NucleoSpin® 96 Plant II (Core Kit) - MACHEREY
Contents
1. Genomic DNA from plant User manual NucleoSpin 96 Plant II NucleoSpin 96 Plant II Core Kit May 2014 Rev 03 MACHEREY NAGEL MN www mn net com Genomic DNA from plant Table of contents 1 Components 1 1 Kit contents 1 2 Reagents to be supplied by user 5 2 Product description 6 2 1 The basic principle 6 2 2 Kit specifications 6 2 3 Required hardware 7 2 4 Recommended accessories for use of the NucleoSpin 96 Plant II Core Kit 8 2 5 Automated processing on robotic platforms 9 2 6 Storage and homogenization of samples 10 2 7 Elution procedures 11 3 Storage conditions and preparation of working solutions 12 4 Safety instructions 13 5 Protocols 15 5 1 NucleoSpin 96 Plant II centrifuge processing 15 5 2 NucleoSpin 96 Plant II vacuum processing 20 6 Appendix 26 6 1 Troubleshooting 26 6 2 Ordering information 27 6 3 Product use restriction warranty 29 MACHEREY NAGEL 05 2014 Rev 03 3 Genomic DNA from plant 1 Components 1 1 Kit contents NucleoSpin 96 Plant Il 2 x 96 preps 4 x 96 preps 24 x 96 preps REF 740663 2 740663 4 740663 24 Lysis Buffer PL1 125 mL 250 mL 6 x 250 mL Lysis Buffer PL2 100 mL 200 mL 6 x 200 mL Precipitation Buffer PL3 25 mL 50 mL 6 x 50 mL Binding Buffer PC 125 mL 250 mL 6 x 250 mL Wash Buffer PW1 100 mL 2x 100 mL 12 x 100 mL nee 100 mL 2x 100 mL 12 x 100 mL Elution Buffer PE 60 mL 125 mL 6 x 125 mL RNase A lyophilized 30 mg 2x 30mg 12 x 30 mg Nuc2. Binding Plate Do not moisten the rims of the individual wells while dispensing the samples Optional Seal openings of the binding plate with a Gas permeable Foil Bind DNA to silica membrane Place the NucleoSpin Plant II Binding Plate stacked on an MN Square well Block in the rotor buckets Centrifuge at 5 600 6 000 x g for 5 min Typically lysates will pass through the columns within 1 min The centrifugation process can be extended to 20 min if the lysates have not passed completely Wash silica membrane Add 400 uL PW1 to each well of the NucleoSpin Plant II Binding Plate Seal plate with a Gas permeable Foil and centrifuge again at 5 600 6 000 x g for 2 min Place NucleoSpin Plant Il Binding Plate on a new MN Square well Block Add 700 uL PW2 to each well of the NucleoSpin Plant II Binding Plate Seal plate with a Gas permeable Foil and centrifuge again at 5 600 6 000 x g for 2 min MACHEREY NAGEL 05 2014 Rev 03 19 NucleoSpin 96 Plant II centrifuge processing Add 700 pL PW2 to each well of the NucleoSpin Plant II Binding Plate Seal plate with a Gas permeable Foil and centrifuge again at 5 600 6 000 x g for 2 min Place NucleoSpin Plant Il Binding Plate on a new MN Square well Block Note For critical ethanol sensitive applications it is recommended to prolong the centrifugation time up to 15 min or incubate at higher temperature Remove the adhesive foil and place the NucleoSpi
3. 65 C for 30 min Depending on plant sample and available methods Buffer PL1 and RNase A may be added to the plant material before homogenization by the appropriate mechanical method Proceed with step 3 2b Cell lysis using Buffer PL2 and PL3 Add 400 uL Buffer PL2 and 10 uL RNase A to each sample Close tubes again using new Cap Strips supplied Mix by vigorous shaking for 15 30 s Spin briefly for 30 s at 1 500 x g to collect any sample from the Cap Strips Incubate samples at 65 C for 30 min Depending on plant sample and available methods Buffer PL2 and RNase A may be added to the plant material before homogenization by the appropriate mechanical method Open tubes add 100 pL Buffer PL3 close tubes mix thoroughly and incubate for 5 min on ice to precipitate SDS completely 18 MACHEREY NAGEL 05 2014 Rev 03 NucleoSpin 96 Plant II centrifuge processing Clear lysate by centrifugation Centrifuge the samples for 20 min at full speed 5 600 6 000 x g Remove Cap Strips Adjust binding conditions Pre dispense 450 uL Binding Buffer PC to each well of a MN Square well Block Add 400 uL cleared lysate of each sample and mix by repeated pipetting up and down Mix at least 3 times Transfer lysate to NucleoSpin Plant II Binding Plate Place NucleoSpin Plant Il Binding Plate on a MN Square well Block Transfer samples from the previous step into the wells of the NucleoSpin Plant II
4. Protocol step 1 Homogenize samples Suitable consumables not supplied with the core kits Rack of Tube Strips with Cap Strips Remarks 4 Adjust binding conditions Square well Block or Round well Block or MN Square well Block For mixing cleared lysate with Buffer PC 7 Wash silica MN Wash Plates MN Wash Plate membrane minimizes the risk of cross contamination vacuum processing 8 Elute DNA Rack of Tubes Strips with Cap Strips or Round well Block or Round well Block Low zg centrifugation only i i MACHEREY NAGEL 05 2014 Rev 03 Genomic DNA from plant 2 5 Automated processing on robotic platforms NucleoSpin 96 Plant Il can be used fully automated on many common laboratory workstations For the availability of scripts and general considerations about adapting NucleoSpin 96 Plant Il on a certain workstation please contact MN Full processing under vacuum enables complete automation without the need for centrifugation steps for drying of the binding membrane or for elution The risk of cross contamination is reduced by optimized vacuum settings during the elution step and by the improved shape of the outlets of the NucleoSpin Plant II Binding Plate Drying of the NucleoSpin Plant II Binding Plates under vacuum is sufficient because the bottom of the plate is protected by the MN Wash Plate during the washing steps As a result it is recomm
5. beads Homogenization should be done thoroughly until the plant material is ground to a fine powder In most cases this can be achieved by vigorous shaking for 3 x 60 s with occasional freezing in liquid nitrogen This problem can also be avoided by lyophilizing the material This way it will be easier to grind the material Extraction of DNA from plant material during lysis was not sufficient To obtain higher yields of DNA the incubation time in lysis buffer can be prolonged up to overnight Suboptimal lysis buffer was used beer e Lysis efficiencies of Buffer PL1 CTAB and Buffer PL2 SDS DNA yield is are different and depend on the plant species Try both buffers low in a side by side purification to find the best detergent system to lyse your plant material Sample contains too much RNA Add 10 uL of RNase A solution to the Lysis Buffer PL1 or PL2 before heat incubation If this is not successful add the enzyme to the cleared supernatant of step 3 and incubate for 30 min at 60 C Sub optimal Elution The DNA can be either eluted in higher volumes up to 300 uL or by repeating the elution step up to three times Remember that the elution buffer must be preheated to 70 C prior to elution Also check the pH of the elution buffer used which should be in a range of pH 8 8 5 To ensure correct pH use supplied elution Buffer PE DNA is Sample was contaminated with DNase degraded Check bench pipettes and storage of sample in order
6. mn net com Weiterf hrende Informationen finden Sie in den Sicherheitsdatenbl ttern www mn net com MACHEREY NAGEL 05 2014 Rev 03 15 NucleoSpin 96 Plant II centrifuge processing 5 Protocols 5 1 NucleoSpin 96 Plant Il centrifuge processing For hardware requirements refer to section 2 3 For detailed information on each step see page 18 For use of the NucleoSpin 96 Plant Il Core Kit REF 740468 4 refer to section 2 4 regarding recommended accessories Before starting the preparation Check if Buffer PW2 and RNase A were prepared according to section 3 Set incubator or oven to 65 C Equilibrate Buffer PE to 70 C Protocol at a glance 1 Homogenize samples Up to 100 mg wet or 20 mg lyophilized plant tissue 5 600 6 000 x g 2 min 2a Celllysis using Buffer PL1 500 uL PL1 10 pL RNase A Mix 65 C 30 min Proceed with step 3 2b Celllysis using Buffer PL2 400 uL PL2 and PL3 10 pL RNase A Mix 65 C 30 min 100 uL PL3 Mix and incubate on ice for 5 min Proceed with step 3 16 MACHEREY NAGEL 05 2014 Rev 03 NucleoSpin 96 Plant II centrifuge processing 3 Clear lysate by centrifugation 4 Adjust binding conditions Mix 450 uL PC with 400 uL cleared lysate 5 600 6 000 x g 20 min 5 Transfer lysate to NucleoSpin Plant II Binding Plate 6 Bind DNA to silica membrane 5 600 6 000 x g of the NucleoSpin Plant II 2 min B
7. samples Suitable methods include grinding with pestle and mortar in the presence of liquid nitrogen or using steel beads We also recommend the use of other commercial homogenizers bead mills etc Methods to homogenize samples Commercial homogenizers for example Crush Express for 96 well homogeni zation Saaten Union Resistenzlabor GmbH D 33818 Leopoldsh he Tissue Striker www KisanBiotech com or Geno Grinder 2000 can be used 10 MACHEREY NAGEL 05 2014 Rev 03 Genomic DNA from plant 2 7 Samples can be disrupted using bead based homogenization tools for exam ple GenoGrinder http www spexcsp com or for Germany www c3 analysen technik de or Mixer Mill MM400 http www retsch com products milling ball mills mm 400 Please refer to instrument manufacturers recommendations for suitable plates or tubes for homogenization Homogenizing samples by VA steel beads diameter 3 mm Put 4 5 beads and plant material together into a 15 ml plastic tube Falcon chill the tube in liquid nitrogen and vortex for about 30 seconds e g with a Multi Pulse Vortexer Sch tt Labortechnik GmbH Postfach 3454 D 37024 G ttingen Germany Repeat this chilling and vortexing procedure until the entire plant material is ground to a powder Chill the tube once more and remove the beads by rolling them out gently or remove them with a magnet Keep the material frozen throughout the whole homogenization procedure Do not add nitr
8. to avoid DNase contamination MACHEREY NAGEL 05 2014 Rev 03 27 Genomic DNA from plant Problem Possible cause and suggestions Sample contains DNA degrading contaminants e g phenolic com pounds secondary metabolites N Repeat washing step with Buffer PW1 DNA purity is low Elution buffer contains EDTA e EDTA can disturb subsequent reactions Use of water or supplied Elution Buffer PE is highly recommended 6 2 Ordering information Product REF Pack of NucleoSpin 96 Plant Il 740663 2 1 x 96 preps 740663 4 4x 96 preps 740663 24 24 x 96 preps NucleoSpin 96 Plant II Core Kit 740468 4 4x 96 preps NucleoSpin 8 Plant II 740669 12 x 8 preps 740669 5 60 x 8 preps NucleoSpin 8 Plant II Core Kit 740467 4 48 x 8 preps Buffer PL1 740918 125 mL Buffer Set PL2 PL3 740919 1 set 100 mL Buffer PL2 25 mL Buffer PL3 Buffer PC 740937 125 mL Buffer PW1 740938 125 mL Buffer PW2 Concentrate 740939 50 mL for 250 mL Buffer PW2 RNase A lyophilized 740505 100 mg 740505 50 50 mg Proteinase K 740506 100 mg 28 MACHEREY NAGEL 05 2014 Rev 03 Genomic DNA from plant Product REF Pack of MN Square well Block 740476 4 740476 24 24 MN Wash Plate 740479 4 740479 24 24 Rack of Tube Strips 740477 4 sets 1 set consists of 1 rack 12 strips with 8 740477 24 24 sets tubes each and 12 Cap Strips Cap Strips 740478 48 740478 24 288 Gas permeable Foil 740675 50 Self adhering Foil 740676
9. 50 NucleoVac 96 Vacuum Manifold 740681 1 NucleoVac Vacuum Regulator 740641 1 Visit www mn net com for more detailed product information MACHEREY NAGEL 05 2014 Rev 03 29 Genomic DNA from plant 6 3 Product use restriction warranty NucleoSpin 96 Plant II Core Kit components are intended developed designed and sold FOR RESEARCH PURPOSES ONLY except however any other function of the product being expressly described in original MACHEREY NAGEL product leaflets MACHEREY NAGEL products are intended for GENERAL LABORATORY USE ONLY MACHEREY NAGEL products are suited for QUALIFIED PERSONNEL ONLY MACHEREY NAGEL products shall in any event only be used wearing adequate PROTECTIVE CLOTHING For detailed information please refer to the respective Material Safety Data Sheet of the product MACHEREY NAGEL products shall exclusively be used in an ADEQUATE TEST ENVIRONMENT MACHEREY NAGEL does not assume any responsibility for damages due to improper application of our products in other fields of application Application on the human body is STRICTLY FORBIDDEN The respective user is liable for any and all damages resulting from such application DNA RNA PROTEIN purification products of MACHEREY NAGEL are suitable for IN VITRO USES ONLY ONLY MACHEREY NAGEL products specially labeled as IVD are also suitable for IN VITRO diagnostic use Please pay attention to the package of the product IN VITRO diagnostic products are expres
10. 50 uL Binding Buffer PC to each well of a MN Square well Block Add 400 uL cleared lysate of each sample and mix by repeated pipetting up and down Mix at least 3 times Transfer lysate to NucleoSpin Plant II Binding Plate Place waste tray into manifold base Insert spacers labeled MTP MULTI 96 PLATE notched side up into NucleoVac and place the MN Wash Plate on them Close manifold and place NucleoSpin Plant II Binding Plate on top of the manifold Transfer samples from the previous step into the wells of the NucleoSpin Plant II Binding Plate Do not moisten the rims of the individual wells while dispensing the samples Bind DNA to silica membrane Apply vacuum of 0 2 to 0 4 bar to allow samples to pass through the membrane Flow through rate should be about 1 2 drops per second Adjust vacuum strength accordingly Finally release the vacuum Reduction of atmospheric pressure MACHEREY NAGEL 05 2014 Rev 03 25 NucleoSpin 96 Plant II vacuum processing 7 Wash silica membrane Add 400 pL PW1 to each well of the NucleoSpin Plant Il Binding Plate and apply vacuum of 0 2 to 0 4 bar until the buffer has passed the membrane completely Release the vacuum Add 700 uL PW2 to each well of the NucleoSpin Plant II Binding Plate and apply vacuum of 0 2 to 0 4 bar until the buffer has passed the membrane completely Release the vacuum Add 700 pL PW2 to each well of the NucleoSpin
11. DNA can be extracted with Iysis buffers containing chaotropic salts denaturing agents and detergents The standard isolation ensures the lysis of plant material with the CTAB Lysis Buffer PL1 which is specially developed for plants In addition an SDS based lysis buffer Buffer PL2 is provided as an alternative Buffer PL2 requires subsequent protein precipitation with potassium acetate Lysates should be cleared by centrifugation in order to remove polysaccharides contaminations and residual cellular debris The clear supernatant is mixed with Binding Buffer PC to create conditions for optimal binding to the silica membrane in the binding plate After washing with two different buffers Buffer PW1 and Buffer PW2 DNA can be eluted in low salt Buffer PE or water and is ready to use for subsequent analysis and processing 2 2 Kit specifications NucleoSpin 96 Plant II is designed for the isolation of genomic DNA from plant material e NucleoSpin 96 Plant II allows parallel purification of multiples of 96 samples each with up to 100 mg sample per well wet weight Depending on the individual sample NucleoSpin 96 Plant II shows yields in the range of 1 30 ug DNA maximum column capacity is about 30 ug with an AseolAogo ratio between 1 80 and 1 90 and typical concentrations of 100 200 ng uL The amount of DNA that can be expected per mg of sample extracted depends on the size and ploidy of the genome For example 100 mg fresh wheat w
12. Plant Il Binding Plate and apply vacuum of 0 2 to 0 4 bar until the buffer has passed the membrane completely Release the vacuum Remove MN Wash Plate and waste tray Reassemble the vacuum manifold and dry the membrane by applying maximum vacuum 0 6 bar for 10 minutes 8 Elute DNA Insert spacers MICROTUBE RACK into the vacuum manifold base Place the Rack of Tube Strips into the manifold base Close the manifold and insert the NucleoSpin Plant II Binding Plate into the manifold top Dispense 100 uL pre heated Buffer PE 70 C to each well of the NucleoSpin Plant II Binding Plate Dispense the buffer directly onto the membrane Incubate at room temperature for 2 min Apply vacuum of 0 4 bar until the elution buffer has passed the membrane completely For optimal yield it is recommended to repeat this step once incubation of Buffer PE on the membrane not required Yields will be 10 20 higher when eluting with 2 x 100 uL Buffer PE depending on the total amount of DNA However the concentration of DNA will be much lower than with 100 uL Note Elution can be done with TE buffer at least pH 8 0 as well Elution efficiency will decrease when using elution buffers with pH s 8 0 26 MACHEREY NAGEL 05 2014 Rev 03 Genomic DNA from plant 6 Appendix 6 1 Troubleshooting Problem Possible cause and suggestions Homogenization of plant material was not sufficient For most species we recommend grinding with steel
13. aces Due to the dead volume of the silica membrane please note that the difference between the dispensed elution volume and the recovered elution buffer is approximately 45 uL recovered elution volume dispensed elution volume 45 uL 12 MACHEREY NAGEL 05 2014 Rev 03 Genomic DNA from plant 3 Storage conditions and preparation of working solutions Attention Buffer PL1 contains CTAB Buffer PL2 contains SDS Buffers PC and PW1 contain chaotropic salt Wear gloves and goggles CAUTION Buffers PC and PW1 contain guanidine hydrochloride which can form highly reactive compounds when combined with bleach sodium hypochlorite DO NOT add bleach or acidic solutions directly to the sample preparation waste Store RNase A at 4 C on arrival storage at 4 C may cause precipitation of salts in different buffers All other components can be stored at room temperature 18 25 C and are stable for up to 1 year Before starting any NucleoSpin 96 Plant II protocol prepare the following Lysis Buffer PL2 Check for precipitated SDS especially after storage at temperatures below 20 C If necessary incubate the bottle for several minutes at 30 40 C and mix well until the precipitate is redissolved completely Wash Buffer PW2 Add the indicated volume of ethanol 96 100 to Buffer PW2 Concentrate before first use Store Buffer PW2 at room temperature 18 25 C for up to one year RNase A Add the given volume of wat
14. ample 2 x 100 UL Finally combine eluates and measure yield Alternatively use preheated Elution Buffer PE 70 C Preheat elution buffer to increase yield After loading half of the preheated elution buffer 50 uL onto the membrane incubate the NucleoSpin Plant II Binding Plate for 3 min at 60 70 C Centrifuge for elution as indicated Repeat the elution step once Highly concentrated eluates Using a minimal elution volume about 50 uL about 70 80 of bound nucleic acids can be eluted resulting in highly concentrated eluates MACHEREY NAGEL 05 2014 Rev 03 11 Genomic DNA from plant Elution may also be performed with Tris EDTA buffer TE with a pH equal or higher than 8 This will increase DNA stability during long term or multi use storage at 4 C or ambient temperature by inhibiting omnipresent DNases However EDTA interferes depending on the final concentration with certain downstream applications For optimal performance of isolated DNA in downstream applications we recommend eluting with the supplied elution buffer and storing it especially long term at 20 C Several freeze thaw cycles will not interfere with most downstream applications Performance of long range PCR e g gt 10 kbp or the detection limit of trace amounts of DNA species may be reduced after multiple freeze thaw cycles or prolonged storage of eluted DNA at 4 C or room temperature This is due to shearing of DNAor adsorption to surf
15. cket height 85 mm Regarding waste collection suitable consumables e g MN Square well Blocks are necessary and they are not included in the kit For the most convenient handling without the need of emptying and reusing the MN Square well Blocks we recommend using six MN Square well Blocks if two 96 well plates are processed at once see ordering information Alternatively it is possible to empty the MN Square well Blocks after every centrifugation step reducing the amount of MN Square well Blocks needed Vacuum processing The NucleoSpin 96 Plant II kit can be used with the NucleoVac 96 Vacuum Manifold see ordering information When using NucleoSpin 96 Plant II with less than 96 samples Self adhering PE Foil see ordering information should be used in order to MACHEREY NAGEL 05 2014 Rev 03 7 Genomic DNA from plant close and protect non used wells of the NucleoSpin Plant II Binding Plate and thus guarantee proper vacuum Establish a reliable vacuum source for the NucleoVac 96 Vacuum Manifold The manifold may be used with a vacuum pump house vacuum or water aspirator We recommend a vacuum of 0 2 to 0 4 bar reduction of atmospheric pressure The use of the NucleoVac Vacuum Regulator see ordering information is recommended Alternatively adjust the vacuum so that during the purification the sample flows through the column with a rate of 1 2 drops per second Depending on the amount of sample being use
16. d the vacuum times may need to be increased for complete filtration Additionally a suitable centrifuge for sample preparation steps may be required For general consumables and equipment needed please see section 1 2 2 4 Recommended accessories for use of the NucleoSpin 96 Plant II Core Kit The NucleoSpin 96 Plant Il Core Kit provides all necessary buffers enzymes and NucleoSpin Binding Plates Accessories e g lysis plates waste collection plates elution plates or tubes are not provided with the Core Kit The reduced kit composition along with a large variety of separately available accessories allow optimal adjustment of the kit to individual user needs The user can select additional consumables according to his requirements for highest flexibility The NucleoSpin 96 Plant Il Core Kit provides buffers RNase A and NucleoSpin Binding Plates only Accessory plates e g elution plates are not provided with the core kit The user can individually select additional consumables from a variety of suitable accessory plates according to his requirements for highest flexibility For use of NucleoSpin 96 Plant Il Core Kit follow the standard protocols see section 5 1 or 5 2 respectively Recommended accessories for use of the NucleoSpin 96 Plant Il Core Kit are avail able from MACHEREY NAGEL For ordering information please refer to section 6 2 8 MACHEREY NAGEL 05 2014 Rev 03 Genomic DNA from plant
17. e gloves eye protection Schutzhandschuhe Augenschutz tragen P 301 312 IF SWALLOWED Call a POISON CENTER doctor if you feel unwell BEI VERSCHLUCKEN Bei Unwohlsein GIFTINFORMATIONSZENTRUM Arzt anrufen P 302 352 IF ON SKIN Wash with plenty of water BEI KONTAKT MIT DER HAUT Mit viel Wasser waschen P 304 340 IF INHALED Remove victim to fresh air and keep at rest in a position comfort able for breathing BEI EINATMEN An die frische Luft bringen und in einer Position ruhigstellen die das Atmen erleichtert P 305 351 338 IF IN EYES Rinse continuously with water for several minutes Remove contact lenses if present and easy to do continue rinsing Bei Kontakt mit den Augen Einige Minuten lang behutsam mit Wasser sp len Vorhandene Kontaktlinsen nach M glichkeit entfernen Weiter sp len P 330 Rinse mouth Mund aussp len P 333 313 If skin irritation occurs Get medical advice attention Bei Hautreizung rztlichen Rat einholen rztliche Hilfe hinzuziehen P 337 313 Get medical advice attention Bei anhaltender Augenreizung rztlichen Rat einholen rztliche Hilfe hinzuziehen P 342 311 If experiencing respiratory symptoms Call a POISON CENTER doctor Bei Symptomen der Atemwege GIFTINFORMATIONSZENTRUM Arzt anrufen P 403 235 Store in a well ventilated place Keep cool K hl an einem gut bel fteten Ort aufbewahren For further information please see Material Safety Data Sheets www
18. ended to integrate the MN Wash Plate into the automated procedure to protect against these wash buffer residues The MN Frame see ordering information can be used to position the disposable MN Wash Plate inside the vacuum chamber This also reduces the risk of cross contamination as common metal adaptors tend to get contaminated by gDNA In addition thorough cleaning of the vacuum chamber is recommended after each run to prevent forming of gDNA containing aerosols Visit MN online at www mn net com or contact your local MACHEREY NAGEL distributor for technical support regarding hardware software setup instructions and selection of the protocol Several application notes of the NucleoSpin 96 Plant Il kit on various liquid handling instruments can also be found at www mn net com at Bioanalysis Literature 2 6 Storage and homogenization of samples We recommend using young plant samples and keeping the plants in the dark for about 12 h before collecting samples if possible in order to reduce the polysaccharide content Plant samples can be stored frozen under ethanol or lyophilized In many cases lyophilized dried material can be processed more easily and gives higher yield However keep in mind that dried samples may reduce the amount of starting material by the factor 5 for example 20 mg dried plant leaves vs 100 mg fresh weight As plant tissue is very robust the lysis procedure is most effective with well homogenized powdered
19. er indicated on the vial see below to lyophilized RNase A Store the RNase A solution at 4 C for up to 3 months For longer storage up to 1 year the RNase A solution should be divided into small aliquots and stored at 20 C NucleoSpin NucleoSpin NucleoSpin NucleoSpin 96 Plant II 96 Plant Il 96 Plant Il 96 Plant II Core Kit 2 x 96 preps 4x96 preps 24x96 preps 4x96 preps REF 740663 2 740663 4 740663 24 740468 4 Wash 100 mL 2x 100 mL 12 x 100 mL 2 x 100 mL Buffer PW2 Add 400 mL Add 400 mL Add 400 mL Add 400 mL Concentrate ethanol ethanol to ethanol to ethanol to each bottle each bottle each bottle RNase A 30 mg 2x 30 mg 12 x 30 mg 2x 30mg lyophilized Add 2 5 mL Add 2 5 mL Add 2 5 mL Add 2 5 mL H O H O H O H O to each vial to each vial to each vial MACHEREY NAGEL 05 2014 Rev 03 13 Genomic DNA from plant 4 Safety instructions The following components of the NucleoSpin 96 Plant Il and NucleoSpin 96 Plant Il Core kits contain hazardous contents Wear gloves and goggles and follow the safety instructions given in this section GHS classification Only harmful features need not be labeled with H and P phrases until 125 mL or 125 g Mindergef hrliche Eigenschaften m ssen bis 125 mL oder 125 g nicht mit H und P S tzen gekennzeichnet werden Component Hazard contents GHS symbol Hazard Precaution phrases phrases Inhalt Gefahrstoff GHS Symbol H S tze P S tze PC Guan
20. er PW2 and RNase A were prepared according to section 3 Set incubator or oven to 65 C Equilibrate Buffer PE to 70 C Protocol at a glance 1 Homogenize samples Up to 100 mg wet or 20 mg lyophilized plant tissue 5 600 6 000 x g 2 min 2a Celllysis using Buffer PL1 500 uL PL1 10 pL RNase A Mix 65 C 30 min Proceed with step 3 2b Celllysis using Buffer PL2 400 uL PL2 and PL3 Mix 65 C 30 min 100 pL PL3 Mix and incubate on ice for 5 min Proceed with step 3 3 Clear lysate by centrifugation 5 000 6 000 x g 20 min Reduction of atmospheric pressure MACHEREY NAGEL 05 2014 Rev 03 21 NucleoSpin 96 Plant II vacuum processing 4 Adjust binding conditions Mix 450 pL PC with 400 uL cleared lysate 5 Transfer lysate to NucleoSpin Plant II Binding Plate 6 Bind DNA to silica membrane 0 2 to 0 4 bar of the NucleoSpin Plant Il 2 min Binding Plate 7 Wash and dry silica membrane 400 pL PW1 700 pL PW2 700 pL PW2 0 4 bar 1 min each step Remove MN Wash Plate Dry silica membrane 10 min maximum vacuum 8 Elute DNA 100 pL PE incubate 2 min 0 4 bar 2 min Repeat once 22 MACHEREY NAGEL 05 2014 Rev 03 NucleoSpin 96 Plant Il vacuum processing Setup of vacuum manifold Binding Washing steps Step 4 Place the NucleoSpin Binding Plate on top of the manifold lid Step 3 Place the manifold lid on top of the manifo
21. idinium hydrochlo Warning 226 302 210 233 ride 24 36 ethanol 301 312 330 35 55 403 235 Guanidiniumhydrochlorid Achtung 24 36 Ethanol 35 55 PW1 Guanidinium hydrochlo Warning 226 302 210 233 280 ride 36 50 isopropa 319 301 312 nol 20 50 305 351 338 Guanidiniumhydrochlorid Achtung 330 337 313 36 50 Isopropanol 403 235 20 50 RNase A RNase A Iyophilized Danger 317 334 261 RNase A Iyophilisiert lt gt Gefahr 304 340 3424311 3014312 280 3024352 3334313 Hazard phrases H 226 Flammable liquid and vapour Fl ssigkeit und Dampf entz ndbar H 302 Harmful if swallowed Gesundheitssch dlich bei Verschlucken H 317 May cause an allergic skin reaction Kann allergische Hautreaktionen verursachen H 319 Causes serious eye irritation Verursacht schwere Augenreizung H 334 May cause allergy or asthma symptoms or breathing difficulties if inhaled Kann bei Einatmen Allergie asthmaartige Symptome oder Atembeschwerden verursa chen 14 MACHEREY NAGEL 05 2014 Rev 03 Genomic DNA from plant Precaution phrases P 210 Keep away from heat hot surfaces sparks open flames and other ignition sources No smoking Von Hitze heiBen Oberflachen Funken offenen Flammen sowie anderen Ztindquellenarten fernhalten Nicht rauchen P 233 Keep container tightly closed Beh lter dicht verschlossen halten P 261 Avoid breathing dust Einatmen von Staub vermeiden P 280 Wear protectiv
22. inding Plate 7 Wash and dry silica membrane 400 uL PW1 5 600 6 000 x g 2 min 700 pL PW2 5 600 6 000 x g 2 min 700 pL Pw2 5 600 6 000 x g 10 min 8 Elute DNA 100 uL PE 70 C incubate 2 min 5 600 6 000 x g 2 min Repeat once MACHEREY NAGEL 05 2014 Rev 03 NucleoSpin 96 Plant Il centrifuge processing Detailed protocol For hardware requirements refer to section 2 3 For use of the NucleoSpin 96 Plant Il Core Kit REF 740468 4 refer to section 2 4 regarding recommended accessories Before starting the preparation Check if Buffer PW2 and RNase A were prepared according to section 3 Set incubator or oven to 65 C Equilibrate Buffer PE to 70 C Homogenize samples Fill up to 100 mg wet plant tissue or up to 20 mg dried material for example lyophilized plant tissue into each tube of the Tube Strips Add one 3 mm diameter steel bead to each tube Close the tubes with Cap Strips Freeze samples in liquid nitrogen Disrupt cells by vigorous shaking using a mixer mill Centrifuge at 5 600 x g for 2 min and remove Cap Strips For further processing use either Buffer PL1 2a or Buffers PL2 PL3 2b 2a Cell lysis using Buffer PL1 Add 500 uL Buffer PL1 and 10 uL RNase A to each sample Close tubes again using new Cap Strips supplied Mix by vigorous shaking for 15 30 s Spin briefly for 30 s at 1 500 x g to collect any sample from the Cap Strips Incubate samples at
23. ith a hexaploid genome 1 7 x 10 bp contain 30 ug DNA whereas the same amount of Arabidopsis with a smaller diploid genome 1 9 x 10 bp yields only 3 ug DNA The eluted DNA is ready to use in subsequent reactions like PCR restriction analysis etc NucleoSpin 96 Plant Il can be processed under vacuum or in a centrifuge Two lysis buffers based on CTAB PL1 or SDS PL2 are provided NucleoSpin 96 Plant Il can be used manually with the NucleoVac 96 Vacuum Manifold see ordering information or other vacuum devices 6 MACHEREY NAGEL 05 2014 Rev 03 Genomic DNA from plant Kit specifications at a glance Parameter NucleoSpin 96 Plant II Technology Silica membrane technology Format 96 well plates Processing Manual or automated vacuum or centrifugation Sample material 20 100 mg plant tissue plant cells wet weight Fragment size 50 bp approx 50 kpb Typical yield 1 30 ug A seo Aoeo 1 8 1 9 Elution volume 100 200 uL Preparation time 60 min plate excl lysis Binding capacity 30 ug 2 3 Required hardware NucleoSpin 96 Plant II can be processed under vacuum or with centrifugation Certain hardware for processing is required Centrifugation For centrifugation a microtiterplate centrifuge is required This centrifuge must be able to accomodate the NucleoSpin Plant II Binding Plate stacked on a Round or Square well Block and reach accelerations of 5 600 6 000 x g is required bu
24. ld base Step 2 Place the MN Wash Plate in the manifold Step 1 Insert spacers MTP MULTI 96 PLATE in the manifold base Final setup Elution step Step 4 Place the NucleoSpin Binding Plate on top of the manifold lid Step 3 Place the manifold lid on top of the manifold base Step 2 Place the Rack of Tube Strips in the manifold Step 1 Insert spacers MICROTUBE RACK in the manifold base Final setup MACHEREY NAGEL 05 2014 Rev 03 23 NucleoSpin 96 Plant II vacuum processing Detailed protocol For hardware requirements refer to section 2 3 For detailed information regarding the vacuum manifold setup see page 23 For use of the NucleoSpin 96 Plant II Core Kit REF 740468 4 refer to section 2 4 regarding recommended accessories Before starting the preparation Check if Buffer PW2 and RNase A were prepared according to section 3 Set incubator or oven to 65 C Equilibrate Buffer PE to 70 C For detailed information regarding the vacuum manifold set up see page 23 Homogenize samples Fill up to 100 mg wet plant tissue or up to 20 mg dried for example lyophilized plant tissue into each tube of the Tube Strips Add one 3 mm diameter steel bead to each tube Close the tubes with Cap Strips Freeze samples in liquid nitrogen Disrupt cells by vigorous shaking using a mixer mill Spin at 5 600 x g for 2 min and remove Cap Strips For further
25. leoSpin Plant Il 2 4 24 Binding Plate dark green rings MN Wash Plate 2 4 24 Rack of Tube Strips 4 8 32 for lysis and elution Cap Strips 24 48 288 MN Square well Block 6 12 72 Gas permeable Foil 10 20 120 User manual 1 1 1 1 The kit for 24 x 96 preparations REF 740663 24 consists of 6 x REF 740663 4 2 For preparation of working solutions and storage conditions see section 3 Composition of Elution Buffer PE 5 mM Tris HCl pH 8 5 4 1 rack 12 strips with 8 tubes each Cap Strips included 4 MACHEREY NAGEL 05 2014 Rev 03 Genomic DNA from plant 1 1 Kit contents continued NucleoSpin 96 Plant Il Core Kit 4 x 96 preps REF 740468 4 Lysis Buffer PL1 250 mL Lysis Buffer PL2 200 mL Precipitation Buffer PL3 50 mL Binding Buffer PC 250 mL Wash Buffer PW1 2 x 100 mL Wash Buffer PW2 Concentrate 2 x 100 mL Elution Buffer PE 125 mL RNase A lyophilized 2 x 30 mg NucleoSpin Plant II Binding Plate 4 dark green rings User manual 1 1 2 Reagents to be supplied by user 96 100 ethanol 1 For preparation of working solutions and storage conditions see section 3 2 Composition of Elution Buffer PE 5 mM Tris HCl pH 8 5 MACHEREY NAGEL 05 2014 Rev 03 5 Genomic DNA from plant 2 Product description 2 1 The basic principle The NucleoSpin 96 Plant Il kit is designed for the isolation of genomic DNA from plant materials After the plant samples have been homogenized the
26. n Plant II Binding Plate into an incubator for 20 min at 37 C to evaporate residual ethanol Elute DNA Place NucleoSpin Plant Il Binding Plate on the Rack of Tube Strips Dispense 100 uL pre heated Buffer PE 70 C to each well of the NucleoSpin Plant II Binding Plate Dispense the buffer directly onto the membrane Optional Incubate for 2 min at 70 C before centrifugation Centrifuge at 5 600 6 000 x g for 2 min Remove the NucleoSpin Plant Il Binding Plate from the Rack of Tube Strips For optimal yield it is recommended to repeat this step once incubation of Buffer PE on the membrane not required Yields will be 10 20 higher when eluting with 2 x 100 uL Buffer PE depending on the total amount of DNA However the concentration of DNA will be much lower than with 100 uL Note Elution can be done with TE buffer at least pH 8 0 as well Elution efficiency will decrease when using elution buffers with pH s 8 0 20 MACHEREY NAGEL 05 2014 Rev 03 NucleoSpin 96 Plant II vacuum processing 5 2 NucleoSpin 96 Plant Il vacuum processing For hardware requirements refer to section 2 3 For detailed information regarding the vacuum manifold setup see page 23 For detailed information on each step see page 24 For use of the NucleoSpin 96 Plant II Core Kit REF 740468 4 refer to section 2 4 regarding recommended accessories Before starting the preparation Check if Buff
27. n products and brands is only a kind of information i e it does not offend against trademarks and brands and can not be seen as a kind of recommendation or assessment Regarding these products or services we can not grant any guarantees regarding selection efficiency or operation MACHEREY NAGEL 05 2014 Rev 03 31
28. ns and descriptions of this MACHEREY NAGEL product appearing in MACHEREY NAGEL published catalogues and product literature are MACHEREY NAGEL s sole representations concerning the product and warranty No other statements or representations written or oral by MACHEREY NAGEL s employees agent or representatives except written statements signed by a duly authorized officer of MACHEREY NAGEL are authorized they should not be relied upon by the customer and are not a part of the contract of sale or of this warranty Product claims are subject to change Therefore please contact our Technical Service Team for the most up to date information on MACHEREY NAGEL products You may also contact your local distributor for general scientific information Applications mentioned in MACHEREY NAGEL literature are provided for informational purposes only MACHEREY NAGEL does not warrant that all applications have been tested in MACHEREY NAGEL laboratories using MACHEREY NAGEL products MACHEREY NAGEL does not warrant the correctness of any of those applications Last updated 07 2010 Rev 03 Please contact MACHEREY NAGEL GmbH amp Co KG Tel 49 0 24 21 969 270 e mail tech bio mn net com Trademarks disclaimer NucleoSpin is a registered trademark of MACHEREY NAGEL GmbH amp Co KG All used names and denotations can be brands trademarks or registered labels of their respective owner also if they are not special denotation To mentio
29. ogen to the tube This leads to sticking and loss of plant material attached to the beads High throughput homogenization Add the plant tissue to the individual tubes of the Tube Strips Add one 3 mm stainless steel bead to each tube and close the individual tubes with Cap Strips Freeze the sample in liquid nitrogen and insert the Rack of Tube Strips in a suitable homogenization tool e g mixer mill For disruption shake the samples for 60 90 s at 30 Hz or until a homogenous plant powder has been formed If necessary repeat shaking once Fresh plant material can also be homogenized with lysis buffer however homogenization of fresh plant material with lysis buffer may cause shearing of DNA For frozen plant material thawing should be avoided during the homogenization Samples should be frozen in liquid nitrogen before homogenization Lyophilized or silica gel dried material can be homogenized with or without lysis buffer Homogenization of lyophilized tissue with lysis buffer may result in higher yield but also may cause shearing of DNA Elution procedures It is possible to adjust the elution method and the volume of the elution buffer to the specific application of interest In addition to the standard method recovery rate about 80 90 described in the protocols there are 3 modifications possible High yields 90 100 of bound nucleic acids can be eluted by performing two elution steps with volumes as indicated in the protocol for ex
30. processing use either Buffer PL1 2a or Buffers PL2 PL3 2b 2a Cell lysis using Buffer PL1 Add 500 uL Buffer PL1 and 10 uL RNase A to each sample Close tubes again using new Cap Strips supplied Mix by vigorous shaking for 15 30 s Spin briefly for 30 s at 1 500 x g to collect any sample from the Cap Strips Incubate samples at 65 C for 30 min Depending on plant sample and available methods Buffer PL1 and RNase A may be added to the plant material before homogenization by the appropriate mechanical method Proceed with step 3 2b Cell lysis using Buffer PL2 and PL3 Add 400 uL Buffer PL2 and 10 uL RNase A to each sample Close tubes again using new Cap Strips supplied Mix by vigorous shaking for 15 30 s Spin briefly for 30 s at 1 500 x g to collect any sample from the Cap Strips Incubate samples at 65 C for 30 min Reduction of atmospheric pressure 24 MACHEREY NAGEL 05 2014 Rev 03 NucleoSpin 96 Plant II vacuum processing Depending on plant sample and available methods Buffer PL2 and RNase A may be added to the plant material before homogenization by the appropriate mechanical method Open tubes add 100 pL Buffer PL3 close tubes mix thoroughly and incubate for 5 min on ice to precipitate SDS completely Clear lysate by centrifugation Centrifuge the samples for 20 min at full speed 5 600 6 000 x g Remove Cap Strips Adjust binding conditions Pre dispense 4
31. siy marked as IVD on the packaging IF THERE IS NO IVD SIGN THE PRODUCT SHALL NOT BE SUITABLE FOR IN VITRO DIAGNOSTIC USE ALL OTHER PRODUCTS NOT LABELED AS IVD ARE NOT SUITED FOR ANY CLINICAL USE INCLUDING BUT NOT LIMITED TO DIAGNOSTIC THERAPEUTIC AND OR PROGNOSTIC USE No claim or representations is intended for its use to identify any specific organism or for clinical use included but not limited to diagnostic prognostic therapeutic or blood banking It is rather in the responsibility of the user or in any case of resale of the products in the responsibility of the reseller to inspect and assure the use of the DNA RNA protein purification products of MACHEREY NAGEL for a well defined and specific application MACHEREY NAGEL shall only be responsible for the product specifications and the performance range of MN products according to the specifications of in house quality control product documentation and marketing material This MACHEREY NAGEL product is shipped with documentation stating specifications and other technical information MACHEREY NAGEL warrants to meet the stated specifications MACHEREY NAGEL s sole obligation and the customer s sole remedy is limited to replacement of products free of charge in the event products fail to perform as warranted Supplementary reference is made to the general business terms and conditions of MACHEREY NAGEL which are printed on the price list Please contact us if you wish to ge
32. t an extra copy There is no warranty for and MACHEREY NAGEL is not liable for damages or defects arising in shipping and handling transport insurance for customers excluded or out of accident or improper or abnormal use of this product defects in products or 30 MACHEREY NAGEL 05 2014 Rev 03 Genomic DNA from plant components not manufactured by MACHEREY NAGEL or damages resulting from such non MACHEREY NAGEL components or products MACHEREY NAGEL makes no other warranty of any kind whatsoever and SPECIFICALLY DISCLAIMS AND EXCLUDES ALL OTHER WARRANTIES OF ANY KIND OR NATURE WHATSOEVER DIRECTLY OR INDIRECTLY EXPRESS OR IMPLIED INCLUDING WITHOUT LIMITATION AS TO THE SUITABILITY REPRODUCTIVITY DURABILITY FITNESS FOR A PARTICULAR PURPOSE OR USE MERCHANTABILITY CONDITION OR ANY OTHER MATTER WITH RESPECT TO MACHEREY NAGEL PRODUCTS In no event shall MACHEREY NAGEL be liable for claims for any other damages whether direct indirect incidental compensatory foreseeable consequential or special including but not limited to loss of use revenue or profit whether based upon warranty contract tort including negligence or strict liability arising in connection with the sale or the failure of MACHEREY NAGEL products to perform in accordance with the stated specifications This warranty is exclusive and MACHEREY NAGEL makes no other warranty expressed or implied The warranty provided herein and the data specificatio
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