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1. repeat the loading step Wash silica membrane optional For some plant material washing with chaotropic salts can further improve the purity by removing protein and other contaminants It can however lower the final yield by up to 15 Add 400 ul wash buffer PW1 to the NucleoSpin Plant II C column Centrifuge for 1 min at 11 000 x g and discard flow through Wash and dry silica membrane Add 700 ul buffer PW2 to the NucleoSpin Plant II column Centrifuge for 1 min at 11 000 x g and discard flow through Make sure ethanol was added to the PW2 concentrate Add another 200 ul buffer PW2 to the NucleoSpin Plant Il column Centrifuge for 2 min at 11 000 x g in order to remove wash buffer completely and dry the silica membrane MACHEREY NAGEL 10 2006 Rev 01 load sample 1 min 11 000 xg optional 400 pl PW1 1 min 11 000 x g 700 pl PW2 1 min 11 000 xg 200 pl PW2 2 min 11 000 xg NucleoSpin Plant II 8 Elute highly pure DNA 50 ul PE 10 C 5min Place the NucleoSpin Plant II column into a new 1 5 ml centrifuge tube not provided 1 min 11 000 xg Pipette 50 ul buffer PE heated to 70 C onto the membrane Incubate the NucleoSpin Plant II column for 5 min at 70 C Centrifuge for 1 min at 11 000 x g to elute the DNA 50 ul PE eS 70 C 5 min Repeat this step with another 50 ul of buffer PE heated dain to 70 C and elute into the same tube 41 0
2. 0 x g MACHEREY NAGEL 10 2006 Rev 01 19 Genomic DNA from Plant 6 2 Ordering information Product Cat No Pack of NucleoSpin Plant Il 740770 10 10 preps NucleoSpin Plant II 740770 50 50 preps NucleoSpin Plant II 740770 250 250 preps NucleoSpin collecting tubes 2 ml 140690 1099 NucleoSpin Filter For filtration of cell homogenates 740606 50 Buffer PL1 740927 125 ml Buffer Set PL2 PL3 100 ml Buffer PL2 and 25 ml Buffer PL3 740928 1 set Buffer PC 740937 125 ml Buffer PW1 740938 125 ml Buffer PW2 Concentrate For 250 ml Buffer PW2 49399 50 mi RNase A 740505 100 mg RNase A 740505 50 50 mg 6 3 Product use restriction warranty NucleoSpin Plant Il kits components were developed designed and sold for research purposes only They are suitable for in vitro uses only No claim or representation is intended for its use to identify any specific organism or for clinical use diagnostic prognostic therapeutic or blood banking It is rather the responsibility of the user to verify the use of the NucleoSpin Plant II kits for a specific application range as the performance characteristic of this kit has not been verified to a specific organism 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 prod
3. 00 x g MACHEREY NAGEL 10 2006 Rev 01 15 NucleoSpin Plant II 5 2 Support protocol for genomic DNA from fungi 1 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 2 Cell lysis Place the sample into a 1 5 ml reaction tube Add 150 mg siliconized glass beads or sea sand and 200 ul buffer PL1 Homogenize sample using a micro pistil and vortex regularly Add an additional 100 ul buffer PL1 and continue to homogenize the sample If the lysis buffer volume is not large enough more lysis buffer PL1 can be added Note that binding buffer PC has to be increased proportionally in step 4 Optional If a high RNA or protein content is present we recommend to add 10 pl RNase A and or proteinase K 5 10 mg ml stock solution see ordering information to the PL1 lysis solution in order to minimize contaminants Add 100 ul chloroform Vortex for 10 s and separate phases by centrifu gation for 5 min at 11 000 x g Pipette the top aqueous layer into a new 1 5 ml centrifuge
4. Genomic DNA from Plant User manual NucleoSpin Plant II October 2006 Rev 01 MACHEREY NAGEL MN Protocol at a glance Rev 01 Genomic DNA Purification from Plant 1 Homogenize samples Mini NucleoSpin Plant II 100 mg bd 400 pl PL1 2 Cell lysis E 10 ul RNase A 65 C 10 min alternatively 300 pl PL2 10 ul RNase A 65 C 10 min 75 ul PL3 0 C 5 min 3 Filtration Clarification eee 3 g of lysate a 4 Adjust DNA binding 450 pl PC conditions 1 min 5 Bind DNA 11 000 x g 6 Wash silica membrane optional 400 ul PW1 1 min 11 000 x g fm 700 pl Pw2 1 min 11 000 xg 200 pl PW2 1 min 11 000 x g 7 Dry silica membrane Drying is performed by the centrifugation during the 2 7 washing step 8 Elute highly pure DNA 50 pl PE 70 C 5 min 1 min 11 000 x g 50 pl PE 70 C 5 min 1 min 11 000 x g MACHEREY NAGEL GmbH amp Co KG e Neumann Neander Str 6 8 e D 52355 D ren Germany Tel 49 0 24 21 969 270 Fax 49 0 24 21 969 279 e e mail tech bio mn net com Genomic DNA from Plant Table of contents 1 Kit contents 2 Product description 2 1 The basic principle 2 2 About this user manual 2 3 Treatment of different plant samples 2 4 Kit specifications 2 5 Storage and homogenization of samples 2 6 Elution procedures Storage conditions and preparation of working solutions Safety instructi
5. Y 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 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 Please contact MACHEREY NAGEL Germany Tel 49 0 24 21 969 270 e mail TECH BlIO mn net com MACHEREY NAGEL 10 2006 Rev 01 21
6. at 65 C 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 3 Filtration Clarification of crude lysate Place a NucleoSpin Filter column violet ring into a new NucleoSpin collecting tube 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 column If not all liquid has passed the filter repeat the centri fugation step If a pellet is visible in the flow through transfer the clear supernatant to a new 1 5 ml tube not provided Alternatively centrifuge the crude lysate for 5 min at 11 000 x g and transfer the supernatant to a new tube 4 Adjust DNA binding conditions Add 450 ul binding buffer PC and mix by pipetting up and down thoroughly 5 times or by vortexing MACHEREY NAGEL 10 2006 Rev 01 alternatively 300 pl PL2 10 ul RNase A 65 C 10 min 75 pl PL3 0 C 5 min 2 min 11 000 x g 450 pl PC 13 NucleoSpin Plant II Bind DNA Place a NucleoSpin Plant II column green ring into a new NucleoSpin collecting tube and load the sample Centrifuge for 1 min at 11 000 x g and discard the flow through The maximum loading capacity of the NucleoSpin Plant II column is 700 ul For higher sample volumes
7. e starting any NucleoSpin Plant II protocol prepare the following e 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 e Buffer PW2 Add the given volume of ethanol 96 100 to buffer PW2 concentrate before first use Store buffer PW2 at room temperature 20 25 C for up to one year e RNase A Add the given volume of water 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 Plant Il 10 preps 50 preps 250 preps 740770 10 740770 50 740770 250 Buffer PW2 6 ml 25 ml 50 ml concentrate add 24 ml ethanol add100mlethanol add 200 ml ethanol 1 5 mg 6 mg 2x 15mg RNase A dissolve in dissolve in dissolve in 150 ul H20 600 ul H20 1500 ul H20 each 10 MACHEREY NAGEL 10 2006 Rev 01 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 this section Component Hazard Hazard Risk Safety Contents Symbol Phrases Phrases BY guanidine x Xn Flammable Harmful if swallowed R 10 22 S 7 16 hydrochl
8. le in the presence of liquid nitrogen to obtain optimal yields After homogenization and treatment of the sample with lysis buffer mixtures can be cleared easily either with a NucleoSpin Filter provided 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 Pre cool mortar and pestle using liquid nitrogen Grind frozen sample thoroughly until a fine powder results and refill mortar occasionally with liquid nitrogen to keep the sample frozen Use a pre cooled spatula to transfer the sample in pre cooled 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 Schutt 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 stat
9. lied elution buffer PE 5 mM Tris HCl pH 8 5 18 MACHEREY NAGEL 10 2006 Rev 01 Genomic DNA from Plant Problem Possible cause and suggestions Sample was too viscous due to too much sample material or material carryover Centrifuge large amounts of sample material before loading it Sapa onto the NucleoSpin Filter see e Make sure cleared lysate is absolutely free of resuspended ant matter before loading it onto the NucleoSpin Plant II column column is clogged e Increase centrifugation speed e Use more lysis buffer PL1 or PL2 Sample was contaminated with DNase e Preheat elution buffer to 70 C for 5 min to eliminate DNase contamination This precaution is not necessary for buffers supplied by MACHEREY NAGEL which are delivered free of DNA is RNase and DNase degraded Centrifugation speed was too high e Centrifuge at a maximum speed of 11 000 x g Higher velocities may lead to shearing of the DNA Sample contains contaminants like phenolic compounds or secondary metabolites e Use optional washing step with wash buffer PW1 and repeat this step if necessary Elution buffer contains EDTA ae quality e EDTA may disturb subsequent reactions Use water or the is low supplied elution buffer PE 5 mM Tris HCl pH 8 5 for elution Salt or ethanol carryover e Make sure the last two wash steps were done with wash buffer PW2 according to the manual and the membrane was dried at least for 2 min at gt 11 00
10. lis leaf herbarium sample y v Cleisostoma racemiferum ain dite ete v not tested 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 spec barley leaf v v Isatis kotchyana leaf herbarium sample v v Laurus azorica laurel leaf v not tested Lupinus spec lupin leaf v v Lycopersicon esculentum tomato stem v v Myagrum perfoliatum leaf herbarium sample v v Oryza sativa rice leaf v v Persea feru caerulea leaf v not tested Pteridium spec leaf v not tested Pterocarya fraxiniofolia leaf v not tested Rosa spec rose leaf v v Rubus fruticosus blackberry leaf v v Sameraria nummularia leaf herbarium sample y v Secale spec rye leaf y v Stereochilus sp leaf silica gel dried v not tested Taucheria lasiocarpum leaf herbarium sample v v Trachycarpus takil leaf v not tested Trichoglottis sp leaf silica gel dried v not tested 6 MACHEREY NAGEL 10 2006 Rev 01 Genomic DNA from Plant Table 1 Plant species tested with NucleoSpin Plant II Lysis buffer Plant species Plant tissue organ successfully tested PL1 PL2 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 coarsley v y fungal mycel not specified v not tested green algae not specified v not tested 2 4 Kit specifications e NucleoSpin Plant II kits a
11. olysaccharides 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 different buffers in subsequent washing steps 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 in subsequent reactions 2 2 About this user manual Experienced users who are performing the isolation of genomic DNA from plant using a NucleoSpin Plant II isolation kit may refer to the Protocol at a glance instead of this user manual The Protocol at a glance is designed to be used only as a supple mental tool for quick referencing while performing the purification procedure First time users are strongly advised to read this user manual 2 3 Treatment of different plant samples Plants are very heterogeneous and contain a lot of different metabolites like polyphenols polysaccharides or acidic components which can lead to suboptimal extraction or subsequent processing of DNA Therefore we offer two different lysis buffers for the optimal processing high yields and a very good 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 buffer PL2 is provided with the kit which requi
12. ons risk and safety phrases NucleoSpin Plant II 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 Appendix 6 1 Troubleshooting 6 2 Ordering information 6 3 Product use restriction warranty MACHEREY NAGEL 10 2006 Rev 01 18 18 20 20 Genomic DNA from Plant 1 Kit contents NucleoSpin Plant Il 250 preps 740770 250 50 preps 740770 50 10 preps 740770 10 Buffer PL1 5 ml 25 ml 125 ml Buffer PL2 4 ml 20 ml 100 ml Buffer PL3 1 ml 5 ml 25 ml Buffer PC 6 ml 30 ml 125 ml Buffer PW1 6 ml 30 ml 125 ml Buffer PW2 concentrate om Zaim Sml Buffer PE 5ml 15 ml 30 ml RNase A 1 5 mg 6 mg 2x15mg r NucleoSpin Filters 10 50 250 violet ring NucleoSpin Plant II 10 50 250 Columns green ring NucleoSpin Collecting Tubes 2 ml 20 190 eee Protocol 1 1 1 For preparation of working solutions and storage conditions see section 3 4 MACHEREY NAGEL 10 2006 Rev 01 Genomic DNA from Plant 2 Product description 2 1 The basic principle After the plant samples have been homogenized the DNA can be extracted with lysis buffers containing chaotropic salts denaturing agents and detergents Lysis mixtures should be cleared by filtration using the NucleoSpin Filters provided with the kits alternatively by centrifugation in order to remove p
13. or homogenizers are only useful to disrupt soft plants in the presence of lysis buffer Keep homogenizer submerged at all times to reduce foaming MACHEREY NAGEL 10 2006 Rev 01 Genomic DNA from Plant 2 6 Elution procedures The standard elution procedure is already optimized to yield 80 90 by eluting two fold at elevated temperatures However if even higher yields high concentration or maximum speed are required the elution procedure can be adapted as follows e Complete yield 90 100 of the bound nucleic acids can be eluted by performing two 100 ul elution steps instead of two 50 ul steps or performing a third elution step with 50 ul which means a total of three 50 ul elution steps Keep the 5 minutes incubation at 70 C e High concentration 70 80 of the bound nucleic acids can be eluted highly concentrated by performing only one 50 ul elution step Keep the 5 minutes incubation at 70 C e Fast elution 70 80 of the bound nucleic acids can be eluted by eluting only once with 100 ul elution buffer and cutting down the incubation time from 5 to 1 min MACHEREY NAGEL 10 2006 Rev 01 9 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 e All kit components can be stored at room temperature 20 25 C and are stable for up to one year Befor
14. oride Irritating to eyes and skin 36 38 ethanol lt 40 Pw guanidine x Xn Flammable Harmful if swallowed R 10 22 S 7 16 25 hydrochloride Irritating to eyes and skin 36 38 isopropanol lt 25 RNASEA RNase A x gn May cause sensitization by R 42 43 S 22 24 lyophilized inhalation and skin contact Risk Phrases R10 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 S 16 Keep away from sources of ignition No Smoking S 22 Do not breathe dust S 24 Avoid contact with the skin S 25 Avoid contact with the eyes Label not necessary if quantity below 125 g or ml according to 67 548 EEC Art 25 1999 45 EC Art 12 and German GefStoffV 42 and TRGS 200 7 1 MACHEREY NAGEL 10 2006 Rev 01 11 NucleoSpin Plant II 5 NucleoSpin Plant II protocols 5 1 Standard protocol for genomic DNA from plant Before starting with the preparation set incubators or water baths to 65 C Before elution equilibrate elution buffer PE to 70 C Note The NucleoSpin Plant II kits include two different lysis buffers for optimal results with most common plant species Lysis buffer is based on the established CTAB procedure positively charged detergent buffer is based on SDS negatively charged detergent Please refer to section 2 3 for chosing the optimal lysis buffer system fo
15. r your individual plant sample 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 5 samples Proceed with cell lysis using buffer PL1 step 2a or alternatively buffer PL2 step 2b 2a SIDES MI L ad Transfer the resulting powder to a new tube and add 400 ul buffer PL1 Vortex the mixture thoroughly 400 pl PL1 If the lysis buffer volume is not large enough the plant powder can be resuspended in additional buffer PL1 Note that the volume of binding buffer PC has to be increased proportionally in step 4 Add of 10 pl RNase A solution to the lysis mixture and 10 pl mix sample RNase A Incubate the suspension for 10 min at 65 C 65 C 10 min For some plant material it might be advantageous to increase the incubation time to 30 60 min Proceed with step 3 12 MACHEREY NAGEL 10 2006 Rev 01 NucleoSpin Plant II YA mCell lysis using buffer PL2 Transfer the resulting powder to a new tube and add 300 ul buffer PL2 Vortex the mixture thoroughly If the lysis buffer volume is not large enough the plant powder can be resuspended in additional buffer PL2 Note that the volumes of precipitation buffer PL3 step 2b and binding buffer PC step 4 have to be increased proportionally Add of 10 pl RNase A solution to the lysis mixture and mix sample Incubate the suspension for 10 min
16. re designed for the isolation of genomic DNA from plant tissue and other biological samples like soil using two optimized lysis buffer systems based on the established CTAB and SDS methods e NucleoSpin Filters are included for conveniently clearing the lysate before loading it onto the NucleoSpin Plant II columns e RNase A is included to remove RNA and to allow photometric quantification of pure genomic DNA e The optimized binding buffer PC and the optional chaotropic wash buffer PW1 completely remove proteins RNA metabolites and other PCR inhibitors e The eluted pure DNA is ready to use in subsequent reactions like PCR restriction analysis Southern blotting etc Table 2 Kit specifications at a glance Parametere NucleoSpin Plant II amea eor wai Typical yield 1 30 ug Elution volume 100 ul Binding capacity 50 ug Time prep 30 min Column type mini MACHEREY NAGEL 10 2006 Rev 01 7 2 5 Genomic DNA from Plant Storage and homogenization of 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 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 pest
17. res subsequent protein precipitation by potassium acetate buffer PL3 provided For some plant species buffers PL1 and PL2 can be used with similar results However for most plant material the lysis efficiency is different due to the negative charge of SDS and the positive charge of CTAB 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 10 2006 Rev 01 5 Genomic DNA from Plant Table 1 gives an overview about customer data on different plant species that have been tested using NucleoSpin Plant Il It indicates plant species that were successfully tested and the corresponding buffer system that was used Important For a large variety of plant species both lysis buffers will give good results Use the table only for rough orientation and guideline which buffer system has already been tested In order to obtain optimal results with your individual sample material we recommend to test both buffers in parallel to check which system will be suited best Table 1 Plant species tested with NucleoSpin Plant II Lysis buffer Plant species Plant tissue organ successfully tested PL1 PL2 Abies alba fir needle y v Amorphophallus titanum leaf v not tested Apium graveolens celery corm v v Arabidopsis thaliana leaf v not tested Boreava orienta
18. s we recommend grinding with steel beads or mortar and pestle see section 2 5 For disruption of the cell wall it is important to homogenize the plant material thoroughly until the sample is ground to a fine powder e Instead of freezing in liquid nitrogen the sample can also be lyophilized and easily ground at room temperature Suboptimal lysis buffer was used e Lysis efficiencies of buffers PL1 CTAB and 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 e Cell lysis might be insufficient and too much DNA might get lost during lysate clarification if e g dry material soaks up too much lysis buffer Use more lysis buffer and increase the volume of binding buffer PC proportionally DNA yield is low Suboptimal binding buffer volume was used e Increase binding buffer PC proportionally if more lysis buffer was used Extraction of DNA from plant material during lysis was insufficient e Increase incubation time in lysis buffer up to overnight Suboptimal Elution e 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 70 C for at least 5 minutes e 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 supp
19. tube The chloroform extraction step is optional but highly recommended Incubate for 10 min at 65 C For some fungi material it might be advantageous to increase the incubation time to 30 60 min Proceed with section 5 1 step 3 16 MACHEREY NAGEL 10 2006 Rev 01 NucleoSpin Plant II 5 3 Support protocol for soil compost dung and animal excrements Note This protocol requires an additional extraction buffer which is not provided in the kit 2 M NaCl 20 mM EDTA 100 mM Tris Cl 2 w v CTAB 2 w v Polyvinylpyrrolidon PVP pH 8 0 1 Homogenize sample Weigh 5 g soil or 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 Cell lysis 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 wl of the clear supernatant into a new 1 5 ml centrifuge tube Proceed with section 5 1 step 3 MACHEREY NAGEL 10 2006 Rev 01 17 Genomic DNA from Plant 6 Appendix 6 1 Troubleshooting Problem Possible cause and suggestions Homogenization of plant material was not sufficient e For most specie
20. ucts free of charge in the event products fail to perform as warranted Supplementary reference is made to the general 20 MACHEREY NAGEL 10 2006 Rev 01 Genomic DNA from Plant business terms and conditions of MACHEREY NAGEL which are printed on the price list Please contact us if you wish an extra copy MACHEREY NAGEL does not warrant against 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 against defects in products or components not manufactured by MACHEREY NAGEL or against 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 MACHERE

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