NucleoSpin Plant II Genomic DNA Purification User Manual
Contents
1. Elute DNA Place the NucleoSpin Plant II Column into a new 1 5 mL microcentrifuge tube not provided Pipette 50 uL Buffer PE 65 C onto the membrane Incubate the NucleoSpin Plant I Column for 5 min at 65 C Centrifuge for 1min at 11 000 x g to elute the DNA Repeat this step with another 50 uL Buffer PE 65 C and elute into the same tube Note To achieve maximum yield or higher concentrations refer to section 2 6 for alternative elution procedures 50 uL PE 65 C 5 min 11 000x g 1 min 50 uL PE 65 C 5 min 11 000 x g 1 min 18 MACHEREY NAGEL 12 2010 Rev 05 NucleoSpin Plant II 5 2 Support protocol for genomic DNA from fungi Attention Additional reagents and equipment necessary Ethanol 96 100 Chloroform Micro pistill Siliconized glass beads or sea sand Homogenize sample Wash 50 200 mg mycelium fresh weight or material from a fruiting body of macro fungi in ethanol Mycelium can be obtained from a liquid culture or scraped off with or without agar from the surface of a solid medium Cover sample completely with ethanol and mix carefully Short washing in ethanol is sufficient in most cases although incubation overnight sometimes increases DNA yield Long term storage in ethanol is also possible Remove ethanol by pipetting and squeezing the mycelium Cell lysis Place the sample into a 1 5 mL microcentrifuge tube not provided Add 1502. Genomic DNA from Plant User Manual NucleoSpin Plant II NucleoSpin Plant Il Midi NucleoSpin Plant Il Maxi This product is distributed by Clontech Laboratories Inc A Takara Bio Company 1290Terra Bella Ave Mountain View CA 84043 www clontech com AEG Website for more details 5 For ordering information j clickchere ae 1 800 662 2566 PENARI orders clontech com PR113798 For technical support 1 800 662 2566 Clontech tech clontech com December 2010 Rev 05 MACHEREY NAGEL MN Genomic DNA Purification from Plant Protocol at a glance Rev 05 NucleoSpin Plant II 1 Homogenize c c c samples 100 mg 400 mg 1500 mg 2 Celllysis 400 uL 17 m EE em EE 10 uL RNase A 25 uL RNase A 100 uL RNase A 65 C 10 min 65 C 10 min 65 C 10 min ALTERNATIVELY ALTERNATIVELY ALTERNATIVELY 300 uL 15m E 5 3 mL ETE 10 uL RNase A 25 uL RNase A 100 uL RNase A 65 C 10 min 65 C 15 min 65 C 20 min on ice 5 min on ice 5 min on ice 5 min 3 Filtration ages 211 000 x g 4 500 x g 7 4 500 x g onlysere 2 min 10 min n 10 min V WV Adjust DNA binding 450 uL PC 2 3 mL PC 10 mLPC conditions 5 Bind DNA l z11 000xg 4 500xg 4 500xg 1 min ce 2 min 2 min 6 Wash and dry silica EB 200w EJ imPw EM mrw membrane z11 000xg 4 500 x g 4 500 x g 1 min 2 min 2 min i EJ 704 EJ 50v EM 00v 211 000xg 4 500 x g 4 500
3. MACHEREY NAGEL 12 2010 Rev 05 15 NucleoSpin Plant II 2b Red NP EH Re rad Transfer the resulting powder to a new tube and add 300 uL Buffer PL2 Vortex the mixture thoroughly Note If the sample can not be resuspended easily because for example the plant powder is soaking up too much buffer additional Buffer PL2 can be added Note that the volumes of RNase A Buffer PL3 step 2b and Buffer PC step 4 have lo be increased proportionally Add 10 pL RNase A solution and mix sample thoroughly Incubate the suspension for 10 min at 65 C Note For some plant material it might be advantageous to increase the incubation time to 30 60 min Add 75 uL Buffer PL3 mix thoroughly and incubate for 5 minutes on ice to precipitate SDS completely Proceed with step 3 Filtration Clarification of crude lysate Place a NucleoSpin Filter violet ring into a new Collection Tube 2 mL and load the lysate onto the column Centrifuge for 2 min at 11 000 x g collect the clear flow through and discard the NucleoSpin Filter If not all liquid has passed the filter repeat the centrifuga tion step If a pellet is visible in the flow through transfer the clear supernatant to a new 1 5 mL microcentrifuge tube not provided Alternatively centrifuge the crude lysate for 5 min at 11 000 x g and transfer the supernatant to a new tube or pass the precleared supernatant through the NucleoSpin Filter to remove solid
4. NucleoSpin NucleoSpin Plant Il Plant II Midi Plant Il Maxi Sample size Up to 100 mg Up to 400 mg Up to 1500 mg wet weight wet weight wet weight Up to 20 mg Up to 80 mg Up to 300 mg dry weight dry weight dry weight Typical yield 1 30 ug 10 100 ug 50 300 ug Elution volume 100 uL 400 uL 2000 uL Binding capacity 50 ug 200 ug 2500 ug Time prep 30 min 90 min 90 min MACHEREY NAGEL 12 2010 Rev 05 Genomic DNA from Plant 2 3 Storage of plant samples Plant samples can be stored in ethanol lyophilized or frozen Fresh material can be kept at 4 C for one day but should be frozen at 20 C for longer storage 2 4 Homogenization of plant samples As plant tissue is very robust the lysis procedure is most effective with well homogenized powdered samples Suitable methods include any type of commercial homogenizers rotor stator homogenizer or bead mills using steel or glass beads However we recommend grinding with a mortar and pestle in the presence of liquid nitrogen to obtain optimal yields After homogenization and treatment of the sample with lysis buffer the crude lysate can be cleared easily either with NucleoSpin Filters or by centrifugation Methods to homogenize samples Grinding with mortar and pestle in the presence of liquid nitrogen Freeze plant material in liquid nitrogen and do not let the sample thaw at any time during homogenization Precool mortar and pestle using liquid nitrogen Grind froz
5. Proceed with step 3 MACHEREY NAGEL 12 2010 Rev 05 25 NucleoSpin Plant Il Maxi 2b RecA Eerie Transfer the resulting powder to a new tube and add 5 3 mL Buffer PL2 Vortex the mixture thoroughly Note If the sample can not be resuspended easily because for example the plant powder is soaking up too much buffer additional Buffer PL2 can be added Note that the volumes of RNase A Buffer PL3 step 2b and Buffer PC step 4 have to be increased proportionally Add 100 pL RNase A solution and mix sample thoroughly Incubate the suspension for 20 min at 65 C Note For some plant material it might be advantageous to increase the incubation time to 30 60 min Add 700 uL Buffer PL3 mix thoroughly and incubate for 5 min on ice to precipitate SDS completely Proceed with step 3 Filtration Clarification of crude lysate Transfer the lysate to a NucleoSpin Filter XL Centrifuge for 10 min at 4 500 x g collect the clear flow through and discard the NucleoSpin Filter XL If not all liquid has passed the filter repeat the centrifugation step If a pellet is visible in the flow through transfer the clear supernatant to a new 50 mL microcentrifuge tube not provided Alternatively centrifuge the crude lysate for 5min at 4 500 x g and transfer the supernatant to a new tube or pass the precleared supernatant through the NucleoSpin Filter XL to remove solid particles completely Adjust DNA b
6. 1 Standard protocol for genomic DNA from plant Before starting the preparation Check if Wash Buffer PW2 and RNaseA were prepared according to section 3 Preheat Elution Buffer PE to 65 C Note The NucleoSpin Plant II kits include two different lysis buffers for optimal results with most common plant species Please refer to section 2 5 for choosing the optimal lysis buffer system for your individual plant sample and for information on how to process even more sample material than recommended in the following protocol 1 Homogenize sample Homogenize up to 100 mg wet weight or up to 20 mg dry weight lyophilized plant material for homogenization Homogenize methods see section 2 4 samples Proceed with cell lysis using Buffer PL1 step 2a or alternatively Buffer PL2 step 2b 2a KADASTA du Transfer the resulting powder to a new tube and add 400 uL PL1 400 uL Buffer PL1 Vortex the mixture thoroughly Note If the sample can not be resuspended easily because for example the plant powder is soaking up too much buffer additional Buffer PL1 can be added Note that the volumes of RNase A step 2a and Buffer PC step 4 have to be increased proportionally Add 10 pL RNase A solution and mix sample thoroughly 10 uL RNase A Incubate the suspension for 10 min at 65 C 65 C Note For some plant material it might be advantageous to 10 min increase the incubation time to 30 60 min Proceed with step 3
7. 2 g dung into a petri dish Add extraction buffer until the sample is completely soaked Heat the sample in a microwave oven 400 W for a few seconds until the extraction buffer is foaming Extraction buffer may be added to keep the sample in a slushy state 2 Celllysis Transfer sample into a bead mill or mortar Add 0 5 mL sea sand and disrupt the sample 3 Filtration Clarification of lysate Transfer the homogenized sample into a centrifuge tube e g Sorvall SS34 and centrifuge for 10 min at 5 000 x g Pipette 300 pL of the clear supernatant into a new 1 5 mL microcentrifuge tube not provided Proceed with section 5 1 step 3 20 MACHEREY NAGEL 12 2010 Rev 05 NucleoSpin Plant Il Midi 6 NucleoSpin Plant Il Midi protocol Before starting the preparation Check if Wash Buffer PW2 and RNaseA were prepared according to section 3 Preheat Elution Buffer PE to 65 C A centrifuge with a swing out rotor and appropriate buckets capable of reaching 4 500 x g is required Note The NucleoSpin Plant Il Midi kits include two different lysis buffers for optimal results with most common plant species Please refer to section 2 5 for choosing the optimal lysis buffer system for your individual plant sample and for information on how to process even more sample material than recommended in the following protocol 1 Homogenize sample Homogenize samples weight lyophilized plant material for homo
8. Genomic DNA from Plant 1 Components 1 1 Kit contents NucleoSpin Plant Il 10 preps 50 preps 250 preps REF 740770 10 740770 50 740770 250 Lysis Buffer PL1 5mL 25 mL 125 mL Lysis Buffer PL2 4 mL 20 mL 100 mL Precipitation Buffer PL3 1mL 5mL 25 mL Binding Buffer PC 6 mL 30 mL 125 mL Wash Buffer PW1 6 mL 30 mL 125 mL Wash Buffer PW2 6 mL 25 mL 50 mL Concentrate Elution Buffer PE 5mL 15 mL 30 mL RNase A lyophilized 1 5 mg 6 mg 2x15 mg NucleoSpin Filters 10 50 250 violet rings NucleoSpin Plant II 10 50 250 Columns green rings Collection Tubes 2 mL 20 100 500 User Manual 1 1 1 For preparation of working solutions and storage conditions see section 3 Composition of Elution Buffer PE 5 mM Tris HCl pH 8 5 4 MACHEREY NAGEL 12 2010 Rev 05 Genomic DNA from Plant 1 1 Kit contents continued NucleoSpin Plant II Midi NucleoSpin Plant Il Maxi 20 preps 10 preps REF 740771 20 740772 10 Lysis Buffer PL1 2x25mL 75mL Lysis Buffer PL2 2x20mL 60 mL Precipitation Buffer PL3 5mL 15mL Binding Buffer PC 2x30mL 125 mL Wash Buffer PW1 30 mL 50 mL Wash Buffer PW2 25 mL 50 mL Concentrate Elution Buffer PE 15 mL 30 mL RNase A lyophilized 6 mg 10 mg NucleoSpin Filters L XL 20 10 plus Collection Tubes NucleoSpin Plant II 20 10 Midi Maxi Columns plus Collection Tubes Collection Tubes 20 10 15 mL 50 mL User Manual For preparation of working solutions and storage conditio
9. 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 N VITRO USES ONLY ONLY MACHEREY NAGEL products specially labeled as IVD are also suitable for N VITRO diagnostic use Please pay attention to the package of the product N VITRO diagnostic products are expressly marked as IVD on the packaging IF THERE IS NO IVD SIGN THE PRODUCT SHALL NOT BE SUITABLE FOR N 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 dia
10. particles completely Adjust DNA binding conditions Add 450 uL Buffer PC and mix thoroughly by pipetting up and down 5 times or by vortexing 300 pL PL2 10 uL RNase A 65 C 10 min 75 uL PL3 on ice 5 min 11 000 x g 2 min 450 uL PC 16 MACHEREY NAGEL 12 2010 Rev 05 NucleoSpin Plant II Bind DNA Place a NucleoSpin Plant Il Column green ring into a new Collection Tube 2 mL and load a maximum of 700 uL of the sample Centrifuge for 1 min at 11 000 x g and discard the flow through The maximum loading capacity of the NucleoSpin Plant Il Column is 700 uL For higher sample volumes repeat the loading step Wash and dry silica membrane Add 400 uL Buffer PW1 to the NucleoSpin Plant Il Column Centrifuge for 1 min at 11 000 x g and discard flow through Note Although washing with Buffer PW1 increases purity it can in some cases slightly reduce the final yield Add 700 uL Buffer PW2 to the NucleoSpin Plant II Column Centrifuge for 1 min at 11 000 x g and discard flow through Add another 200 pL Buffer PW2 to the NucleoSpin Plant Il Column Centrifuge for 2 min at 11 000 x g in order to remove wash buffer and dry the silica membrane completely Load lysate 11 000 x g 1 min 400 uL PW1 11 000 x g 1 min 700 uL Pw2 11 000 x g 1 min 200 uL PW2 11 000 x g 2 min MACHEREY NAGEL 12 2010 Rev 05 17 NucleoSpin Plant II
11. soaks up too much lysis buffer Use more lysis buffer and increase the volume of Binding Buffer PC proportionally Suboptimal binding buffer volume was used Increase Binding Buffer PC proportionally if more lysis buffer was used Extraction of DNA from plant material during lysis was insufficient Increase incubation time in lysis buffer up to overnight Suboptimal Elution The DNA can either be eluted in higher volumes or by repeating the elution step up to three times Incubate NucleoSpin Plant II Column with elution buffer at 65 C for atleast 5 minutes Also check the pH of the elution buffer which should be in the range of pH 8 0 8 5 To ensure correct pH use supplied Elution Buffer PE 5 mM Tris HCl pH 8 5 MACHEREY NAGEL 12 2010 Rev 05 29 Genomic DNA from Plant Problem Possible cause and suggestions Sample was too viscous due to too much sample material or material carry over m Centrifuge large amounts of sample material before loading oe it onto the NucleoSpin Filter or Filter L XL ilter or NucleoSpin Make sure the cleared lysate is absolutely free of resuspended Plant Il Column matter before loading it onto the NucleoSpin Plant II or Plant is clogged II Midi Maxi Column Increase centrifugation speed and time Use more Lysis Buffer PL1 or PL2 Sample was contaminated with DNase If another elution buffer than Buffer PE is used make sure it is free of DNase activity for example b
12. x g 1 min y 5 2 min 2 min E 200 wt Pw EM i mpwe EM 5 z11 000xg 4 500xg 4 500 x g 2 min 10 min 10 min 7 Elute DNA 50 uL PE 200 uL PE 1000 uL PE 65 C 5 min i 65 C 5 min 65 C 5 min 211 000xg 4 500 x g 4 500 x g 1 min s 2 min 2 min Repeat elution step Repeat elution step MACHEREY NAGEL GmbH amp Co KG Neumann Neander Str 6 8 D 52355 D ren Germany Tel 49 0 24 21 969 270 www mn net com e mail tech bio mn net com Repeat elution step Genomic DNA from Plant Table of contents 1 Components 1 1 Kit contents 1 2 Reagents consumables and equipment to be supplied by user 1 8 About this User Manual 2 Product description 2 1 The basic principle 2 2 Kit specifications 2 3 Storage of plant samples 2 4 Homogenization of plant samples 2 5 Lysis of plant samples 2 6 Elution procedures 3 Storage conditions and preparation of working solutions 4 Safety instructions risk and safety phrases 5 NucleoSpin Plant Il protocols 5 1 Standard protocol for genomic DNA from plant 5 2 Support protocol for genomic DNA from fungi 5 3 Support protocol for soil compost dung and animal excrements 6 NucleoSpin Plant II Midi protocol 7 NucleoSpin Plant Il Maxi protocol 8 Appendix 8 1 Troubleshooting 8 2 Ordering information 8 3 Product use restriction warranty oO 0 rR BR O o oN N N 13 14 15 15 19 20 21 25 29 29 31 32 MACHEREY NAGEL 12 2010 Rev 05
13. 25 uL Buffer PE Resulting yield and concentration is shown as solid and dotted lines respectively 100 0 40 80 1 0 35 80 4 3 0 30 _ 704 Z 3 A X 604 poas E 2 504 pozo E X o 40 4 kA L A 015 2 ul 010 20 NE PI L 0 05 0 0 00 0 100 200 300 400 500 600 700 Elution volume pL Figure 2 NucleoSpin Plant II Midi elution profile Genomic DNA from 400 mg fresh wheat leaves was purified and eluted once A or twice B with 100 600 uL Buffer PE Resulting yield and concentration is shown as solid and dotted lines respectively MACHEREY NAGEL 12 2010 Rev 05 11 Genomic DNA from Plant 0 16 N o eo co o 0 14 zi o eo 0 12 ES P o 0 10 zik N e 0 08 Qo el L 0 06 DNA yield ug 3 eo l 7 B 0 04 DNA concentration ug L B o 0 02 N o T r r r r 0 0 500 1000 1500 2000 2500 3000 Elution volume uL o Figure 3 NucleoSpin Plant Il Maxi elution profile Genomic DNA from 1000 mg fresh wheat leaves was purified and eluted once A or twice B with 500 2500 uL Buffer PE Resulting yield and concentration is shown as solid and dotted lines respectively A two fold elution generally yields more DNA than just one elution with the same total buffer volume This is most important for small buffer volumes However large volumes or eluting two times results in a lower DNA concentration
14. REY 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 specifications 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 n
15. The standard elution procedure is already optimized to yield 80 90 by eluting two fold at elevated temperatures However if even higher yields a higher concentration or maximum speed is required the elution procedure can be adapted Table 3 Elution parameters Procedure NucleoSpin NucleoSpin NucleoSpin of exp yield Plant Il Plant Il Midi Plant Il Maxi Standard elution 50 uL 50 uL 200 uL 200 uL 1000 uL 1000 uL 85 90 65 C 5 min 65 C 5 min 65 C 5 min Maximum yield 100 uL 100 uL 400 uL 400 uL 2000 uL 2000 uL 95 100 65 C 5 min 65 C 5 min 65 C 5 min High concentration 25 uL 25 uL 100 uL 100 uL 500 uL 500 uL 7596 65 C 5 min 65 C 5 min 65 C 5 min Fast elution 100 uL 400 uL 2000 uL 60 7096 RT 65 C 1 5 min RT 65 C 1 5 min RT 65 C 1 5 min 12 MACHEREY NAGEL 12 2010 Rev 05 Genomic DNA from Plant 3 Storage conditions and preparation of working solutions Attention Buffers PL1 PL2 PC and PW1 contain guanidine hydrochloride and or detergents like CTAB or SDS Wear gloves and goggles All kit components can be stored at room temperature 18 25 C and are stable for at least one year Before starting any NucleoSpin Plant Il 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 u
16. charge of SDS and the positive charge of CTAB Table 2 gives an overview about customer data on different plant species and the corresponding buffer system that has been tested successfully using NucleoSpin Plant Il Important For a large variety of plant species both lysis buffers allow good results Use the table only for a rough orientation and guideline which buffer system has already been tested In order to find optimal lysis conditions when using a certain plant sample for the first time it is recommended to do side by side preparations of one batch of homogeneously ground material with both lysis buffers MACHEREY NAGEL 12 2010 Rev 05 9 Genomic DNA from Plant Table 2 Plant species tested with NucleoSpin Plant II Lysis buffer Plant species Plant tissue organ successfully tested PL1 PL2 Abies alba fir Needle v v Amorphophallus titanum Leaf v Not tested Apium graveolens celery Corm v v Arabidopsis thaliana Leaf v v Boreava orientalis Leaf herbarium sample v v Cleisostoma racemiferum Inflorescence rachis silica v Not tested gel dried Doritis pulcherrima Leaf silica gel dried v Not tested Eichornia azurea Leaf v Not tested Encephalartos natalensis Leaf v Not tested Galium aparine Leaf v v Hordeum sp barley Leaf v v Isatis kotschyana Leaf herbarium sample v v Laurus azorica laurel Leaf v Not tested Lupinus sp lupin Leaf v v Lycopersicon esculentum Stem v v tomato Myagrum perfolia
17. ected per mg of sample depends on the size and ploidy of the genome For example 100 mg fresh wheat with a hexaploid genome 1 7 x 10 bp contains 30 ug DNA whereas the same amount of Arabidopsis with a smaller diploid genome 1 9 x 10 bp only yields 3 ug DNA Thus it might be advantageous to process even more than the recommended sample mass up to 5 fold to obtain a reasonable DNA yield However to ensure a complete lysis all lysis buffer volumes of protocol step 2 have to be increased proportionally and multiple loading steps are necessary Additional lysis buffers PL1 PL2 PL3 RNase have to be purchased separately see ordering information Choosing the optimal lysis buffer system Plants are very heterogeneous and contain varying amounts of polyphenols acidic components or polysaccharides which can lead to suboptimal DNA extraction or performance in downstream applications Therefore we offer two different lysis buffers for optimal processing high yields and an excellent DNA quality with most common plant species The standard protocol uses Lysis Buffer PL1 which is based on the established CTAB procedure Additionally the SDS based Lysis Buffer PL2 is provided which requires subsequent protein precipitation by potassium acetate Precipitation Buffer PL3 For some plant species Lysis Buffers PL1 and PL2 can be used with similar results However for most plant material the lysis efficiency is different due to the negative
18. en sample thoroughly to a fine powder and refill mortar occasionally with liquid nitrogen to keep the sample frozen Use a precooled spatula to transfer the sample in precooled tubes Make sure no liquid nitrogen is transferred or all nitrogen has evaporated before closing the tube VA steel beads diameter 7 mm sample available on request Put 4 5 beads and plant material 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 www schuett labortechnik de Repeat the chilling and vortexing procedure until the entire plant material is ground to a fine powder Chill the tube once more and remove the beads by rolling them out gently or using a magnet Keep the material frozen throughout the whole homogenization procedure Do not add nitrogen to the tube since this leads to sticking and loss of plant material attached to the beads Rotor stator homogenizers are only useful to disrupt soft plants in the presence of lysis buffer Keep homogenizer submerged at all times to reduce foaming 8 MACHEREY NAGEL 12 2010 Rev 05 Genomic DNA from Plant 2 5 Lysis of plant samples Increasing the amount of starting material The standard protocols of NucleoSpin Plant II Midi Maxi kits allow processing of 10 1500 mg of plant material This usually yields 1 300 ug of high quality DNA However the amount of DNA that can be exp
19. ette 1000 uL Buffer PE 65 C onto the membrane Incubate the NucleoSpin Plant II Maxi Column for 5 min at 65 C Centrifuge for 2 min at 4 500 x g to elute the DNA Repeat this step with another 1000 uL Buffer PE 65 C and elute into the same tube Note To achieve maximum yield or higher concentrations refer to section 2 6 for alternative elution procedures 1000 pL PE 65 C 5 min 4 500 x g 2 min V 1000 uL PE C5 65 C 5 min 4 500 x g 2 min 28 MACHEREY NAGEL 12 2010 Rev 05 Genomic DNA from Plant 8 Appendix 8 1 Troubleshooting Problem Possible cause and suggestions DNA yield is low Homogenization of plant material was not sufficient For most species we recommend grinding with steel beads or mortar and pestle see section 2 4 For disruption of the cell wall it is important to homogenize the plant material thoroughly until the sample is ground to a fine powder Instead of freezing in liquid nitrogen the sample can also be lyophilized and easily ground at room temperature Suboptimal lysis buffer was used Lysis efficiencies of Buffer PL1 CTAB and Buffer PL2 SDS are different and depend on the plant species Try both buffers in a side by side purification to find the best detergent System to lyse your plant material Suboptimal lysis buffer volume was used Cell lysis might be insufficient and too much DNA might get lost during lysate clarification if e g dry material
20. genization Homogenize up to 400 mg wet weight or up to 80 mg dry methods see section 2 4 c Proceed with cell lysis using Buffer PL1 step 2a or alternatively Buffer PL2 step 2b 2a Re Ge ie zinc ud Transfer the resulting powder to a new tube and add 4 1 7 mL PL1 1 7 mL Buffer PL1 Vortex the mixture thoroughly Note If the sample can not be resuspended easily because e g the plant powder is soaking up too much buffer additional Buffer PL1 can be added Note that the volumes of RNase A step 2a and Buffer PC step 4 have to be increased proportionally Add 25 pL RNase A solution and mix sample thoroughly 25 uL RNase A Incubate the suspension for 15 min at 65 C 65 C Note For some plant material it might be advantageous to 10 min increase the incubation time to 30 60 min Proceed with step 3 MACHEREY NAGEL 12 2010 Rev 05 21 NucleoSpin Plant Il Midi 2b Red EH RE rad Transfer the resulting powder to a new tube and add 1 5 mL Buffer PL2 Vortex the mixture thoroughly Note If the sample can not be resuspended easily because for example the plant powder is soaking up too much buffer additional Buffer PL2 can be added Note that the volumes of RNase A Buffer PL3 step 2b and Buffer PC step 4 have lo be increased proportionally Add 25 pL RNase A solution and mix sample thoroughly Incubate the suspension for 15 min at 65 C Note For some plant material it might be advanta
21. geous to increase the incubation time to 30 60 min Add 200 pL Buffer PL3 mix thoroughly and incubate for 5 min on ice to precipitate SDS completely Proceed with step 3 Filtration Clarification of crude lysate Transfer the lysate to a NucleoSpin Filter L Centrifuge for 10 min at 4 500 x g collect the clear flow through and discard the NucleoSpin Filter L If not all liquid has passed the filter repeat the centrifugation step If a pellet is visible in the flow through transfer the clear supernatant to a new 15 mL microcentrifuge tube not provided Alternatively centrifuge the crude lysate for 5 min at 4 500 x g and transfer the supernatant to a new tube or pass the precleared supernatant through the NucleoSpin Filter L to remove solid particles completely Adjust DNA binding conditions Add 2 3 mL Buffer PC to the cleared lysate and mix immediately by vortexing for 30 s lt 1 5 mL PL2 25 uL RNase A 65 C 10 min 200 uL PL3 on ice 5 min 4 500 x g 10 min 2 3 mL PC Vortex 30 s 22 MACHEREY NAGEL 12 2010 Rev 05 NucleoSpin Plant Il Midi Bind DNA Load sample on a NucleoSpin Plant Il Midi Column Centrifuge for 2 min at 4 500 x g and discard the flow through The maximum loading capacity of the NucleoSpin Plant II Midi Column is 5 mL For higher sample volumes repeat the loading step Wash and dry silica membrane Add 1 mL Buffer PW1
22. gnostic 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 get 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 32 MACHEREY NAGEL 12 2010 Rev 05 Genomic DNA from Plant components not manufactured by MACHEREY NAGEL or damages resulting from such non MACHE
23. h Buffer PW2 and RNaseA were prepared according to section 3 Preheat Elution Buffer PE to 65 C A centrifuge with a swing out rotor and appropriate buckets capable of reaching 4 500 x g is required Note The NucleoSpin Plant Il Maxi kits include two different lysis buffers for optimal results with most common plant species Please refer to section 2 5 for choosing the optimal lysis buffer system for your individual plant sample and for information on how to process even more sample material than recommended in the following protocol Homogenize sample Homogenize up to 1500 mg wet weight or up to 300 mg dry weight lyophilized plant material for homogenization Homogenize methods see section 2 4 samples Proceed with cell lysis using Buffer PL1 step 2a or alternatively Buffer PL2 step 2b 2a Cell lysis using Buffer PL1 Transfer the resulting powder to a new tube and add 6 mL 6 mL PL1 Buffer PL1 Vortex the mixture thoroughly Note If the sample can not be resuspended easily because for example the plant powder is soaking up too much buffer additional Buffer PL1 can be added Note that the volumes of RNase A step 2a and Buffer PC step 4 have to be increased proportionally Add 100 uL RNase A solution and mix sample thoroughly 100 pL RNase A Incubate the suspension for 20 min at 65 C 65 C Note For some plant material it might be advantageous to 10 min increase the incubation time to 30 60 min
24. inding conditions Add 10 mL Buffer PC to the cleared lysate and mix immediately by vortexing for 30 s C 5 3 mL PL2 100 pL RNase A 65 C 10 min 700 uL PL3 on ice 5 min 4 500 x g 10 min 10mL PC Vortex 30 s 26 MACHEREY NAGEL 12 2010 Rev 05 NucleoSpin Plant Il Maxi Bind DNA Load sample on a NucleoSpin Plant Il Maxi Column Centrifuge for 2 min at 4 500 x g and discard the flow through The maximum loading capacity of the NucleoSpin Plant II Maxi Column is 15 mL For higher sample volumes repeat the loading step Wash and dry silica membrane Add 4 mL Buffer PW1 to the NucleoSpin Plant II Maxi Column Centrifuge for 2 min at 4 500 x g and discard flow through Note Although washing with Buffer PW1 increases purity it can in some cases slightly reduce the final yield Add 10 mL Buffer PW2 to the NucleoSpin Plant II Maxi Column Centrifuge for 2 min at 4 500 x g and discard flow through Add another 2 mL Buffer PW2to the NucleoSpin Plant II Maxi Column Centrifuge for 10 min at 4 500 x g in order to remove wash buffer and dry the silica membrane completely Load sample e 4 500 x g 2 min 4 mL PW1 4 500 x g 2 min 10 mL PW2 4 500 x g 2 min 2 mL PW2 4 500 x g 10 min MACHEREY NAGEL 12 2010 Rev 05 27 Genomic DNA from Plant Elute DNA Place the NucleoSpin Plant Il Maxi Column into a new Collection Tube 50 mL Pip
25. mg siliconized glass beads or sea sand and 200 uL Buffer PL1 Homogenize sample using a micro pistil and vortex regularly Add additional 100 uL Buffer PL1 and continue to homogenize the sample Note If the sample can not be handled easily because e g the sample material is soaking up too much buffer additional Buffer PL1 can be added Note that the volume of Buffer PC step 4 has to be increased proportionally Optional If the sample is rich in RNA or protein we recommend adding 10 uL RNase A and or Proteinase K 5 10 mg mL stock solution see ordering information respectively to the PL1 lysis solution in order to minimize contaminants Incubate for 10 min at 65 C Add 100 uL chloroform Vortex for 10 s and separate phases by centrifugation for 15 min at 20 000 x g Pipette the top aqueous layer into a new 1 5 mL microcentrifuge tube not provided For some fungi it might be advantageous to increase the incubation time to 30 60 min Proceed with section 5 1 step 3 MACHEREY NAGEL 12 2010 Rev 05 19 NucleoSpin Plant II 5 3 Support protocol for soil compost dung and animal excrements Attention Additional equipment necessary Bead mill e g Pulverisette O Fritsch Idar Oberstein or mortar and pestle Sea sand siliconized Extraction buffer 2 M NaCl 20 mM EDTA 100 mM Tris HCl 2 w v CTAB 2 w v Polyvinylpyrrolidon MW 40 000 pH 8 0 1 Homogenize sample Weigh 5 g soil or
26. ns see section 3 Composition of Elution Buffer PE 5 mM Tris HCl pH 8 5 MACHEREY NAGEL 12 2010 Rev 05 Genomic DNA from Plant 1 2 Reagents consumables and equipment to be supplied by user Reagents 96 100 ethanol Consumables 1 5 mL microcentrifuge tubes NucleoSpin Plant Il or 15 50 mL tubes NucleoSpin Plant II Midi Maxi for elution Disposable tips Equipment Manual pipettors Centrifuge for microcentrifuge tubes NucleoSpin Plant Il or an appropriate centrifuge with swing out rotors capable of reaching 4 500 x g for 15 mL 50 mL tubes NucleoSpin Plant II Midi Maxi Thermal heating block or water bath for incubation and preheating of Elution Buffer PE to 65 C Equipment for sample disruption and homogenization see section 2 4 Personal protection equipment lab coat gloves goggles 1 3 About this User Manual It is strongly recommended reading the detailed protocol sections of this User Manual if the NucleoSpin Plant II Midi Maxi kit is used for the first time Experienced users however my refer to the Protocol at a glance instead The Protocol at a glance is designed to be used only as a supplemental tool for quick referencing while performing the purification procedure All technical literature is available on the internet at www mn net com 6 MACHEREY NAGEL 12 2010 Rev 05 Genomic DNA from Plant 2 2 1 Product description The basic principle The plant sam
27. ntil the precipitate is re dissolved completely Wash Buffer PW2 Add the given volume of ethanol 96 10096 as indicated on the bottle or in the table below to Buffer PW2 Concentrate before first use Mark the label of the bottle to indicate that the ethanol is added Buffer PW2 at is stable at room temperature 18 25 C for at least one year RNase A Add the given volume of water as indicated on the vial and in the table 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 Plant Il 10 preps 50 preps 250 preps REF 74070 10 740770 50 740770 250 Wash Buffer PW2 6 mL 25 mL 50 mL Concentrate add 24 mL add 100 mL add 200 mL ethanol ethanol ethanol RNase A 1 5 mg 6 mg 2x15mg dissolve in dissolve in dissolve in 150 uL H O 600 uL H O 1500 uL H O each NucleoSpin Plant Il Midi NucleoSpin Plant Il Maxi 20 preps 10 preps REF 740771 20 740772 10 Wash Buffer PW2 25 mL 50 mL Concentrate add 100 mL ethanol add 200 mL ethanol RNase A 6 mg 10 mg dissolve in 600 uL H O dissolve in 1100 uL H O MACHEREY NAGEL 12 2010 Rev 05 13 Genomic DNA from Plant 4 Safety instructions risk and safety phrases The following components of the NucleoSpin Plant II kits contain hazardous contents Wear gloves and goggles and follow the safety instructions given in thi
28. ot 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 9 mn net com MACHEREY NAGEL 12 2010 Rev 05 33
29. ples are homogenized by mechanical treatment Then the DNA can be extracted with Lysis Buffers PL1 or PL2 containing chaotropic salts denaturing agents and detergents Crude lysates should be cleared by centrifugation and or filtration using the NucleoSpin Filters provided with the kits in order to remove polysaccharides contaminations and residual cellular debris The clear flow through is mixed with Binding Buffer PC to create conditions for optimal binding of DNA to the silica membrane After loading this mixture onto the spin column contaminants are washed away using Wash Buffers PW1 and PW2 The genomic DNA can finally be eluted with low salt Elution Buffer PE 5 mM Tris HCl pH 8 5 or nuclease free water and is ready to use for subsequent reactions 2 2 Kit specifications NucleoSpin Plant Il kits are designed for the isolation of genomic DNA from plant tissue using two optimized lysis buffer systems based on the established CTAB and SDS methods NucleoSpin Filters are included for conveniently clearing the crude lysates RNase A is included to remove RNA and to allow photometric quantification of pure genomic DNA The optimized Binding Buffer PC and the chaotropic Wash Buffer PW1 completely remove proteins RNA metabolites and other PCR inhibitors The eluted DNA is ready to use for subsequent reactions like PCR restriction analysis Southern Blot etc Table 1 Kit specifications at a glance Parameter NucleoSpin
30. s section Component Hazard Risk Safety contents phrases phrases PC Guanidine x Xn Flammable Harmful R 10 22 S 7 16 hydrochloride if swallowed 36 38 ethanol Irritating to eyes lt 40 and skin PW1 Guanidine x Xn Flammable Harmful R 10 22 S 7 16 25 hydrochloride if swallowed 36 38 isopropanol Irritating to eyes 25 and skin RNase A RNase A x Xn May cause sensitiza R 42 43 S 22 24 lyophilized tion by inhalation and skin contact Risk phrases R 10 Flammable R22 Harmful if swallowed R 36 38 Irritating to eyes and skin R 42 43 May cause sensitization by inhalation and skin contact Safety phrases S7 Keep container tightly closed S16 Keep away from source of ignition No Smoking S22 Do not breathe dust S24 Avoid contact with the skin 25 Avoid contact with the eyes Hazard labeling not neccessary if quantity per bottle below 25 g or mL certificate of exemption according to 67 548 EEC Art 25 1999 45 EC Art 12 and German GefStoffV 8 20 3 and TRGS 200 7 1 For further information see Material Safety Data Sheet Hazard labeling not neccessary if quantity per bottle below 125 g or mL certificate of exemption according to 67 548 EEC Art 25 1999 45 EC Art 12 and German GefStoffV 8 20 3 and TRGS 200 7 1 For further information see Material Safety Data Sheet 14 MACHEREY NAGEL 12 2010 Rev 05 NucleoSpin Plant II 5 NucleoSpir Plant Il protocols 5
31. to the NucleoSpin Plant Il Midi Column Centrifuge for 2 min at 4 500 x g and discard flow through Note Although washing with Buffer PW1 increases purity it can in some cases slightly reduce the final yield Add 3 mL Buffer PW2 to the NucleoSpin Plant Il Midi Column Centrifuge for 2 min at 4 500 x g and discard flow through Add another 1 mL Buffer PW2 to the NucleoSpin Plant Il Midi Column Centrifuge for 10 min at 4 500 x g in order to remove wash buffer and dry the silica membrane completely Load sample 4 500 x g 2 min 1 mL PW1 4 500 x g 2 min 3 mL PW2 4 500 x g 2 min 1 mL PW2 4 500 x g 10 min MACHEREY NAGEL 12 2010 Rev 05 23 NucleoSpin Plant Il Midi Elute DNA Place the NucleoSpin Plant II Midi Column into a new Collection Tube 15 mL Pipette 200 uL Buffer PE 65 C onto the membrane Incubate the NucleoSpin Plant Il Midi Column for 5 min at 65 C Centrifuge for 2 min at 4 500 x g to elute the DNA Repeat this step with another 200 uL Buffer PE 65 C and elute into the same tube Note To achieve maximum yield or higher concentrations refer to section 2 6 for alternative elution procedures V 200 uL PE 65 C 5 min 4 500 x g 2 min 200 uL PE 65 C 5 min 4 500 x g 2 min 24 MACHEREY NAGEL 12 2010 Rev 05 NucleoSpin Plant Il Maxi 7 NucleoSpin Plant Il Maxi protocol Before starting the preparation Check if Was
32. tum Leaf herbarium sample v v Oryza sativa rice Leaf v v Persea feru caerulea Leaf v Not tested Pteridium sp Leaf v Not tested Pterocarya fraxiniofolia Leaf v Not tested Rosa sp rose Leaf v v Rubus fruticosus blackberry Leaf v v Sameraria nummularia Leaf herbarium sample v v Secale sp rye Leaf v v Stereochilus sp Leaf silica gel dried v Not tested Tauscheria lasiocarpum Leaf herbarium sample v v Trachycarpus takil Leaf v Not tested Trichoglottis sp Leaf silica gel dried v Not tested Triticum aestivum wheat Leaf v v Vigna radiata mung bean Root v v Zea mays maize Leaf v v Zea mays maize Grain dried ground v v coarsely Fungal mycel not specified v Not tested Green algae not specified v Not tested 10 MACHEREY NAGEL 12 2010 Rev 05 Genomic DNA from Plant 2 6 Elution procedures The following graphs show DNA yields solid line and the resulting DNA concentrations dotted line in dependence on elution buffer volume Elution is done with either one elution step A or two subsequent elution steps with the indicated elution buffer volume each B 25 0 45 L 0 40 20 0 35 0 30 a r 0 25 r 0 20 DNA yield ug o r 0 15 DNA concentration ug pL F 0 10 0 05 0 25 50 75 100 125 Elution volume pL Figure 1 NucleoSpin Plant Il elution profile Genomic DNA from 100 mg fresh wheat leaves was purified and eluted once A or twice B with 10 1
33. y addition of 1 mM DNA is EDTA or heating the buffer to 70 C for 10 min degraded Centrifugation speed was too high Centrifuge at a maximum speed of 11 000xg Higher velocities may lead to shearing of the DNA Elution buffer contains EDTA EDTA may disturb subsequent reactions Use water or the supplied Elution Buffer PE 5 mM Tris HCl pH 8 5 for elution DNA quality is low Salt or ethanol carry over Make sure the last two wash steps were done with Wash Buffer PW2 and the membrane was dried according to the protocol 30 MACHEREY NAGEL 12 2010 Rev 05 Genomic DNA from Plant 8 2 Ordering information Product REF Pack of NucleoSpin Plant II 740770 10 50 250 10 50 250 preps NucleoSpin Plant II Midi 740771 20 20 preps NucleoSpin Plant Il Maxi 740772 10 10 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 740505 50 50 mg 740505 100 mg Proteinase K 740506 100 mg NucleoSpin Filters 740606 50 for filtration of cell homogenates Collection Tubes 2 mL 740600 1000 MACHEREY NAGEL 12 2010 Rev 05 31 Genomic DNA from Plant 8 3 Product use restriction warranty NucleoSpin Plant II Midi Maxi kit components are intended developed designed and sold FOR RESEARCH PURPOSES ONLY except however any other function of the product
Download Pdf Manuals
Related Search