl Buffer AW2 to the DNeasy Mini spin column and centrifuge for 2 min at 20 000 x g 14 000 rpm to
4. to 65 C to redissolve before adding ethanol Do not heat Buffer AW1 after ethanol has been added E Preheat Buffer AP1 to 65 C This heating is necessary for the DNeasy Plant Maxi procedure and will also dissolve any precipitate that may have formed in Buffer API E Buffer AW2 and Buffer AW1 are supplied as concentrates Before using for the first time add the appropriate amount of ethanol 96 100 as indicated on the bottle to obtain a working solution HE Preheat a water bath or heating block to 65 C Procedure 1 For disruption using the TissueRuptor follow step 2 for disruption using the TissueLyser follow steps 3 7 Alternatively plant or fungal tissue can be ground to a fine powder under liquid nitrogen using a mortar and pestle Transfer the tissue powder and liquid nitrogen to an appropriately sized tube and allow the liquid nitrogen to evaporate Do not allow the sample to thaw Proceed immediately to step 8 26 DNeasy Plant Handbook 10 2012 2 TissueRuptor procedure Place the sample material lt 1 g wet weight or lt 0 2 g lyophilized tissue into a 15 ml centrifuge tube Add liquid nitrogen to the tube and freeze the sample for 30 s Keep the sample submerged in liquid nitrogen and disrupt at full speed until the sample is homogenized Allow the liquid nitrogen to evaporate and proceed immediately to step 8 Disruption time depends on the starting material used Keep the disruption time as short as possible t
5. 96 plate a Varying amount of starting Adjust the amounts of starting materials material across the DNeasy accordingly 96 plate 44 DNeasy Plant Handbook 10 2012 Comments and suggestions b Racks of collection microtubes It is essential to turn the racks of collection not turned during disruption microtubes during disruption in the when using the Tissuelyser Tissuelyser to ensure all samples are evenly disrupted see Disruption and homogenization using the Tissuelyser System page 15 c Nonuniform sample disruption Ensure that all samples are uniformly when using alternative disruption disrupted methods DNeasy Plant Handbook 10 2012 45 Appendix A Determination of Yield Purity and Length of DNA Determination of yield and purity The concentration and purity of DNA can be determined by measuring the absorbance at 260 nm Azs and 280 nm Aso in a spectrophotometer To ensure accuracy make sure the absorbance readings fall into the linear range of your method e g between 0 1 and 1 0 for spectrophotometric OD Sample dilution should be adjusted accordingly An absorbance of 1 0 at 260 nm corresponds to 50 pg of DNA per milliliter Ao 1 50 pg ml We recommend scanning absorbance from 220 320 nm as this will indicate whether other factors are interfering with absorbance at 260 and 280 nm DNA samples from plant tissue offen contain copurified polysaccharides and other metabolites which can interfere with
6. 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 2006 2012 QIAGEN all rights reserved www giagen com Australia techservice au qiagen com Austria techservice at qiagen com Belgium techservice bnl giagen com Brazil suportetecnico brasil giagen com Canada techservice ca qiagen com China techservice cn qiagen com Denmark techservice nordic giagen com Finland techservice nordic qiagen com France techservice fr giagen com Germany techservice de qiagen com Hong Kong techservice hk qiagen com India techservice india qiagen com Ireland techservice uk qiagen com Italy techservice it giagen com Japan techservice jp giagen com Korea South techservice kr giagen com Luxembourg techservice bnI giagen com Mexico techservice mx qiagen com The Netherlands techservice bnl giagen com Norway techservice nordic qiagen com Singapore techservice sg qiagen com Sweden techservice nordic giagen com Switzerland techservice ch qiagen com UK techservice uk giagen com USA techservice us qiagen com 1074217 10 2012 QIAGEN Sample amp Assay Technologies
7. DNA does not perform well in downstream experiments a Ethanol carryover Ensure that during the second wash with Buffer AW2 the DNeasy spin column or DNeasy 96 plate is centrifuged at 20 000 x g 14 000 rpm for 2 min Mini at 3000 5000 x g for 10 min Maxi or at full speed for 15 min DNeasy 96 to dry the membrane After centrifugation remove the DNeasy spin column or DNeasy 96 plate carefully from the collection tubes so the column or plate does not come into contact with the flow through as this will result in carryover of ethanol b Salt carryover Ensure that Buffer AW2 is at room temperature before use c Insufficient excess DNA used in Optimize the amount of DNA used in the downstream application downstream application if necessary Downstream applications can be adversely affected by insufficient or excess DNA d DNeasy 96 protocols Eluate is In future preparations include an additional slightly green or yellow wash with 800 pl ethanol 96 100 after washing with Buffer AW2 If 2 wash steps are performed the first centrifugation step should be 5 min at 5600 x g after which the flow through should be discarded from the S Block before performing the second wash step Ensure that residual ethanol is removed during the second wash step by centrifuging for 15 min at maximum speed Slight color in the eluate will not necessarily interfere with downstream applications DNeasy 96 protocols Yields vary across DNeasy
8. Plant Maxi Kit with up to 1 g wet weight fresh frozen or lyophilized plant tissue following the specifications in Table 1 The Purification of Total DNA from Fresh Plant Tissue DNeasy 96 Protocol page 30 is for use with the DNeasy 96 Plant Kit with fresh plant tissue following the specifica tions in Table 1 This protocol is optimized for use with the Tissuelyser System for effi cient and convenient high throughput disruption of plant tissue The Purification of Total DNA from Frozen or Lyophilized Plant Tissue DNeasy 96 Protocol page 35 is for use with the DNeasy 96 Plant Kit with frozen or lyophilized plant tissue following the specifications in Table 1 This protocol is optimized for use with the Tissuelyser System for efficient and convenient high throughput disruption of plant tissue Table 1 Specifications for DNeasy Plant Kits DNeasy Plant Kit Mini Maxi 96 amount well Maximum amount of 100 mg wet 1 g wetweight 50 mg wet weigth starting material weight 0 2 g dry 10 mg dry weigth 20 mg dry weight weight DNA binding capacity 50 pg 500 pg 50 pg Maximum loading volume 700 pl 15 ml I ml Elution volume 50 400 pl 500 2000 100 200 pl Typical yields 3 30 pg 30 260 pg 1 15 pg Preparation time lt 1 hour lt 2 hours lt 2 hours 192 samples Exceeding the recommended amount of starting material will reduce yield and purity t Typically the binding capacity will not be reached since the D
9. and allowed to cool precipitates may form when the solution is added to the disrupted leaf material If this occurs redissolve the precipitates by incubating the racks of collection microtubes in a water bath at 65 C for 10 20 min Place a heavy plate over the racks during incubation to prevent the caps from coming off Remove all water that gt 2S has entered the racks before centrifuging in the following step 8 Prior to opening the microtubes make sure that the racks were knocked against a sZ bench to remove any tissue powder from the caps step 11 The microtubes may N 3 become brittle at low temperature Take care not to break the connection between ie the tubes when removing the caps Volume for 2 x 96 Volume per sample samples Buffer AP1 preheated to 80 C 400 pl 90 ml RNase A 100 mg ml 1 pl 225 pl Reagent DX 1 pl 225 pl 15 excess mixture is included in these calculations to allow for pipetting errors t When using lyophilized tissue without using liquid nitrogen preheat Buffer AP to 65 C Reagent DX is viscous 38 DNeasy Plant Handbook 10 2012 13 Seal the microtubes with new caps provided ensure that the microtubes are properly sealed to avoid leakage during shaking Place a clear cover over each rack of collection microtubes and shake the racks vigorously up and down for 15 s To collect any solution from the caps centrifuge the collection microtubes Allow the centrifuge
10. the microtubes Ensure that no liquid nitrogen remains but do not allow the leaf material to thaw Remove the clear cover DNeasy 96 wn ak Fr rs 2 a ec N o E LL During freezing in liquid nitrogen the leaf material and bead in each collection microtube may stick together hindering disruption in the Tissuelyser This step ensures that the beads are free for optimal disruption Keep the clear covers from the collection microtube racks for use in step 8 When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information consult the appropriate safety data sheets SDSs available from the product supplier 36 DNeasy Plant Handbook 10 2012 5 Sandwich each rack of collection microtubes between adapter plates and fix into TissueLyser clamps as described in the Tissuelyser User Manual Work quickly so that the plant material does not thaw Note Ensure that the microtubes are properly sealed with caps IMPORTANT Do not allow the Tissuelyser adapter plates to come into contact with liquid nitrogen IMPORTANT Two plate sandwiches must be clamped to the Tissuelyser to provide balance To process 96 samples or less 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 6 Grind the samples for 1 min at 20 Hz IMPORTANT Prolonging th
11. tip Options include the Matrix Impact cordless electronic multichannel pipet which has a unique adjustable tip spacing system allowing the user to transfer liquid directly from sample tubes to 96 well plates We recommend using extended tips with a maximum volume of 1250 pl with the Matrix multichannel pipet available from Matrix cat no 8255 for tips with filters or 8252 for tips without filters These multichannel pipets and pipet tips can be purchased from Matrix Technologies Corporation www matrixtechcorp com E Reagent reservoirs for multichannel pipets HE Freezer or cold room at 20 C This is not a complete list of suppliers and does not include many important vendors of biological supplies DNeasy Plant Handbook 10 2012 13 Important Notes Collection and storage of starting material After harvesting if plant tissue will not be used immediately it should be frozen in liquid nitrogen It can then be stored at 80 C for later processing Ground tissue powder can also be stored at 80 C Alternatively tissue can be dried or Iyophilized after harvesting to allow storage at room temperature 15 25 C To ensure DNA quality samples should be completely dried within 24 hours of collection If possible it is preferable to collect young materials e g leaves needles since they contain more cells per weight and therefore result in higher yields In addition young leaves and needles contain smaller amounts of pol
12. 00 mg and determine the amount that provides the highest DNA yield and purity Note The maximum amount of starting material that can be used with the collection microtubes provided in the DNeasy 96 Plant Kit is 50 mg wet weight Other disruption vessels should be used for more than 50 mg starting material 14 DNeasy Plant Handbook 10 2012 Table 2 Approximate wet weights of leaf material Sample Size Approximate wet weight mg Leaf punch 1 5 cm diameter 25 75 Leaf surface area 12 cm e g 4 x 3 cm 170 510 DNA yields vary depending on genome size ploidy and age of sample Yields typically range from 3 30 pg per 100 mg of wet weight sample Disruption and homogenization using the TissueRuptor Homogenization using the TissueRuptor system is carried out by first installing the adapter on the motor drive and placing a disposable probe into the adapter see the TissueRuptor User Manual Fresh or frozen samples can be disrupted after freezing in liquid nitrogen without Buffer AP1 Alternatively fresh material can be directly disrupted in Buffer AP1 without using liquid nitrogen but this may cause shearing of high molecular weight DNA We do not recommend disrupting frozen material in lysis buffer as this can result in low yields and degraded DNA Especially hard tissues such as roots or seeds could cause the disposable probes to break and may not be well suited for use with the TissueRuptor These tissues can be
13. 012 DNeasy 96 Plant Procedure Plant leaf tissue Disruption Ready to use DNA Collect plant tissue Lyse and precipitate polysaccharides Prepare cleared lysate and add binding buffer Bind amp wash Elute into elution microtubes The DNeasy membrane combines the binding properties of a silica based membrane with simple microspin technology or with the QIAGEN 96 Well Plate Centrifugation System see ordering information page 50 DNA adsorbs to the DNeasy membrane in the presence of high concentrations of chaotropic salt which remove water from hydrated molecules in solution Buffer conditions in DNeasy Plant procedures are designed to enable specific adsorption of DNA to the silica membrane and optimal removal of carbohydrates polyphenolics and other plant metabolites 10 DNeasy Plant Handbook 10 2012 Description of protocols Different protocols in this handbook provide detailed instructions to use DNeasy Kits for purification of genomic DNA from plant tissue The protocol Purification of Total DNA from Plant Tissue Mini Protocol page 22 is for use with the DNeasy Plant Mini Kit with up to 100 mg wet weight fresh frozen or lyophilized plant tissue following the specifications in Table 1 The protocol Purification of Total DNA from Plant Tissue Maxi Protocol page 26 is for use with the DNeasy
14. 96 Protocol For DNeasy Mini protocol E Microcentrifuge tubes 1 5 ml E Microcentrifuge with rotor for 2 ml tubes HE Ice Do not use denatured alcohol which contains other substances such as methanol or methylethylketone 12 DNeasy Plant Handbook 10 2012 For DNeasy Maxi protocol EB 15 ml and 50 ml centrifuge tubes The use of disposable polypropylene tubes is recommended Tubes used for the lysis step should be capable of withstanding the g forces involved in centrifugation and should also be compatible with liquid nitrogen E Laboratory centrifuge capable of 3000 5000 x g equipped with a swing out rotor All centrifugation steps are carried out in a conventional laboratory centrifuge e g SIGMA 6 16 series from SIGMA Laborzentrifugen GmbH www sigma zentrifugen de using a swinging bucket rotor DNeasy Maxi spin columns and QlAshredder Maxi spin columns fit into the 50 ml centrifuge tubes provided These tubes are compatible with almost all laboratory centrifuges and rotors In the unlikely event that these tubes do not fit your rotor the spin columns can also be used with any other commercially available 50 ml polypropylene or glass tubes E Spatula E ice For DNeasy 96 protocols M Centrifuge 4 16S or 4 16KS with Plate Rotor 2 x 96 see page 17 BE Multichannel pipet with extended tips For efficient processing we recommend the use of an electric multichannel pipet with a capacity of at least 1 ml per pipet
15. A Plant Kit For 360 preps 5 Rod Covers 5 Tube 941517 360 MagAttract Suspension G Buffers and Reagents BioSprint 96 DNA Plant For 576 automated preps on the 941557 Kit 576 BioSprint 96 workstation Large 96 Rod Covers 96 Well Microplates MP S Blocks MagAttract Suspension G Buffers and Reagents MagAttract 96 DNA Plant MagAttract Suspension and buffers 67163 Core Kit 24 for 24 x 96 preps RNeasy Plant Mini Kit 20 For 20 RNA minipreps 20 RNeasy 74903 Mini Spin Columns 20 QlAshredder Mini Spin Columns Collection Tubes 1 5 ml and 2 ml RNase Free Reagents and Buffers RNeasy Plant Mini Kit 50 For 50 RNA minipreps 50 RNeasy 74904 Mini Spin Columns 50 QlAshredder Mini Spin Columns Collection Tubes 1 5 ml and 2 ml RNase Free Reagents and Buffers For up to date licensing information and product specific 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 Larger kit sizes available please inquire DNeasy Plant Handbook 10 2012 51 Notes 52 DNeasy Plant Handbook 10 2012 Notes DNeasy Plant Handbook 10 2012 53 Notes 54 DNeasy Plant Handbook 10 2012 Trademarks QIAGEN BioSprint DNeasy MagAttract RNeasy TissueRuptor QIAGEN Group Impact Matrix Matrix Technologies Corp SIGMA SIGMA Laborzentrifuge
16. Handbook 10 2012 DNeasy 96 Plant Kit DNeasy 96 Plant Kit 6 Catalog no 69181 Number of preps 6x96 DNeasy 96 Plates 6 S Blocks 2 Collection Microtubes 1 2 ml racked 12x96 Collection Microtube Caps 4 x 120 x 8 Elution Microtubes RS racked and caps 6x96 AirPore Tape Sheets 3 x 2S Buffer AP1 2 x 140 ml Buffer P3 2 x 50 ml Buffer AW 1 concentrate 151 ml Buffer AW2 concentrate 2x 81 ml Buffer AE 128 ml RNase A 100 mg ml 2 x 440 pl Reagent DX 1 ml 96 Well Plate Registers 6 Quick Start Protocol 1 information label before use to obtain a working solution Storage Reusable see Appendix B page 48 for cleaning instructions Contains a chaotropic salt Not compatible with disinfectants containing bleach See page 6 for safety Buffer AW1 and Buffer AW2 are supplied as concentrates Add ethanol 96 100 according to the bottle DNeasy Maxi spin columns should be stored dry at 2 8 C upon arrival and are stable for 1 year under these conditions All other components of DNeasy Plant Kits including RNase A stock solution should be stored dry at room temperature 15 25 C and are stable for 1 year under these conditions For storage longer than 1 year or if ambient temperatures often exceed 25 C we recommend keeping the RNase A stock solution at 2 8 C DNeasy Plant Handbook 10 2012 5 Intended Use DNeasy Plant Kits are intended for molecular biology applications These products are no
17. NA content of the recommended amounts of starting material will not exceed 30 pg Mini 300 pg Maxi or 50 pg 96 DNeasy Plant Handbook 10 2012 11 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 safety data sheets SDSs available from the product supplier For all protocols M Equipment for disruption and homogenization We recommend using the TissueRuptor or using the Tissuelyser with the following accessories Protocol TissueLyser accessories DNeasy Mini Adaptor Set 2 x 24 cat no 69982 Tungsten Carbide Beads 3 mm cat no 69997 1 5 ml or 2 ml safe lock microcentrifuge tubes DNeasy Maxi Grinding Jar Set S Steel cat no 69985 DNeasy 96 Adaptor Set 2 x 96 cat no 69984 Tungsten Carbide Beads 3 mm cat no 69997 Collection Microtubes included with DNeasy 96 Plant Kit Stainless Steel Beads 5 mm cat no 69989 can also be used We recommend the use of tungsten carbide beads as these perform better and more consistently than stainless steel beads Pipets and pipet tips Water bath or heating block for heating at 65 C and 80 C Vortexer Ethanol 96 100 Liquid nitrogen not required when processing lyophilized plant material nor when processing fresh plant material in the protocol Purification of Total DNA from Fresh Plant Tissue DNeasy
18. OD readings Absorbance scans should show a symmetric peak at 260 nm and have an overall smooth shape Figure 2 Purity is determined by calculating the ratio of absorbance at 260 nm to absorbance at 280 nm Pure DNA has an Aggo Avgo ratio of 1 7 1 9 0 5 1 Pine 2 Tobacco 0 4 2 x x 3 Oak Absorbance 220 260 320 Wavelenght nm Figure 2 UV scan of DNeasy purified DNA diluted 1 5 in water If present in the same sample both DNA and RNA will be measured with a spectrophotometer If DNA alone is to be quantified in a sample that also contains RNA a fluorimeter must be used DNA purified using DNeasy Plant procedures is free of RNA contamination since an RNase digestion step is included in the procedure Average DNA yields are shown in Table 6 46 DNeasy Plant Handbook 10 2012 Table 6 Average DNA yields obtained with DNeasy Plant Kits Source Yield pg DNA per 100 mg Arabidopsis Arabidopsis thaliana 3 4 Barley Hordeum vulgare 8 12 Fir Abies alba 6 10 Maize Zea mays 15 20 Oak Quercus robur 10 15 Pine Pinus sylvestris 20 25 Potato Solanum tuberosum 4 6 Rape Brassica napus 2 4 Spinach Spinacia oleracea 5 10 Tobacco Nicotiana tabacum 20 25 Tomato Lycopersicon esculentum 10 15 Wheat Triticum aestivum 25 30 DNA yields vary due to genome size ploidy age of sample etc All material was collected as young leaves or needles Determination of length The precise length of genomic DNA should be det
19. October 2012 DNeasy Plant Handbook DNeasy Plant Mini Kit DNeasy Plant Maxi Kit For purification of total cellular DNA from plant cells and tissues or fungi DNeasy 96 Plant Kit For high throughput purification of DNA from plant tissue QIAGEN Sample and Assay Technologies QIAGEN is the leading provider of innovative sample and assay technologies enabling the isolation and detection of contents of any biological sample Our advanced high quality products and services ensure success from sample to result QIAGEN sets standards in Purification of DNA RNA and proteins Nucleic acid and protein assays microRNA research and RNAi Automation of sample and assay technologies Our mission is to enable you to achieve outstanding success and breakthroughs For more information visit www giagen com Contents Kit Contents 4 Storage 5 Intended Use 6 Safety Information 6 Quality Control 6 Introduction 7 Principle and procedure 7 Description of protocols 11 Equipment and Reagents to Be Supplied by User 12 Important Notes 14 Collection and storage of starting material 14 Sample size 14 Disruption and homogenization using the TissueRuptor 15 Disruption and homogenization using the Tissuelyser System 15 Centrifugation DNeasy 96 procedures 17 Lysate filtration with QlAshredder DNeasy Mini and Maxi procedures 19 Elution 19 DNA storage 21 Protocols E Purification of Total DNA from Plant Tissue Mini Protocol 22 E Pur
20. Source 50 mg Elution volume Total DNA yield Concentration young leaves pl pg ng pl Wheat 2 x 50 16 6 166 2 x 100 20 4 102 Lupin 2x50 1 5 15 2x 100 2 0 10 Because the column has a certain residual volume the volume of eluate recovered is always less than the volume of buffer used for elution the actual yield is therefore less than the theoretical yield Preventing dilution of the eluate The first eluate can contain up to 70 of the total DNA yield Therefore to prevent dilution of the first eluate the second elution can be performed separately To elute separately the second eluate should be collected in a separate tube Composition of elution buffer Buffer AE is 10 mM Tris Cl 0 5 mM EDTA pH 9 0 The pH of Buffer AE is optimal for DNA elution from the DNeasy membrane Elution with buffer of pH lower than 9 0 may reduce DNA yield For long term storage of DNA we recommend eluting in Buffer AE because DNA stored in water is subject to acid hydrolysis DNA storage DNA is stable for several weeks when stored at 4 C in Buffer AE For long term storage freezing at 20 C is recommended DNeasy Plant Handbook 10 2012 21 wn ft i ale m Mini Protocol Protocol Purification of Total DNA from Plant Tissue Mini Protocol Important points before starting MH If using the DNeasy Plant Mini Kit for the first time read Important Notes page 14 M Ensure that you are familia
21. Things to do before starting page 35 A white precipitate may form upon addition of Buffer AW1 This precipitate does not interfere with the DNeasy 96 Plant procedure or any subsequent application 20 Close the collection microtubes with new caps provided ensure that the tubes are properly sealed to prevent leakage during shaking Place a clear cover over each rack of collection microtubes and shake the racks vigorously up and down for 15 s To collect any solution from the caps centrifuge the collection microtubes Allow the centrifuge to reach 3000 rpm and then stop the centrifuge DNeasy 96 wn ak J 2 D ec N o E LL Do not prolong this step Note To ensure optimal DNA yields it is important to shake the racks of collection microtubes vigorously up and down with both hands for the full 15 s The genomic DNA will not be sheared by vigorous shaking 21 Place two DNeasy 96 plates on top of S Blocks provided Mark the DNeasy 96 plates for later sample identification 22 Remove and discard the caps from the collection microtubes Carefully transfer 1 ml of each sample to each well of the DNeasy 96 plates Take care not to wet the rims of the wells to avoid aerosols during centrifugation Do not transfer more than 1 ml per well Note Lowering pipet tips to the bottoms of the wells may cause sample overflow and cross contamination Therefore remove one set of caps at a time and begin dr
22. Tissuelyser System page 15 More foam will form in the tubes that were outermost during the initial disruption step IMPORTANT Merely rotating the entire plate sandwich so that the QIAGEN logos are upside down when reinserted into the mixer mill is not sufficient since the same samples that were outermost during the initial disruption will remain outermost in the second disruption step Grind the samples for another 1 5 min at 30 Hz IMPORTANT Prolonging the disruption time may result in shearing of DNA Remove the plate sandwiches from the TissueLyser and remove the adapter plates from each rack of collection microtubes To collect any solution from the caps centrifuge the collection microtubes Allow the centrifuge to reach 3000 rpm and then stop the centrifuge Do not prolong this step Remove and discard caps Add 130 pl Buffer P3 to each collection microtube Close the microtubes carefully with new caps provided ensure that the microtubes are properly sealed to avoid leakage during shaking Place a clear cover saved from step 1 over each rack of collection microtubes and shake the racks vigorously up and down for 15 s To collect any solution from the caps centrifuge the collection microtubes Allow the centrifuge to reach 3000 rpm and then stop the centrifuge Do not prolong this step Note To ensure optimal DNA yields it is important to shake the racks of collection microtubes vigorously up and down with both hands for t
23. aids the precipitation of proteins and inhibitors of downstream applications following addition of Buffer P3 17 Centrifuge the racks of collection microtubes for 5 min at 6000 rpm Compact pellets will form but some particles may float Be careful not to transfer any of these particles in the following step EE DNeasy Plant Handbook 10 2012 39 18 Remove and discard the caps Carefully transfer 400 pl of each supernatant to new racks of collection microtubes provided ensuring that the new tubes are in the correct orientation Do not discard the pellets as they contain the tungsten carbide beads which can be recovered and reused see Appendix B page 48 Do not transfer more than 400 yl of the supernatant as otherwise the capacity of the DNeasy 96 plates and the S Blocks used in subsequent steps will be exceeded If less than 400 pl supernatant is recovered adjust the amount of Buffer AW1 in step 19 accordingly Collection microtubes are connected in strips of 8 To avoid transferring particulate matter it is helpful to remove the strips from the rack so that the contents of the microtubes are visible and to use a multichannel pipet on its lowest speed setting Save the used collection microtubes to recover the tungsten carbide beads at a later stage see Appendix B page 48 19 Add 1 5 volumes typically 600 pl of Buffer AW1 to each sample Note Ensure that ethanol has been added to Buffer AW prior to use See
24. amples directly into the collection microtubes Freeze or lyophilize the harvested samples before starting the protocol DNeasy Plant Handbook 10 2012 35 96 so yNa mal O N arl a wn 7 Cc Procedure 1 Place sample material lt 50 mg wet weight or lt 10 mg lyophilized tissue into each tube in 2 collection microtube racks Unless a different optimal amount of starting material has been previously determined do not use more than 50 mg wet weight or 10 mg lyophilized tissue per sample see Sample size page 14 IMPORTANT Do not allow frozen sample material to thaw during handling and weighing To prevent samples from thawing keep the racks of collection microtubes on a bed of dry ice Keep the clear covers from the collection microtube racks for use in step 4 Use the plate register cards provided to record the position of each sample in the racks 2 Add one tungsten carbide bead to each collection microtube Seal the microtubes with the caps provided 3 Cool the racks of collection microtubes in liquid nitrogen Ensure that the microtubes remain tightly closed When using lyophilized tissue the microtubes do not need to be frozen in liquid nitrogen Continue with step 4 4 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
25. ated from the rotor number and speed and appears automatically in the RCF field It is also possible to enter the RCF value 5796 x g manually in the RCF field after selecting RCF in the same way 4 Select Time by turning the knob Press once and by turning the knob again set the time as recommended in the particular protocol step Confirm entry by pressing the knob 5 For the Centrifuge 4 16KS set the temperature to 40 C 6 Open the lid place the 96 well plates with the metal carriers in the buckets then close the lid The start and lid keys light up 7 Push Start to start the centrifuge When the centrifuge is running the lid key will not be lit Each run can be interrupted by pushing Stop 8 At the end of the run the lid key will light up Open the centrifuge lid by pressing the lid key Remove the plates All preset parameters remain after a run has finished 18 DNeasy Plant Handbook 10 2012 Lysate filtration with QlAshredder DNeasy Mini and Maxi procedures In the DNeasy Plant Mini and Maxi procedures cell debris and salt precipitates are removed by centrifugation through a QlAshredder spin column The preparation of a cleared lysate is essential to prevent clogging of the DNeasy spin column in the following step Traditional methods involve removing the debris and precipitates by centrifugation and using the supernatant in subsequent steps However not all particulate matter forms a compact pe
26. awing up the samples as soon as the pipet tips contact the liquid Repeat until all the samples have been transferred to the DNeasy 96 plates 40 DNeasy Plant Handbook 10 2012 23 Seal each DNeasy 96 plate with an AirPore Tape Sheet provided Centrifuge for 4 min at 6000 rpm AirPore Tape prevents cross contamination between samples during centrifugation After centrifugation check that all of the lysate has passed through the membrane in each well of the DNeasy 96 plates If lysate remains in any of the wells centrifuge for a further 4 min 24 Remove the tape Carefully add 800 pl Buffer AW2 to each sample Note Ensure that ethanol has been added to Buffer AW2 prior to use See Things to do before starting page 35 25 Centrifuge for 15 min at 6000 rpm to dry the DNeasy membranes For efficient drying do not reseal the DNeasy 96 plate with AirPore Tape IMPORTANT Residual ethanol in the DNeasy membranes derived from Buffer AW2 may inhibit PCR and must be removed by centrifugation before elution of the DNA Note DNeasy membranes are sometimes slightly colored after this wash step This should not affect the DNeasy 96 Plant procedure A very dark membrane could indicate that too much starting material was used A second wash step with 800 pl ethanol 96 100 may improve DNA quality in these cases Empty the flow through from the S Block before performing this second wash step 26 To elute the DNA place each DNeasy 96 plate in
27. d the centrifuge user manual for operating instructions Do not allow the Tissuelyser adapter plates to come into contact with liquid nitrogen during the procedure This protocol describes processing of 192 samples 2 x 96 If you wish to process 96 or fewer samples provide a balance for the Tissuelyser by assembling a second plate sandwich using a rack of collection microtubes without samples or buffers but containing tungsten carbide beads and fixing this second sandwich into the empty clamp Tungsten carbide beads are reusable See Appendix B page 48 for recovery and cleaning details All centrifugation steps should be performed at room temperature 15 25 C If the Centrifuge 4 1 6KS is used set the temperature to 40 C for all centrifugation steps Things to do before starting Buffer AW2 and Buffer AW1 are supplied as concentrates Before using for the first time add the appropriate amount of ethanol 96 100 as indicated on the bottle to obtain a working solution Buffer AW1 concentrate may form precipitates upon storage If necessary warm to 65 C to redissolve before adding ethanol Do not heat Buffer AW1 after ethanol has been added Preheat Buffer AP1 to 80 C This heating is necessary for the DNeasy 96 Plant procedure and will also dissolve any precipitate that may have formed in Buffer AP1 When using lyophilized tissue without using liquid nitrogen preheat Buffer AP to 65 C If possible harvest plant s
28. disrupted using a mortar and pestle or the Tissuelyser System For optimal results we recommend to keep the disruption time as short as possible Disruption for more than 1 minute may lead to shearing of genomic DNA Note Disruption can be performed without lysis buffer by keeping the sample submerged in liquid nitrogen before and during disruption In this case Buffer AP1 and RNase A stock solution 100mg ml need to be added to the sample immediately after disruption Disruption and homogenization using the TissueLyser System Either fresh frozen or lyophilized plant tissue samples can be processed using the Tissuelyser Fresh material can be directly disrupted in lysis buffer without using liquid nitrogen Alternatively fresh or frozen samples can be disrupted after freezing in liquid nitrogen without lysis buffer Lyophilized tissue can be disrupted without buffer at ambient temperature Disruption of samples in lysis buffer yields DNA ideal for PCR while disruption of samples in liquid nitrogen yields DNA of a higher molecular weight We do not recommend disrupting frozen material in lysis buffer as this results in low yields and degraded DNA DNeasy Plant Handbook 10 2012 15 ee Figure 1 The TissueLyser Il TissueLyser LT and TissueRuptor for sample disruption IMPORTANT When using the Tissuelyser with frozen plant material do not allow the adapter plates to come into contact with liquid nitrogen Note When using th
29. dry the membrane It is important to dry the membrane of the DNeasy Mini spin column since residual ethanol may interfere with subsequent reactions This centrifugation step ensures that no residual ethanol will be carried over during elution Discard flow through and collection tube u gt Qa 3 8 vu j a C o S After washing with Buffer AW2 the DNeasy Mini spin column membrane is usually only slightly colored In the rare case that the membrane remains significantly colored after washing with Buffer AW2 refer to Darkly colored membrane or green yellow eluate after washing with Buffer AW2 in the Troubleshooting Guide on page 43 Note Following the centrifugation remove the DNeasy Mini spin column from the collection tube carefully so the column does not come into contact with the flow through as this will result in carryover of ethanol 18 Transfer the DNeasy Mini spin column to a 1 5 ml or 2 ml microcentrifuge tube not supplied and pipet 100 pl Buffer AE directly onto the DNeasy membrane Incubate for 5 min at room temperature 15 25 C and then centrifuge for 1 min at 26000 x g 28000 rpm to elute Elution with 50 pl instead of 100 yl increases the final DNA concentration in the eluate significantly but also reduces overall DNA yield If larger amounts of DNA gt 20 pg are loaded eluting with 200 pl instead of 100 pl increases yield See Elution page 19 19 Repeat step 18 once A new microce
30. e Tissuelyser Adapter Sets samples on the inside of the adaptor rack move more slowly than samples on the outside To prevent variation in sample homogenization the adaptor sets should be removed from the Tissuelyser and disassembled after the first disruption step For the second disruption step the adaptor sets should be reassembled so that the samples tubes that were outermost in the rack are now innermost Rearranging the racks of collection microtubes in this way ensures that all samples are thoroughly and equally 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 same samples that were outermost during the initial disruption will remain outermost in the second disruption step An instruction manual is provided with the TissueLyser Read this manual carefully before using the Tissuelyser Do not use the Tissuelyser with equipment other than that supplied DNeasy Mini procedure Plant material and a tungsten carbide bead are added to a 2 ml safe lock tube Tubes are placed into the adaptor sets which are fixed into the clamps of the Tissuelyser Disruption is performed in two 1 2 minute high speed 20 30 Hz shaking steps The beads are reusable see Appendix B page 48 for cleaning instructions 16 DNeasy Plant Handbook 10 2012 For optimal operation the Tissuelyser should always be balanced A balance can be prov
31. e disruption time may result in DNA shearing 7 Remove and disassemble the plate sandwiches noting the orientation of the racks of collection microtubes during the first round of disruption Ensure that the collection microtubes are tightly closed 8 Cool the racks of collection microtubes again in liquid nitrogen Place a clear cover 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 microtubes Ensure that no liquid nitrogen remains but do not allow the leaf material to thaw Remove the clear cover IMPORTANT Do not put the adapter plates into liquid nitrogen Disassemble the plate sandwiches as described above and place only the racks of collection microtubes in liquid nitrogen 96 Asbena O N 5 acl 2 5 3 N n When using lyophilized tissue the microtubes do not need to be frozen in liquid nitrogen Continue with step 9 Keep the clear covers from the collection microtube racks for use in step 13 9 Ensure that the collection microtubes are tightly closed 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 Work quickly so that the plant material does not thaw Rotating the racks of collection microtubes in this way ensures that all samples are thoroughly disru
32. ed and homogenized by a rotating blade rotor stator homogenization TissueRuptor disposable probes enable flexible sample disruption in a wide range of volumes and formats Using a separate disposable probe for each sample provides ease of use and prevents cross contamination Either fresh frozen or lyophilized plant tissue samples can be processed using the TissueRuptor Tissues can be disrupted in liquid nitrogen or directly disrupted in lysis buffer without using liquid nitrogen depending on the protocol and downstream application used See Disruption and homogenization using the TissueRuptor page 15 for further details TissueLyser System The Tissuelyser System provides convenient simultaneous disruption and homogenization of multiple biological samples through high speed shaking in plastic tubes with stainless steel tungsten carbide or glass beads The Tissuelyser Il cat no 85300 provides medium to high throughput sample disruption of up to 48 or 192 samples The Tissuelyser LT cat no 85600 provides fast low to medium throughput sample disruption of up to 12 samples Either fresh frozen or lyophilized plant tissue samples can be processed using the Tissuelyser Fresh material can be directly disrupted in lysis buffer without using liquid nitrogen Alternatively fresh or frozen samples can be disrupted after freezing in liquid nitrogen without lysis buffer Lyophilized tissue can be disrupted without buffer at ambie
33. eld see Tables 3 5 If larger amounts Mini gt 20 pg Maxi gt 100 pg of DNA are loaded onto DNeasy Mini or Maxi spin columns eluting with 2 x 200 pl Mini or 2 x 1000 pl Maxi will increase yield see Tables 3 and 4 DNeasy Plant Handbook 10 2012 19 Table 3 DNeasy mini procedure elution volumes and corresponding yields Source 100 mg Elution volume Total DNA yield Concentration young leaves pl pg ng pl Arabidopsis 2x50 3 6 38 2 x 100 3 8 20 2 x 200 4 1 11 Barley 220 US 83 2 x 100 9 5 50 2 x 200 10 0 26 Tobacco 2 x 50 20 5 216 2 x 100 23 2 122 2x200 29 7 78 Because the column has a certain residual volume the volume of eluate recovered is always less than the volume of buffer used for elution the actual yield is therefore less than the theoretical yield Table 4 DNeasy maxi procedure elution volumes and corresponding yields Source 1 g Elution volume Total DNA yield Concentration young leaves pl pg ng pl Maize 2 x 500 92 102 2x750 112 79 2 x 1000 143 72 Fir 2 x 500 63 72 2 32 30 90 65 2 x 1000 93 A8 Rape 2 x 500 21 23 2x750 26 18 2 x 1000 27 14 Because the column has a certain residual volume the volume of eluate recovered is always less than the volume of buffer used for elution the actual yield is therefore less than the theoretical yield 20 DNeasy Plant Handbook 10 2012 Table 5 DNeasy 96 procedure elution volumes and corresponding yields
34. ermined by pulsefield gel electrophoresis PFGE through an agarose gel To prepare the sample for PFGE the DNA should be concentrated by alcohol precipitation and the DNA pellet dried briefly at room temperature 15 25 C for 5 10 minutes Avoid drying the DNA pellet for more than 10 minutes since overdried genomic DNA is very difficult to redissolve Redissolve in approximately 30 pl TE buffer pH 8 0 for at least 30 minutes at 60 C Load 3 5 yg of DNA per well Standard PFGE conditions are as follows E 1 agarose gel in 0 5x TBE electrophoresis buffer Switch intervals 5 40 seconds Run time 17 hours Voltage 170 V When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information consult the appropriate safety data sheets SDSs available from the product supplier DNeasy Plant Handbook 10 2012 47 Appendix B Recovery and Cleaning of Beads and S Blocks Cleaning beads and grinding balls Tungsten carbide and stainless steel beads and grinding balls can be reused Used beads can be recovered from cell debris and cleaned using the procedure below B1 Follow step 1a for DNeasy Mini procedures step 1b for DNeasy Maxi procedures or step Ic for DNeasy 96 procedures Bla DNeasy Mini Close the cap of the 2 ml collection tube and briefly vortex to dislodge the bead and pellet from the bottom of the tube B1b DNeasy Maxi If using grinding jars for the disruption o
35. ets of Adapter Plates for use with 69984 2x96 Collection Microtubes racked on the Tissuelyser Grinding Jar Set S Steel 2 Grinding Jars 10 ml 2 Stainless 69985 2 x 10 ml Steel Grinding Balls 20 mm Tungsten Carbide Beads Tungsten Carbide Beads suitable 69997 3 mm 200 for use with the Tissuelyser System QIAGEN 96 Well Plate Centrifugation System Centrifuge 4 168 Universal laboratory centrifuge with 81500 brushless motor 81510 81525 81520 Centrifuge 4 16KS Refrigerated universal laboratory 81600 centrifuge with brushless motor 81610 81625 81620 Plate Rotor 2 x 96 Rotor for 2 QIAGEN 96 well plates 81031 for use with QIAGEN Centrifuges Accessories Collection Tubes 2 ml 1000 Collection Tubes 2 ml 19201 Collection Microtubes Nonsterile polypropylene tubes 19560 racked 10 x 96 1 2 ml 960 in racks of 96 Collection Microtube Caps Nonsterile polypropylene caps for 19566 120 x 8 collection microtubes 1 2 ml and round well blocks 960 in strips of 8 S Blocks 24 96 well blocks with 2 2 ml wells 19585 24 per case Japan t North America t UK 8 Rest of World 50 DNeasy Plant Handbook 10 2012 Ordering Information Product Contents Cat no AirPore Tape Sheets 50 Microporous tape sheets for 19571 covering 96 well blocks 50 sheets per pack Buffer AW2 324 ml Wash Buffer 2 Concentrate 19072 Concentrate 324 ml Buffer AE 240 ml 240 ml Elution Buffer 19077 Related products BioSprint 15 DN
36. f large sample volumes skip to step 2 Bic DNeasy 96 Seal the collection microtubes with caps Place a clear cover saved from step 1 of DNeasy 96 Plant procedures 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 B2 Empty the contents of the tubes jars into a sieve and rinse the beads thoroughly with water B3 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 B4 Rinse beads thoroughly with distilled water to remove the HCI B5 Dry beads before use Cleaning S Blocks To avoid cross contamination after each use rinse the S Blocks thoroughly in tap water incubate for 1 min at room temperature 15 25 C 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 page 50 for ordering information When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information consult the appropriate safety data sheets SDSs available from the product supplier 48 DNeasy Plant Handbook 10 2012 Ordering Information Product Contents Cat no DNeasy Plant Mini Kit 50 50 DNeasy Mini Spin Columns 69104 50 QlAshredder Mini Spin Columns RNase A Buffers Collection Tubes
37. h a multichannel pipet Volume per sample Volume for 2 x 96 samples Buffer AP1 preheated to 65 C 400 pl 90 ml RNase A 100 mg ml 1 pl 225 pl Reagent DX 1 pl 225 pl 15 excess mixture is included in these calculations to allow for pipetting errors t Reagent DX is viscous Sandwich each rack of collection microtubes between adapter plates and fix into TissueLyser clamps as described in the Tissuelyser User Manual Note 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 samples or less 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 Grind the samples for 1 5 min at 30 Hz IMPORTANT Prolonging the disruption time may result in DNA shearing DNeasy Plant Handbook 10 2012 31 96 Aspanq anss jUD q Ysa y Fresh Plant Tissue DNeasy 96 11 32 Remove and disassemble the plate sandwiches Ensure that the collection microtubes are tightly closed Reassemble the plate sandwiches so that the collection microtubes nearest the Tissuelyser in steps 4 and 5 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 See Disruption and homogenization using the
38. he full 15 s The genomic DNA will not be sheared by vigorous shaking The centrifugation step prevents precipitates from freezing to the caps which would otherwise be difficult to remove after incubation at 20 C step 11 Keep the clear covers from the collection microtube racks for use in step 15 Incubate the racks of collection microtubes for 10 min at 20 C This incubation aids the precipitation of proteins and inhibitors of downstream applications following addition of Buffer P3 DNeasy Plant Handbook 10 2012 12 13 14 15 16 Centrifuge the racks of collection microtubes for 5 min at 6000 rpm Compact pellets will form but some particles may float Be careful not to transfer any of these particles in the following step Remove and discard the caps Carefully transfer 400 pl of each supernatant to new racks of collection microtubes provided ensuring that the new tubes are in the correct orientation Do not discard the pellets as they contain the tungsten carbide beads which can be recovered and reused see Appendix B page 48 Do not transfer more than 400 pl of the supernatant as otherwise the capacity of the DNeasy 96 plates and the S Blocks used in subsequent steps will be exceeded If less than 400 pl supernatant is recovered adjust the amount of Buffer AW1 in step 14 accordingly Collection microtubes are connected in strips of 8 To avoid transferring particulate matter it is helpful to remove the
39. high purity of the DNA The DNeasy membrane ensures complete removal of all inhibitors of PCR and other enzymatic reactions DNA purified using DNeasy Plant Kits is highly suited for use in all downstream applications including PCR RAPD analysis AFLP analysis RFLP analysis Southern blotting microsatellite analysis SNP genotyping and quantitative real time PCR Use of the TissueRuptor or the Tissuelyser for rapid and convenient disruption of plant tissue samples is recommended for the most efficient processing in DNeasy Plant procedures Principle and procedure Disruption of plant tissue Complete and quick disruption of starting material is essential to ensure high DNA yields and to avoid DNA degradation DNeasy Plant procedures are optimized for use with leaf tissues but can also be used to purify DNA from other plant tissues and fungi including seeds and spores However when using tissues other than leaves the disruption method may require optimization to ensure maximum DNA yield and quality DNeasy Plant Kits are compatible with all sample disruption methods Optimal results are obtained using the TissueRuptor homogenizer or the Tissuelyser System DNeasy Plant Handbook 10 2012 7 TissueRuptor homogenizer The TissueRuptor is a handheld rotor stator homogenizer designed for rapid efficient and flexible disruption of a variety of biological samples including plant and animal tissues Samples are simultaneously disrupt
40. ided by assembling a second adaptor using safe lock tubes without samples or buffers but containing the beads and fixing this second adaptor into the empty clamp DNeasy Maxi procedure Plant material is added to the grinding jars The grinding balls are added and the jars are fixed in the clamps of the Tissuelyser Disruption is performed in two 1 2 minute high speed 20 30 Hz shaking steps The grinding jars and balls are reusable see Appendix B page 48 for cleaning instructions For optimal operation the Tissuelyser should always be balanced If using a single grinding jar the balance should consist of a second grinding jar containing a stainless steel ball DNeasy 96 procedures For DNeasy 96 procedures plant material and a tungsten carbide bead are added to each of 192 collection microtubes in 2 racks The racks are placed into the adaptor sets which are fixed into the clamps of the TissueLyser Disruption is performed in two 1 2 minute high speed 20 30 Hz shaking steps The beads are reusable see Appendix B page 48 for cleaning instructions For optimal operation the Tissuelyser should always be balanced A balance can be provided by assembling a second adaptor using a rack of collection microtubes without samples or buffers but containing the beads and fixing this second adaptor into the empty clamp Centrifugation DNeasy 96 procedures Centrifuges 4 16S or 4 16KS DNeasy 96 spin protocols use a streamlined centr
41. ification of Total DNA from Plant Tissue Maxi Protocol 26 Purification of Total DNA from Fresh Plant Tissue DNeasy 96 Protocol 30 E Purification of Total DNA from Frozen or Lyophilized Plant Tissue DNeasy 96 Protocol 35 Troubleshooting Guide 42 Appendix A Determination of Yield Purity and Length of DNA 46 Appendix B Recovery and Cleaning of Beads and S Blocks 48 Ordering Information 49 DNeasy Plant Handbook 10 2012 3 Kit Contents DNeasy Plant Mini and Maxi Kits DNeasy Plant Kits Mini 50 Mini 250 Maxi 6 Maxi 24 Catalog no 69104 69106 68161 68163 Number of preps 50 250 6 24 DNeasy Mini Spin Columns colorless 50 250 DNeasy Maxi Spin Columns colorless 6 24 QlAshredder Mini Spin Columns lilac 50 250 QlAshredder Maxi Spin Columns lilac 6 24 Collection Tubes 2 ml 50 250 Collection Tubes 50 ml 6 24 Buffer AP1 AO ml 200 ml 40 ml 140 ml Buffer P3 20ml 2x50nml 18 ml 50 ml Buffer AW 1 concentrate t 2x19 ml 151 ml 25 ml 151 ml Buffer AW2 concentrate 17ml 2x40ml 26ml 2x68ml Buffer AE 2x12ml 2x60ml 15 ml 60 ml RNase A 100 mg ml 220 pl 5x220pl 1104p 440 pl Quick Start Protocol 1 1 Contains a chaotropic salt Not compatible with disinfectants containing bleach See page 6 for safety information t Buffer AW1 and Buffer AW2 are supplied as concentrates Add ethanol 96 100 according to the bottle label before use to obtain a working solution 4 DNeasy Plant
42. ifugation procedure that enables purification of DNA from up to 2 x 96 samples in parallel for direct use in any downstream application The DNeasy 96 Plant procedure requires use of the QIAGEN 96 Well Plate Centrifugation System comprising the Plate Rotor 2 x 96 and the table top Centrifuge 4 16S or the refrigerated table top Centrifuge 4 16KS see page 50 for ordering information In addition to the Plate Rotor 2 x 96 a wide range of other rotors can be used with these centrifuges Standard table top centrifuges and microtiter plate rotors are not suitable for the DNeasy 96 protocol for 2 reasons the microtiter plate buckets are either not deep enough for the complete DNeasy 96 package or they will not swing out properly and furthermore high g forces gt 5500 x g are required for optimal performance of the DNeasy 96 procedure The speed limit of the Centrifuge 4 168 and the Centrifuge A 16KS 6000 rpm 5796 x g is programmed so that the given g force will not be exceeded All centrifugation steps are performed at room temperature 15 25 C DNeasy Plant Handbook 10 2012 17 IMPORTANT Centrifuges must be properly maintained for optimal performance It is particularly important that the buckets and rotor pins are routinely greased to prevent suboptimal running conditions that may lead to cracking of DNeasy 96 plates For further information about QIAGEN Centrifuges and the Plate Rotor 2 x 96 contact QIAGEN Technical Services or your loca
43. ipitates detergent proteins and polysaccharides 10 Recommended Centrifuge the lysate for 5 min at 20 000 x g 14 000 rpm Some plant materials can generate very viscous lysates and large amounts of precipitates during this step This can result in shearing of the DNA in the next step see Lysate filtration with QlAshredder page 19 In this case optimal results are obtained if the majority of these precipitates are removed by centrifugation for 5 min at 20 000 x g 14 000 rpm After centrifugation apply supernatant to QlAshredder Mini spin column and continue with step 11 wn ft i ale m Mini Protocol 11 Pipet the lysate into the QlAshredder Mini spin column lilac placed in a 2 ml collection tube and centrifuge for 2 min at 20 000 x g 14 000 rpm lt may be necessary to cut the end off the pipet tip to apply the lysate to the QlAshredder Mini spin column The QlAshredder Mini spin column removes most precipitates and cell debris but a small amount will pass through and form a pellet in the collection tube Be careful not to disturb this pellet in step 12 12 Transfer the flow through fraction from step 11 into a new tube not supplied without disturbing the cell debris pellet Typically 450 pl of lysate is recovered For some plant species less lysate is recovered In this case determine the volume for the next step 13 Add 1 5 volumes of Buffer AW1 to the cleared lysate and mix by pipetting
44. l distributor see back cover for contact information Note If the Centrifuge 4 16KS is used set the temperature to 40 C for all centrifugation steps Note Use AirPore Tape Sheets provided to seal DNeasy 96 plates during all centrifugation steps to prevent cross contamination between samples Abbreviated instructions for using the Centrifuge 4 165 and Centrifuge 4 16KS Warning Never run the centrifuge with empty plate carriers placed inside the buckets that is without the collection microtubes or DNeasy 96 plates and S Blocks If unsupported the carriers will collapse under high gforces Therefore remove the carriers during test runs Standard microtiter plates may be centrifuged in the same carriers if the g force does not exceed 500 x g 1 Switch on the centrifuge by pressing the main switch on the back 2 Select the rotor selection list in the display field by turning the knob After pressing the knob turn the knob again to select the rotor bucket combination 09100 09158 for the Plate Rotor 2 x 96 Confirm entry by pressing the knob Entering the rotor number automatically sets the time and speed limits for centrifugation for that particular rotor thus eliminating the danger of the centrifuge over speeding 3 Select Speed by turning the knob Press the knob and by turning the knob again set the speed to 6000 Confirm entry by pressing the knob The corresponding relative centrifugal force RCF is calcul
45. llet making preparation of a cleared lysate by centrifugation very difficult The QlAshredder spin column removes all cell debris and precipitates making the preparation of a cleared lysate rapid and efficient With some starting materials e g oak leaves centrifugation of the entire lysate through the QlAshredder spin column can result in sheared DNA Investigation has shown that this is not due to the pore size of the QlAshredder spin column but rather due to the high viscosity of the lysate and the large amount of precipitates These form a compact layer on the QlAshredder spin column Centrifugation of the lysate through this layer can result in size reduction of the DNA Therefore for certain plant tissues an additional centrifugation step is recommended This additional centrifugation is part of the standard DNeasy Plant Maxi procedure and is included in the DNeasy Plant Mini procedure as an optional step Elution Purified DNA is eluted from the DNeasy spin column or DNeasy 96 plate using either Buffer AE or water Optimal results are obtained by eluting twice The elution volume is typically 2 x 100 pl for the DNeasy Plant Mini Kit and the DNeasy 96 Plant Kit and 2 x 750 pl for the DNeasy Plant Maxi Kit Higher concentrations of DNA If higher concentrations of DNA are required in the eluate reducing the elution volume to 2 x 50 pl Mini or DNeasy 96 or 2 x 500 pl Maxi significantly increases concentration but reduces overall yi
46. m each lot of the DNeasy Plant Kits is tested against predetermined specifications to ensure consistent product quality 6 DNeasy Plant Handbook 10 2012 Introduction DNeasy Plant Kits provide a fast and easy way to purify DNA from plant and fungal tissue Up to 100 mg of tissue can be processed using the DNeasy Plant Mini Kit or up to 1 g of tissue using the DNeasy Plant Maxi Kit The DNeasy 96 Kit is designed for high throughput DNA purification from 50 mg plant tissue per well for some plant tissues up to 100 mg per well can be used Easy to use DNeasy Plant procedures provide pure total DNA genomic mitochondrial and chloroplast for reliable PCR and Southern blotting in less than 1 hour DNeasy Plant Mini Kit or 2 hours DNeasy Plant Maxi Kit or DNeasy 96 Plant Kit Purification requires no phenol or chloroform extraction or alcohol precipitation and involves minimal handling This makes DNeasy Mini and Maxi Kits highly suited for simultaneous processing of multiple samples For higher throughput applications the DNeasy 96 Plant Kit enables simultaneous processing of 96 or 192 samples Puritied DNA is eluted in low salt buffer or water ready for use in downstream applications DNA purified using DNeasy Plant Kits is up to 40 kb in size with fragments of 20 25 kb predominating DNA of this length denatures completely in PCR and shows the highest amplification efficiency Purified DNA has an A3s0 Aaso ratio of 1 7 1 9 indicating
47. n GmbH Limited License Agreement for DNeasy Plant Kits Use of this product signifies the agreement of any purchaser or user of the product to the following terms 1 The product may be used solely in accordance with the protocols provided with the product and this 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 protocols provided with the product this handbook and additional protocols available at www qiagen com Some of these additional protocols have been provided by QIAGEN users for QIAGEN users These protocols have not been thoroughly tested or optimized by QIAGEN QIAGEN neither guarantees them nor warrants that they do not infringe the rights of third parties 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 fo take any steps that could lead to or facilitate any acts prohibited above QIAGEN may enforce the prohibitions of this Limited License
48. n a working solution Buffer AW1 concentrate may form precipitates upon storage If necessary warm to 65 C to redissolve before adding ethanol Do not heat Buffer AW1 after ethanol has been added Preheat Buffer AP1 to 65 C This heating is necessary for the DNeasy 96 Plant procedure and will also dissolve any precipitate that may have formed in Buffer AP1 DNeasy Plant Handbook 10 2012 Procedure 1 Harvest leaves and place up to 50 mg into each tube in 2 collection microtube racks Unless a different optimal amount of starting material has been previously determined do not use more than 50 mg wet weight per sample see Sample size page 14 Most leaf material can be stored at 4 C for at least 24 h prior to processing without affecting DNA yield or quality Keep the clear covers from the collection microtube racks for use in step 10 Use the plate register cards provided to record the position of each sample in the racks Add one tungsten carbide bead to each collection microtube Combine Buffer AP1 RNase A and Reagent DX according to the table below to make a working lysis solution Pipet 400 pl of the working lysis solution into each collection microtube Seal the microtubes with the caps provided It is important to prepare a fresh working lysis solution To allow thorough mixing of the solution combine the components in a tube and vortex to mix then dispense the solution into a reagent reservoir for use wit
49. nt temperature See Disruption and homogenization using the Tissuelyser System page 15 for further details DNA purification In DNeasy Plant procedures see flowcharts plant material is first mechanically disrupted and then lysed by addition of lysis buffer and incubation RNase A in the lysis buffer digests the RNA in the sample After lysis proteins and polysaccharides are salt precipitated In the DNeasy 96 procedures cell debris and precipitates are removed by centrifugation In DNeasy Mini and Maxi procedures cell debris and precipitates are removed in a single step by a brief spin through the QlAshredder column a unique filtration and homogenization unit Binding buffer and ethanol are added to the cleared lysate to promote binding of the DNA to the DNeasy membrane The sample is then applied to a DNeasy spin column or DNeasy 96 plate and centrifuged DNA binds to the membrane while contaminants such as proteins and polysaccharides are efficiently removed by two wash steps Pure DNA is eluted in a small volume of low salt buffer or water DNeasy purified DNA has Azgo Azgo ratios of 1 7 1 9 and absorbance scans show a symmetric peak at 260 nm confirming high purity 8 DNeasy Plant Handbook 10 2012 DNeasy Plant Procedure Plant tissue Grind lyse amp precipitate Centrifuge through QlAshredder Add ethanol amp bind DNA 7 4 4 Wash E Eure ep Ready to use DNA DNeasy Plant Handbook 10 2
50. ntrifuge tube can be used for the second elution step to prevent dilution of the first eluate Alternatively the microcentrifuge tube can be reused for the second elution step to combine the eluates See Elution page 19 Note More than 200 pl should not be eluted into a 1 5 ml microcentrifuge tube because the DNeasy Mini spin column will come into contact with the eluate DNeasy Plant Handbook 10 2012 25 Plant Tissue 2 Q9 2 fe a Protocol Purification of Total DNA from Plant Tissue Maxi Protocol Important points before starting M If using the DNeasy Plant Maxi Kit for the first time read Important Notes page 14 E Ensure that you are familiar with operating the TissueRuptor or the Tissuelyser See Disruption and homogenization using the TissueRuptor page 15 or Disruption and homogenization using the Tissuelyser System page 15 Refer to the TissueRuptor User Manual or the Tissuelyser Handbook for operating instructions E Buffer AP1 may develop a yellow color upon storage This does not affect the procedure E All centrifugation steps are carried out at 3000 5000 x g although 5000 x g is preferable at room temperature 15 25 C in a laboratory centrifuge with a swing out rotor Do not use a fixed angle rotor see Equipment and Reagents to Be Supplied by User page 12 Things to do before starting E Buffer AW1 concentrate may form precipitates upon storage If necessary warm
51. o avoid shearing of genomic DNA Alternatively fresh or lyophilized material can be directly disrupted in lysis buffer after step 8 without using liquid nitrogen but this may cause shearing of high molecular weight DNA We do not recommend disrupting frozen material in lysis buffer as this can result in low yields and degraded DNA aNssi JUDId 3 TissueLyser procedure Place the sample material lt 1 g wet weight or lt 0 2 g lyophilized tissue into a grinding jar together with the stainless steel grinding ball Freeze the grinding jar in liquid nitrogen for approximately 30 s 3 ei 8 When using lyophilized tissue the grinding jars do not need to be frozen in liquid nitrogen 4 Place the grinding jars into the clamps of the TissueLyser Immediately grind the samples for 1 min at 30 Hz 5 Remove the grinding jars and refreeze in liquid nitrogen for 30 s When using lyophilized tissue the samples do not need to be frozen in liquid nitrogen 6 Repeat step 4 7 Transfer the fine powder into a 15 ml centrifuge tube not supplied using a spatula Do not allow the sample to thaw Proceed immediately to step 8 Note The majority of plant tissue is ground to a fine powder after 2 disruption steps however for some materials one disruption step may be sufficient Other tissues such as seeds and roots may require 3 disruption steps Optimization of the disruption procedure may be required for some plant ma
52. o dry the membrane of the DNeasy Maxi spin column since residual ethanol may interfere with subsequent reactions This centrifugation step ensures that no residual ethanol will be carried over during elution After washing with Buffer AW2 the DNeasy Maxi spin column membrane is usually only slightly colored In the rare case that the membrane remains significantly colored after washing with Buffer AW2 refer to Darkly colored membrane or green yellow eluate after washing with Buffer AW2 in the Troubleshooting Guide on page 43 nss j JUDId amp amp ui 5 A o 16 Transfer the DNeasy Maxi spin column to a new 50 ml tube supplied Pipet 0 75 1 ml Buffer AE directly onto the DNeasy Maxi spin column membrane Incubate for 5 min at room temperature and then centrifuge for 5 min at 3000 5000 x g to elute Note Elution may also be performed with 0 5 ml of Buffer AE instead of 0 75 1 ml This increases the final DNA concentration in the eluate but also reduces overall DNA yield See Elution page 19 17 Add another 0 75 1 ml of Buffer AE and repeat the elution step as described in step 16 The first and second eluates may be combined or collected separately For separate collection of the eluates see Elution page 19 DNeasy Plant Handbook 10 2012 29 Fresh Plant Tissue DNeasy 96 Protocol Purification of Total DNA from Fresh Plant Tissue DNeasy 96 Protocol Important points before star
53. pted See Disruption and homogenization using the Tissuelyser System page 15 IMPORTANT Merely rotating the entire plate sandwich so that the QIAGEN logos are upside down when reinserted into the mixer mill is not sufficient since the same samples that were outermost during the initial disruption will remain outermost in the second disruption step 10 Grind the samples for another 1 min at 20 Hz DNeasy Plant Handbook 10 2012 37 11 Remove the plate sandwiches from the Tissuelyser and remove the adapter plates from each rack of collection microtubes Knock the racks against the bench 5 times to ensure that no tissue powder remains in the caps Keep the samples frozen until working lysis solution is added step 12 IMPORTANT The samples should not be allowed to thaw while working lysis solution is being prepared step 12 Store the samples at 20 C until the working lysis solution is ready 12 Combine Buffer AP1 RNase A and Reagent DX according to the table below to make a working lysis solution Carefully remove the caps from the collection microtubes Immediately pipet 400 pl working lysis solution into each collection microtube It is important to prepare a fresh working lysis solution To allow thorough mixing of the solution combine the components in a tube and vortex to mix then dispense the solution into a reagent reservoir for use with a multichannel pipet If the working lysis solution is not used immediately
54. r with operating the TissueRuptor or the Tissuelyser See Disruption and homogenization using the TissueRuptor page 15 or Disruption and homogenization using the Tissuelyser System page 15 Refer to the TissueRuptor User Manual or the Tissuelyser Handbook for operating instructions E Buffer AP may develop a yellow color upon storage This does not affect the procedure E All centrifugation steps are carried out at room temperature 15 25 C in a microcentrifuge Things to do before starting E Buffer API and Buffer AW1 concentrate may form precipitates upon storage If necessary warm to 65 C to redissolve before adding ethanol to Buffer AW 1 Do not heat Buffer AW1 after ethanol has been added E Buffer AW2 and Buffer AW1 are supplied as concentrates Before using for the first time add the appropriate amount of ethanol 96 100 as indicated on the bottle to obtain a working solution E Preheat a water bath or heating block to 65 C Procedure 1 For disruption using the TissueRuptor follow step 2 for disruption using the TissueLyser follow steps 3 6 Alternatively plant or fungal tissue can be ground to a fine powder under liquid nitrogen using a mortar and pestle Transfer the tissue powder and liquid nitrogen to an appropriately sized tube and allow the liquid nitrogen to evaporate Do not allow the sample to thaw Proceed immediately to step 7 SESE EEE Sep SS SSS 22 DNeasy Plant Handbook 10 2012 2 Tiss
55. roceed immediately to step 7 To prevent variation in sample homogenization the adaptor sets should be removed from the Tissuelyser and disassembled after the first disruption step For the second disruption step the adaptor sets should be reassembled so that the tube order is reversed Rotating the racks of tubes in this way ensures that all samples are thoroughly and equally disrupted Note The majority of plant tissue is ground to a fine powder after 2 disruption steps however for some materials one disruption step may be sufficient Other tissues such as seeds and roots may require disruption steps Optimization of the disruption procedure may be required for some plant material 7 Add 400 pl Buffer AP1 and 4 pl RNase A stock solution 100 mg ml to a maximum of 100 mg wet weight or 20 mg dried disrupted plant or fungal tissue and vortex vigorously No tissue clumps should be visible Vortex or pipet further to remove any clumps Clumps of tissue will not lyse properly and will therefore result in a lower yield of DNA In rare cases where clumps cannot be removed by pipetting and vortexing a disposable micropestle may be used Note Do not mix Buffer AP and RNase A before use DNeasy Plant Handbook 10 2012 23 8 Incubate the mixture for 10 min at 65 C Mix 2 or 3 times during incubation by inverting tube This step lyses the cells 9 Add 130 pl Buffer P3 to the lysate mix and incubate for 5 min on ice This step prec
56. sometimes slightly colored after this wash step This should not affect the DNeasy 96 Plant procedure A very dark membrane could indicate that too much starting material was used A second wash step with 800 pl ethanol 96 100 may improve DNA quality in these cases Empty the flow through from the S Block before performing this second wash step To elute the DNA place each DNeasy 96 plate in the correct orientation on a new rack of Elution Microtubes RS provided add 100 pl Buffer AE to each sample and seal the DNeasy 96 plates with new AirPore Tape Sheets provided Incubate for 1 min at room temperature 15 25 C Centrifuge for 2 min at 6000 rpm Elution in 2 x 50 pl instead of 2 x 100 pl increases DNA concentration but decreases the overall DNA yield see Elution page 19 Repeat step 21 with another 100 pl Buffer AE Use new caps provided to seal the Elution Microtubes RS for storage DNeasy Plant Handbook 10 2012 Protocol Purification of Total DNA from Frozen or Lyophilized Plant Tissue DNeasy 96 Protocol Important points before starting If using the DNeasy 96 Plant Kit for the first time read Important Notes page 14 Ensure that you are familiar with operating the Tissuelyser and the QIAGEN 96 Well Plate Centrifugation System See Disruption and homogenization using the Tissuelyser System page 15 and Centrifugation DNeasy 96 procedures page 17 Refer to the Tissuelyser Handbook an
57. strips from the rack so that the contents of the microtubes are visible and to use a multichannel pipet on its lowest speed setting Save the used collection microtubes to recover the tungsten carbide beads at a later stage see Appendix B page 48 Add 1 5 volumes typically 600 pl of Buffer AW1 to each sample Note Ensure that ethanol has been added to Buffer AW 1 prior to use See Things to do before starting page 30 A white precipitate may form upon addition of Buffer AW1 This precipitate does not interfere with the DNeasy 96 Plant procedure or any subsequent application Close the collection microtubes with new caps provided ensure that the tubes are properly sealed to prevent leakage during shaking Place a clear cover over each rack of collection microtubes and shake the racks vigorously up and down for 15 s To collect any solution from the caps centrifuge the collection microtubes Allow the centrifuge to reach 3000 rpm and then stop the centrifuge Do not prolong this step Note To ensure optimal DNA yields it is important to shake the racks of collection microtubes vigorously up and down with both hands for the full 15 s The genomic DNA will not be sheared by vigorous shaking Place two DNeasy 96 plates on top of S Blocks provided Mark the DNeasy 96 plates for later sample identification DNeasy Plant Handbook 10 2012 33 96 span anss jUD q Ysa y Fresh Plant Tissue DNeasy 96 17 20 21
58. t intended for the diagnosis prevention or treatment of a disease All due care and attention should be exercised in the handling of the products We 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 Safety Information When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information please consult the appropriate safety data sheets SDSs These are available online in convenient and compact PDF format at www giagen com safety where you can find view and print the SDS for each QIAGEN kit and kit component CAUTION DO NOT add bleach or acidic solutions directly to waste containing Buffers AW1 and AL Buffer AW1 contains guanidine hydrochloride which can form highly reactive compounds when combined with bleach If liquid containing this buffer 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 24 hour emergency information Emergency medical information in English French and German can be obtained 24 hours a day from Poison Information Center Mainz Germany Tel 49 6131 19240 Quality Control In accordance with QIAGEN s ISO certified Quality Management Syste
59. t lysis 42 Increase the g force and centrifugation time Perform the optional centrifugation step before loading a large amount of the lysate onto the QlAshredder as outlined in step 10 of the DNeasy Plant Mini protocol page 24 In future preparations ensure that no particulate material is transferred following centrifugation through the QlAshredder spin column Mini and Maxi procedures or when supernatants are transferred to new microtubes prior to addition of Buffer AW1 DNeasy 96 procedures Reduce the amount of starting material and or increase the amounts of Buffer AP1 and Buffer P3 Increase the gforce and centrifugation time Ensure that the starting material is completely disrupted See Disruption and homogenization using the TissueRuptor and Disruption and homogenization using the Tissuelyser System page 15 Reduce the amount of starting material and or increase the amounts of Buffer AP1 and Buffer P3 DNeasy Plant Handbook 10 2012 Comments and suggestions c Incorrect binding conditions d DNA still bound to the membrane e Maxi protocol Incorrect centrifugation method DNA sheared a Precipitate has formed in Buffer AP 1 b Mini protocol Debris and precipitates in lysate Make sure that the amount of lysate is accurately determined so that the correct amount of Buffer AW 1 is added to adjust the binding conditions correctly Increase the volume of Buffer AE or water
60. terial 8 Add 5 ml Buffer AP1 preheated to 65 C and 10 pl RNase A stock solution 100 mg ml to a maximum of 1 g wet weight or 0 2 g dried disrupted plant or fungal tissue and vortex vigorously No tissue clumps should be visible Vortex or pipet further to remove any clumps Clumps of tissue will not lyse properly and will therefore result in a lower yield of DNA In rare cases where clumps cannot be removed by pipetting and vortexing a disposable micropestle may be used Note Do not mix Buffer AP1 and RNase A before use 9 Incubate the mixture for 10 min at 65 C Mix 2 or 3 times during incubation by inverting tube This step lyses the cells DNeasy Plant Handbook 10 2012 27 10 Add 1 8 ml Buffer P3 to the lysate mix and incubate for 10 min on ice This step precipitates detergent proteins and polysaccharides 11 Centrifuge the lysate at 3000 5000 x g for 5 min at room temperature 15 25 C A pellet will form but some particles will float 12 Decant the supernatant into a QlAshredder Maxi spin column lilac placed in a 50 ml collection tube and centrifuge at 3000 5000 x g for 5 min at room temperature in a swing out rotor Transfer the flow through in the collection tube without disturbing the pellet into a new 50 ml tube not supplied Record the volume Typically 5 6 ml of lysate is recovered After centrifugation of the sample most of the debris and precipitates will be retained in the filter but there
61. the correct orientation on a new rack of Elution Microtubes RS provided add 100 pl Buffer AE to each sample and seal the DNeasy 96 plates with new AirPore Tape Sheets provided Incubate for 1 min at room temperature 15 25 C Centrifuge for 2 min at 6000 rpm 96 AsbeNna en o N 5 a a 3 n n Elution in 2 x 50 pl instead of 2 x 100 pl increases DNA concentration but decreases the overall DNA yield see Elution page 19 27 Repeat step 26 with another 100 pl Buffer AE Use new caps provided to seal the Elution Microtubes RS for storage DNeasy Plant Handbook 10 2012 41 Troubleshooting Guide This troubleshooting guide may be helpful in solving any problems that may arise For more information see also the Frequently Asked Questions page at our Technical Support Center www 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 qiagen com Comments and suggestions Clogged QlAshredder spin column a Insufficient centrifugation b Mini protocol High viscosity of lysate precipitates Clogged DNeasy membrane a Carryover of particulate material b Lysate too viscous c Insufficient centrifugation Low yield a Insufficient disruption b Insufficien
62. ting If using the DNeasy 96 Plant Kit for the first time read Important Notes page 14 Ensure that you are familiar with operating the Tissuelyser and the QIAGEN 96 Well Plate Centrifugation System See Disruption and homogenization using the Tissuelyser System page 15 and Centrifugation DNeasy 96 procedures page 17 Refer to the Tissuelyser Handbook and the centrifuge user manual for operating instructions This protocol describes processing of 192 samples 2 x 96 If you wish to process 96 or fewer samples provide a balance for the Tissuelyser by assembling a second plate sandwich using a rack of collection microtubes without samples or buffers but containing tungsten carbide beads and fixing this second sandwich into the empty clamp Tungsten carbide beads are reusable See Appendix B page 48 for recovery and cleaning details All centrifugation steps should be performed at room temperature 15 25 C If the Centrifuge 4 16KS is used set the temperature to 40 C for all centrifugation steps DNA can appear as a smear on agarose gels when using this protocol This can be avoided by using the protocol Purification of Total DNA from Frozen or Lyophilized Plant Tissue DNeasy 96 Protocol page 35 Things to do before starting 30 Buffer AW2 and Buffer AW1 are supplied as concentrates Before using for the first time add the appropriate amount of ethanol 96 100 as indicated on the bottle to obtai
63. to 200 pl DNeasy Plant Mini and DNeasy 96 procedures or to 21 ml DNeasy Plant Maxi procedure and incubate on the column for 5 min at room temperature 15 25 C before centrifugation Use a swinging bucket rotor Do not use a fixed angle rotor Ensure that any precipitate that has formed in Buffer AP1 is completely dissolved before use by heating to 65 C if necessary Perform the optional centrifugation step before loading a large amount of the lysate onto the QlAshredder spin column as described in step 10 of the DNeasy Plant Mini protocol page 24 Darkly colored membrane or green yellow eluate after washing with Buffer AW2 a Too much starting material b Mini protocol Insufficient washing of the membrane c Maxi protocol Insufficient washing of the membrane DNeasy Plant Handbook 10 2012 Reduce the amount of starting material in future preps After washing with Buffer AW2 step 17 perform an additional wash with 500 pl ethanol 96 100 Centrifuge for 2 min at 20 000 x g 14 000 rpm to dry the membrane Continue with step 18 of the DNeasy Plant Mini protocol page 25 After washing with Buffer AW2 step 15 perform an additional wash with 12 ml ethanol 90 v v in water Centrifuge for 10 min at 3000 5000 x g and dry the column for 15 30 min at 65 C in an oven to remove residual ethanol Continue with step 16 of the DNeasy Plant Maxi protocol page 29 43 Comments and suggestions
64. to reach 3000 rpm and then stop the centrifuge Do not prolong this step Note To ensure optimal DNA yields it is important to shake the racks of collection microtubes vigorously up and down with both hands for the full 15 s The genomic DNA will not be sheared by vigorous shaking Keep the clear covers from the collection microtube racks for use in step 15 14 Remove and discard caps Add 130 pl Buffer P3 to each collection microtube 15 Close the microtubes carefully with new caps provided ensure that the microtubes are properly sealed to avoid leakage during shaking Place a clear cover over each rack of collection microtubes and shake the racks vigorously up and down for 15 s To collect any solution from the caps centrifuge the collection microtubes Allow the centrifuge to reach 3000 rpm and then stop the centrifuge Do not prolong this step Note To ensure optimal DNA yields it is important to shake the racks of collection microtubes vigorously up and down with both hands for the full 15 s The genomic DNA will not be sheared by vigorous shaking The centrifugation step prevents precipitates from freezing to the caps which would otherwise be difficult to remove after incubation at 20 C step 16 96 Asbena Er O N 5 acl 2 5 3 N n Keep the clear covers from the collection microtube racks for use in step 20 16 Incubate the racks of collection microtubes for 10 min at 20 C This incubation
65. ueRuptor procedure Place the sample material lt 100 mg wet weight or lt 20 mg lyophilized tissue into a 2 ml microcentrifuge tube Add liquid nitrogen to the tube and freeze the sample for 30 s Keep the sample submerged in liquid nitrogen and disrupt for approximately 30 s at full speed Allow the liquid nitrogen to evaporate and proceed immediately to step 7 Alternatively fresh or lyophilized material can be directly disrupted in lysis buffer after step 7 without using liquid nitrogen but this may cause shearing of high molecular weight DNA We do not recommend disrupting frozen material in lysis buffer as this can result in low yields and degraded DNA u gt Qa 3 3 u Bl a A C o o0 3 Tissuelyser procedure Place the sample material lt 100 mg wet weight or lt 20 mg lyophilized tissue into a 2 ml safe lock microcentrifuge tube together with a 3 mm tungsten carbide bead Freeze the tubes in liquid nitrogen for 30 s When using lyophilized tissue the tubes do not need to be frozen in liquid nitrogen 4 Place the tubes into the TissueLyser Adapter Set 2 x 24 and fix into the clamps of the TissueLyser Immediately grind the samples for 1 min at 30 Hz 5 Disassemble the adaptor set remove the tubes and refreeze in liquid nitrogen for 30 s When using lyophilized tissue the tubes do not need to be frozen in liquid nitrogen 6 Repeat step 4 reversing the position of the tubes within the adaptor set P
66. will also be a pellet in the collection tube Avoid disturbing the pellet when transferring the supernatant 13 Add 1 5 volumes of Buffer AW1 to the cleared lysate and mix immediately by vortexing Plant Tissue 2 Q9 2 fo a 5 For example to 5 ml cleared lysate add 7 5 ml Buffer AW1 Reduce the amount of Buffer AW1 accordingly if the volume of lysate is smaller A precipitate may form after the addition of Buffer AW1 but this will not affect the DNeasy procedure Note Ensure that ethanol has been added to Buffer AW 1 See Things to do before starting page 26 Note It is important to pipet Buffer AW1 directly onto the cleared lysate and to mix immediately 14 Pipet the sample maximum 15 ml including any precipitate that may have formed into the DNeasy Maxi spin column placed in a 50 ml collection tube supplied Centrifuge at 3000 5000 x g for 5 min at room temperature in a swing out rotor Discard the flow through Reuse the collection tube in step 15 Flow through fractions contain Buffer AW1 and are therefore not compatible with bleach See page 6 for safety information 28 DNeasy Plant Handbook 10 2012 15 Add 12 ml Buffer AW2 to the DNeasy Maxi spin column and centrifuge for 10 min at 3000 5000 x g to dry the membrane Discard flow through and collection tube Note Ensure that ethanol has been added to Buffer AW2 prior to use See Things to do before starting page 26 It is important t
67. ysaccharides and polyphenolics and are therefore easier to handle When working with fungi harvest mycelium directly from a culture dish or from liquid culture For liquid culture first pellet cells by centrifugation Remove the supernatant completely before disruption and lysis Fresh frozen or freeze dried fungal material can be used Sample size DNeasy Plant procedures are optimized for a maximum of 100 mg Mini 1 g Maxi or 50 mg DNeasy 96 of wet weight starting material Table 2 provides guidelines for wet weights of leaf tissue If using dried starting material the maximum amount which can be processed must be reduced by a factor of approximately 5 Exceeding the recommended maximum amount of starting material will result in inefficient lysis resulting in low yield and purity With some plant species it may be possible to increase the amount of starting material in the DNeasy 96 procedure to increase DNA yield For example increasing the amount of wheat starting material to 100 mg increased the DNA yield by 35 27 4 pg DNA compared with 20 4 pg DNA after two 100 pl elutions Note that DNA yields do not necessarily increase linearly with increased amounts of starting material Furthermore for some plant species impurities may be present in the purified DNA if the amount of starting material is increased To find the optimum amount of starting material for a particular plant species use a range of amounts e g 50 75 and 1

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