Total RNA isolation from plant
Contents
1. Total RNA isolation from plant User manual NucleoSpin RNA Plant February 2011 Rev 06 MACHEREY NAGEL MN Total RNA isolation from plant Protocol at a glance Rev 06 NucleoSpin RNA Plant 1 Homogenize sample 100 mg 2 Lyse cells 350 uL RA1 3 5 uL 8 mercaptoethanol or 350 uL RAP 3 5 uL B mercaptoethanol Mix 3 Filtrate lysate e 11 000 x g 1 min 4 Adjust RNA binding 350 uL 70 ethanol conditions Mix 5 Bind RNA Ss Load sample S 11 000 x g 30s 6 Desalt silica membrane S 350 uL MDB S E 11 000 x g s 1 min 7 Digest DNA 9 Ss 95 uL DNase Eg reaction mixture Z RT 15 min 8 Wash and dry silica F 1 wash 200 uL RA2 membrane LI 2rd wash 600 uL RA3 F 3 wash 250 uL RA3 11 000 x g st nd 1 and 2 C 5 30s 11 000x g rd i c5 2 min 9 Elute highly pure RNA 60 uL RNase free H O 11 000 x g 1 min MACHEREY NAGEL GmbH amp Co Tel 49 24 21 969 270 Fax 49 KG Neumann Neander Str 6 8 52355 D ren Germany 24 21 969 199 tech bio mn net com www mn net com Total RNA isolation from plant Table of contents 1 Components 1 1 Kit contents 1 2 Reagents consumables and equipment to be supplied by user 1 3 About this user manual 2 Product description 2 1 The basic principle 2 2 Kit specifications 2 3 Handling preparation and storage of starting materials 2 4 Elution procedures 2 5 Yields with different sam2. Elute highly pure RNA Elute the RNA in 60 uL RNase free H O supplied and 60 uL centrifuge at 11 000 x g for 1 min RNase free H O If higher RNA concentrations are desired elution can be 2 done with 40 uL Overall yield however will decrease when 11 000 x g using smaller elution volumes imm For alternative elution procedures see section 2 4 MACHEREY NAGEL 02 2011 Rev 06 17 Total RNA isolation from plant 5 2 Support protocol NucleoSpin RNA Plant rDNase digestion in solution Comments on DNA removal The on column rDNase digestion in the standard protocol is already very efficient and thus resulting in a minimal residual DNA content of the purified RNA This DNA will not be detectable in most downstream applications Despite this there are still certain applications which require even lower contents of residual DNA However removal of DNA to a completely undetectable level is challenging and the efficiency of an on column DNA digestion is sometimes not sufficient for downstream applications requiring lowest residual content of DNA A typical example for such a demanding application is an RT PCR reaction in which the primer molecules do not differentiate between cDNA derived from RNA and contaminating genomic DNA Especially if high copy number targets are analyzed e g multi gene family mitochondrial plastidal or plasmid targets from transfections the target gene is of a very low expression l
3. easy homogenization of disrupted starting material rDNase not active Reconstitute and store lyophilized rDNase according to instructions given in section 3 Contamination of RNA with genomic DNA rDNase solution not properly applied Pipette rDNase solution directly onto the center of the silica membrane Too much cell material used Reduce quantity of cells or tissue used MACHEREY NAGEL 02 2011 Rev 06 21 Total RNA isolation from plant DNA detection system too sensitive The amount of DNA contamination is effectively reduced during the on column digestion with rDNase Anyhow it can not be guaranteed that the purified RNA is 10096 free of DNA therefore in very sensitive applications it might still be possible to detect DNA The NucleoSpin RNA II Plant System is checked by the following procedure One million HeLa cells are subjected to RNA isolation according to the protocol RNA eluate is used as template for PCR detection of a 1 kbp fragment in a 30 cycle reaction Generally no PCR Contamination product is obtained while skipping the DNase digest usually of RNA with leads to positive PCR results genomic DNA continued The probability of DNA detection with PCR increases with the number of DNA copies per preparation single copy target plastidial mitochondrial target plasmid transfected into cells decreasing of PCR amplicon size Use larger PCR targets e g gt 500 bp or intron sp
4. large amounts of chaotropic ions This lysis buffer immediately inactivates RNases which are present in virtually all biological materials and creates appropriate binding conditions which favor adsorption of RNA to the silica membrane Contaminating DNA is removed by an rDNase solution which is directly applied onto the silica membrane during the preparation RNase free rDNase is supplied with the kit Washing steps with two different buffers remove salts metabolites and macromolecular cellular components Pure RNA is finally eluted under low ionic strength conditions with RNase free H O supplied The NucleoSpin RNA Plant kit contains two different lysis buffers RA1 guanidinium thiocyanate and RAP guanidinium HCI respectively In most cases use of Buffer RA1 is recommended for lysis due to the stronger denaturing properties of the thiocyanate The presence of peculiar metabolites in a variety of plant tissues or fungi however requires the use of an alternative buffer because they may lead to solidification of the lysate resulting in a non processible slurry In such cases Buffer RAP is the buffer of choice Besides Buffer RA1 and Buffer RAP MACHEREY NAGEL offers alternatively a lysis buffer with a high detergent concentration Buffer RL1 see ordering information The RNA preparation using NucleoSpin RNA Plant kit can be performed at room temperature The eluate however should be treated with care because RNA is very sens
5. 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 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
6. rDNase RNase free at 4 C on arrival stable up to 1 year All other kit components should be stored at room temperature 18 25 C and are stable up to one year Storage at lower temperatures may cause precipitation of salts Check that 70 ethanol is available as additional solution to adjust RNA binding conditions in the Buffer RA1 lysate Before starting any NucleoSpin RNA Plant protocol prepare the following rDNase RNase free Add indicated volume of RNase free H O see table below to the rDNase vial and incubate for 1 min at room temperature Gently swirl the vials to completely dissolve the rDNase Be careful not to mix rDNase vigorously as rDNase is sensitive to mechanical agitation Dispense into aliquots and store at 18 C The frozen working solution is stable for 6 months Do not freeze thaw the aliquots more than three times Wash Buffer RA3 Add the indicated volume of 96 100 ethanol see table below to Buffer RA3 Concentrate Mark the label of the bottle to indicate that ethanol was added Store Wash Buffer RA3 at room temperature 18 25 C for up to one year NucleoSpin RNA Plant 20 preps 50 preps 250 preps REF 740949 10 740949 50 740949 250 Wash Buffer RAS 5mL 12 5 mL 3x25mL Concentrate Add 20 mL ethanol Add 50 mL ethanol Add 100 mL ethanol to each vial rDNase RNase 1 vial size C 1 vial size D 5 vials size D free lyophilized Add 230 uL Add 540 uL Add 540 uL RNase free H O RNase f
7. 00 x g MACHEREY NAGEL 02 2011 Rev 06 15 NucleoSpin RNA Plant 7 Digest DNA Prepare DNase reaction mixture in a sterile 1 5 mL microcentrifuge tube not provided For each isolation add 10 uL reconstituted rDNase see section 3 to 90 uL Reaction Buffer for rDNase Mix by flicking the tube Apply 95 uL DNase reaction mixture directly onto the center of the silica membrane of the column Incubate at room temperature for 15 min Wash and dry silica membrane Add 200 uL Buffer RA2 to the NucleoSpin RNA Plant Column Centrifuge for 30 s at 11 000 x g Place the column into a new Collection Tube 2 mL Buffer RA2 will inactivate the rDNase Add 600 uL Buffer RA3 to the NucleoSpin RNA Plant Column Centrifuge for 30 s at 11 000 x g Discard flow through and place the column back into the Collection Tube Add 250 uL Buffer RAS to the NucleoSpin RNA Plant Column Centrifuge for 2 min at 11 000 x g to dry the membrane completely Place the column into a nuclease free Collection Tube 1 5 mL supplied If for any reason the liquid level in the Collection Tube has reached the NucleoSpin RNA Plant Column after centrifugation discard flow through and centrifuge again 95 pL rDNase reaction mixture RT 15 min 200 pL RA2 11 000 x g 30s C 600 pL RA3 11 000 x g 30s 250 uL RA3 11 000 x g 2 min 16 MACHEREY NAGEL 02 2011 Rev 06 NucleoSpin RNA Plant
8. ACHEREY NAGEL 02 2011 Rev 06 25
9. EN 740385 125 125 mL rDNase Set 740963 1 set NucleoSpin Filters 740606 50 Collection Tubes 2 mL 740600 1000 DISTRIBUTION AND USE OF NUCLEOSPIN RNA DNA BUFFER SET and NUCLEOSPIN TRIPREP IN THE USA IS PROHIBITED FOR PATENT REASONS MACHEREY NAGEL 02 2011 Rev 06 23 Total RNA isolation from plant 6 3 References Vogelstein B and D Gillespie 1979 Preparative and analytical purification of DNA from agarose Proc Natl Acad Sci USA 76 615 619 6 4 Product use restriction warranty NucleoSpin RNA Plant 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
10. Ethanol 1096 Hazard labeling not necessary if quantity per bottle below 125g 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 12 MACHEREY NAGEL 02 2011 Rev 06 Total RNA isolation from plant Risk phrases R S tze R10 R 20 21 22 R22 R 36 38 R 42 43 Flammable Entz ndlich Harmful by inhalation in contact with skin and if swallowed Gesundheitssch dlich beim Einatmen Verschlucken und Ber hrung mit der Haut Harmful if swallowed Gesundheitssch dlich beim Verschlucken Irritating to eyes and skin Reizt die Augen und die Haut May cause sensitisation by inhalation and skin contact Sensibilisierung durch Einatmen und Hautkontakt m glich Safety phrases S S tze S7 S 13 S16 S22 S24 Keep container tightly closed Beh lter dicht geschlossen halten Keep away from food drink and animal feedstuffs Von Nahrungsmitteln Getr nken und Futtermitteln fernhalten Keep away from sources of ignition No Smoking Von Z ndquellen fernhalten Nicht rauchen Do not breathe dust Staub nicht einatmen Avoid contact with the skin Ber hrung mit der Haut vermeiden MACHEREY NAGEL 02 2011 Rev 06 13 NucleoSpin RNA Plant 5 5 1 Protocols Total RNA isolation from plant tissue or filamentous fungi Before start
11. anning primers if possible Use support protocol 5 2 for subsequent rDNase diges tion in solution Carry over of ethanol or salt Do not let the flow through touch the column outlet after the second Buffer RA3 wash Be sure to centrifuge at the corresponding speed for the respective time in order to remove ethanolic Buffer RA3 completely Suboptimal Check if Buffer RA3 has been equilibrated to room performance temperature before use Washing at lower temperatures of RNA in lowers efficiency of salt removal by Buffer RAS downstream experiments Store isolated RNA properly Eluted RNA should always be kept on ice for optimal stability since trace contaminations of omnipresent RNases general lab ware fingerprints dust will degrade the isolated RNA For short term storage freeze at 20 C for long term storage freeze at 70 C 22 MACHEREY NAGEL 02 2011 Rev 06 Total RNA isolation from plant 6 2 Ordering information Product REF Pack of NucleoSpin RNA Plant 740949 20 50 250 20 50 250 NucleoSpin RNA Clean up 740948 10 50 250 10 50 250 NucleoSpin RNA XS 740902 10 50 250 10 50 250 NucleoSpin RNA Clean up XS 740903 10 50 250 10 50 250 NucleoSpin RNA Protein 740933 10 50 250 10 50 250 NucleoSpin RNA DNA j Buffer Set 740944 Suitable for 100 preps NucleoSpin TriPrep 740966 10 50 250 10 50 250 740961 50 mL Burer RAI 740961500 500 mL 740936 50 50 mL Bufer MAR 740936 500 500 mL 740385 50 50 mL Butler H
12. ble for applications like reverse transcriptase PCR RT PCR Northern blotting primer extension or RNase protection assays rDNase is supplied with the kit DNA contaminations are efficiently removed by on column digestion with rDNase Anyhow traces of DNA might be detected in very sensitive applications For most demanding applications a subsequent digestion with rDNase in the eluate is possible The NucleoSpin RNA II RNA Plant system is checked by the following procedure One million HeLa cells are subjected to RNA isolation according to the protocol RNA eluate is used as template for PCR detection of a 1 kbp fragment in a 30 cycle reaction Generally no PCR fragment is obtained if the DNase is applied However a strong PCR fragment is obtained if DNase is omitted The eventuality of DNA detection with PCR increases with 1 the number of DNA copies per preparation single copy target lt plastidial mitochondrial target plasmid transfected into cells 2 decreasing of PCR amplicon size Table 1 Kit specifications at a glance Parameter NucleoSpin RNA Plant Format Mini spin column Sample material lt 100 mg tissue Fragment size gt 200b Typical yield 3 70 ug from 100 mg plant material A seo so 1 9 2 1 Elution volume 60 uL Preparation time 30 min 6 preps Binding capacity 200 ug MACHEREY NAGEL 02 2011 Rev 06 T Total RNA isolation from plant 2 3 Handling preparation and storage of start
13. e almost omnipresent RNases general lab ware fingerprints dust will degrade RNA For short term storage freeze at 20 C for long term storage freeze at 70 C MACHEREY NAGEL 02 2011 Rev 06 9 Total RNA isolation from plant 2 5 Yields with different samples Table 2 Typical yields of total RNA per 50 mg sample Specie Organ Yield Allium cepa onion Germ bud 13 ug Allium sativum garlic Leaf 13 ug Arabidopsis thaliana Thale cress Leaf 15 ug Beta vulgaris sugar beet Leaf 17 ug Brassica napus rapeseed Leaf 9 ug Blossom 9 ug Stalk 7 ug Capsicum annuum red pepper Leaf 8 ug Cucumis melo cucumber Leaf 15 ug Gladiolus spec Leaf 7 ug Hordeum vulgare barley Leaf 3 ug Lactuca sativa lettuce Leaf 4 ug Lycopersicum esculentum tomato Leaf 10 ug Mucor rouxii fungus Mycelium 6 ug Nicotiana tabacum tabacco Leaf 24 ug Root tip 12 ug Stalk 18 ug Blossom 33 ug Secale cereale rye Leaf 12 ug Taraxacum officinale dadelion Leaf 10 ug Thymus herba barona thyme Leaf 15 ug Triticum aestivum wheat Leaf 4 ug Viola tricolor viola Leaf 9 ug Zea mays maize Leaf 18 ug 10 MACHEREY NAGEL 02 2011 Rev 06 Total RNA isolation from plant 3 Storage conditions and preparation of working solutions Attention Buffers RA1 RA2 and MDW contain guanidinium thiocyanate RAP contains guanidinium HCl Wear gloves and goggles Store lyophilized
14. er Reagents 96 100 ethanol to prepare Wash Buffer RA3 70 ethanol to adjust RNA binding conditions Reducing agent B mercaptoethanol or DTT dithiothreithol or TCEP BisTris Bis 2 hydroxyethyl imino tris hydroxymethyl methane as supplement for Lysis Buffer RA1 Consumables 1 5 mL microcentrifuge tubes Sterile RNase free pipette tips Equipment Manual pipettors Centrifuge for microcentrifuge tubes Equipment for sample disruption and homogenization see section 2 3 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 RNA Plant kit is used for the first time Experienced users however may 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 MACHEREY NAGEL 02 2011 Rev 06 5 Total RNA isolation from plant 2 Product description 2 1 The basic principle One of the most important aspects in the isolation of RNA is to prevent degradation of the RNA during the isolation procedure With the NucleoSpin RNA Plant method the cells are first disrupted by grinding in the presence of liquid N Complete denaturation is then achieved by incubation in a solution containing
15. evel the amplicon is relatively small 200 bp DNA digestion in solution can efficiently destroy contaminating DNA However stringent RNase control and subsequent repurification of the RNA in order to remove buffer salts DNase and digested DNA are usually required The high quality recombinant RNase free DNase rDNAse in the NucleoSpin RNA kits facilitates such a digestion in solution in order to remove even traces of contaminating DNA Check if rDNase was prepared according to section 3 A Digest DNA Reaction Setup Add 6 uL Reaction Buffer for rDNase and 0 6 uL rDNase to 60 uL eluted RNA Alternatively premix 100 uL Reaction Buffer for rDNase and 10 uL rDNase and add 1 10 volume to one volume of RNA eluate B Incubate sample Incubate for 10 min at 37 C 18 MACHEREY NAGEL 02 2011 Rev 06 Total RNA isolation from plant C Repurify RNA Repurify RNA with a suitable RNA cleanup procedure for example by use of the NucleoSpin RNA Clean up RNA Clean up XS kit see ordering information or by ethanol precipitation Ethanol precipitation exemplary Add 0 1 volume of 3 M sodium acetate pH 5 2 and 2 5 volumes of 96 100 ethanol to one volume of sample Mix thoroughly Incubate several minutes to several hours at 20 C or 4 C Note Choose long incubation times for sample containing low RNA concentration Short incubation times are sufficient if the sample contains high RNA concentrat
16. inding conditions Discard the NucleoSpin Filter and add 350 pL ethanol 70 96 to the homogenized lysate and mix by pipetting up and down 5 times Alternatively transfer flow through into a new 1 5 mL microcentrifuge tube not provided add 350 uL ethanol 70 96 and mix by vortexing 2 x 5 s After addition of ethanol a stringy precipitate may become visible which will not affect the RNA isolation Be sure to disaggregate any precipitate by mixing and load all of the precipitate on the column as described in step 5 Do not centrifuge the ethanolic lysate before loading it onto the column in order to avoid pelleting the precipitate Bind DNA For each preparation take one NucleoSpin RNA Plant Column light bue ring placed in a Collection Tube and load the lysate Centrifuge for 30 s at 11 000 x g Place the column in a new Collection Tube 2 mL Maximum loading capacity of NucleoSpin RNA Plant Columns is 750 uL Repeat the procedure if larger volumes are to be processed Desalt silica membrane Add 350 uL MDB Membrane Desalting Buffer and centrifuge at 11 000 x g for 1 min to dry the membrane Salt removal will make the following rDNase digest much more effective If the column outlet has come into contact with the flow through for any reason discard the flow through and centrifuge again for 30 s at 11 000 x g 350 uL 70 ethanol Mix Load lysate 30s 11 000 x g 350 uL MDB 1 min 11 0
17. ing materials RNA is not protected against digestion until the sample material is flash frozen or disrupted in the presence of RNase inhibiting or denaturing agents Therefore it is important that samples are flash frozen in liquid N immediately and stored at 70 C Or processed as soon as possible Samples can be stored in Lysis Buffer RA1 after disruption at 70 C for up to one year at 4 C for up to 24 hours or up to several hours at room temperature Frozen samples are stable up to 6 months Frozen samples in Buffer RA1 should be thawed slowly before starting with the isolation of total RNA Wear gloves at all times during the preparation Change gloves frequently Plant tissues are often solid and must therefore be broken up mechanically as well as lysed Depending on the disruption method the viscosity of the lysed sample has to be reduced further for optimal results It is essential for efficient RNA preparation that all the RNA contained in the sample is released from the cells by disruption and that the viscosity of the sample is reduced by homogenization The most commonly used technique for disruption of plant tissues is grinding with a pestle and mortar Grind the sample to a fine powder in the presence of liquid N Take care that the sample does not thaw during or after grinding or weighing and add the frozen powder to an appropriate aliquot of Buffer RA1 respectively RAP containing B mercaptoethanol and mix immediately The broken
18. ing the preparation Check if Wash Buffer RA3 and rDNase were prepared according to section 3 Homogenize sample Grind up to 100 mg tissue under liquid N for handling and preparation methods see section 2 3 Lyse cells Add 350 uL Buffer RA1 and 3 5 uL B mercaptoethanol B ME to 100 mg tissue and vortex vigorously If the lysate solidifies upon addition of Buffer RA1 use 350 uL Buffer RAP instead Note As alternative to B ME the reducing agent DTT or TCEP may be used Use a final concentration of 10 20 mM DTT or TCEP within the Lysis Buffer RA1 or RAP e g add 7 14 uL of a 500 mM DTT or TCEP solution Filtrate lysate Reduce viscosity and clear the lysate by filtration through NucleoSpin Filter violet ring Place NucleoSpin Filter in a Collection Tube 2 mL apply the mixture and centrifuge for 1 min at 11 000 x g Transfer the filtrate to anew 1 5 mL microcentrifuge tube nor provided Important note Do not disturb the pellet of cell debris at the bottom of the collecting tube which may be visible after centrifugation In case of visible pellet formation depending on sample amount and nature transfer supernatant without any formed pellet to a new 1 5 mL microcentrifuge tube not provided c Grind sample 350 pL RA1 3 5 uL B ME yo 350 uL RAP 3 5 uL B ME e 11 000 x g 1 min 14 MACHEREY NAGEL 02 2011 Rev 06 NucleoSpin RNA Plant Adjust RNA b
19. ion Centrifuge for 10 min at maximum speed Wash RNA pellet with 7096 ethanol Dry RNA pellet and resuspend RNA in RNase free H O MACHEREY NAGEL 02 2011 Rev 06 19 Total RNA isolation from plant 6 Appendix 6 1 Troublesho oting Problem Possible cause and suggestions RNase contamination RNA is degraded no RNA obtained Create an RNase free working environment Wear gloves during all steps of the procedure Change gloves frequently Use of sterile disposable polypropylene tubes is recommended Keep tubes closed whenever possible during the preparation Glassware should be oven baked for at least 2 hours at 250 C before use Reagents not applied or restored properly Poor RNA quality or yield Reagents not properly restored Add the indicated volume of RNase free H O to rDNase vial and 96 ethanol to Buffer RA3 Concentrate and mix Reconstitute and store lyophilized rDNase according to instructions given in section 3 Sample and reagents have not been mixed completely Always vortex vigorously after each reagent has been added No ethanol has been added after lysis Binding of RNA to the silica membrane is only effective in the presence of ethanol Kit storage Reconstitute and store lyophilized rDNase according to instructions given in section 3 Store other kit components at room temperature Storage at low temperatures may cause salt precipitation Keep bottles tightly closed in o
20. itive to trace contaminations of RNases often found on general lab ware fingerprints and dust To ensure RNA stability keep RNA frozen at 20 C for short term or 70 C for long term storage Simultaneous isolation of RNA and DNA NucleoSpin RNA DNA Buffer Set The NucleoSpin RNA DNA Buffer Set see ordering information is a support set for RNA and DNA isolation in conjunction with NucleoSpin RNA II NucleoSpin RNA XS NucleoSpin RNA Plant or NucleoSpin RNA Protein This patented technology enables successive elution of DNA and RNA from one NucleoSpin Column with low salt buffer and water respectively DNA and RNA are immediately ready for downstream applications DISTRIBUTION AND USE OF THE NUCLEOSPIN RNA DNA BUFFER SET IN THE USA IS PROHIBITED FOR PATENT REASONS 6 MACHEREY NAGEL 02 2011 Rev 06 Total RNA isolation from plant 2 2 Kit specifications NucleoSpin RNA Plant is recommended for the isolation of total RNA from plant cells and tissues or filamentous fungi Generally 1 10 of the eluate of total RNA prepared from 10 mg of plant tissue is sufficient as template for RT PCR If possible intron spanning primers should be used for RT PCR Hands on time for RNA preparation from plant tissue with NucleoSpin RNA Plant is less than 30 min NucleoSpin Filters for homogenization and reduction of lysate viscosity are included in the kit The kit allows purification of up to 70 ug of pure RNA suita
21. nection 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 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 24 21 969 270 tech bio 2 mn net com M
22. ples 3 Storage conditions and preparation of working solutions 4 Safety instructions risk and safety phrases 5 Protocols 5 1 Total RNA isolation from plant tissue or filamentous fungi 5 2 Support protocol NucleoSpin RNA Plant rDNase digestion in solution 6 Appendix 6 1 Troubleshooting 6 2 Ordering information 6 3 References 6 4 Product use restriction warranty aon RA o O 0 0 o 11 12 14 14 18 20 20 23 24 24 MACHEREY NAGEL 02 2011 Rev 06 Total RNA isolation from plant 1 Components 1 1 Kit contents NucleoSpin RNA Plant 20 preps 50 preps 250 preps REF 740949 20 740949 50 740949 250 Lysis Buffer RA1 10 mL 25 mL 125 mL Lysis Buffer RAP 10 mL 25 mL 125 mL Wash Buffer RA2 15 mL 15 mL 80 mL Wash Buber RAS 5mL 12 5 mL 3x25mL Concentrate Membrane Desalting Buffer MDB 10 mL 25 mL 125 mL Reaction Buffer for rDNase 3 mL 7 mL 30 mL rDNase RNase free 1 vial 1 vial 5 vials lyophilized size C size D size D RNase free H O 5mL 15 mL 65 mL iiem NucleoSpin Filters 20 50 250 violet rings NucleoSpin RNA Plant Columns light blue rings 20 50 250 plus Collection Tubes Collection Tubes 2 mL 60 150 750 Collection Tubes 1 5 mL 20 50 250 User manual 1 1 1 For preparation of working solutions and storage conditions see section 3 4 MACHEREY NAGEL 02 2011 Rev 06 Total RNA isolation from plant 1 2 Reagents consumables and equipment to be supplied by us
23. rder to prevent evaporation or contamination 20 MACHEREY NAGEL 02 2011 Rev 06 Total RNA isolation from plant lonic strength and pH influence A absorption as well as ratio A oso aso For adsorption measurement use 5 mM Tris pH 8 5 as diluent Please see also Manchester K L 1995 Value of A A ratios for measurement of purity of nucleic acids Biotechniques 19 208 209 Wilfinger W W Mackey K and Chomczyski P 1997 Effect of pH and ionic strength on the spectrophotometric assessment Poor RNA of nucleic acid purity Biotechniques 22 474 481 quality or yield continued Sample material Sample material not stored properly Whenever possible use fresh material If this is not possible flash freeze the samples in liquid N Samples should always be kept at 70 C Never allow tissues to thaw before addition of Buffer RA1 Perform disruption of samples in liquid N Insufficient disruption and or homogenization of starting material Ensure thorough sample disruption and use NucleoSpin Filters for easy homogenization of disrupted starting material Sample material Too much starting material used Overloading may lead to decreased overall yield Reduce amount of sample material Clogged or use larger volume of Buffer RA1 NucleoSpin Column Insufficient disruption and or homogenization of starting material Ensure thorough sample disruption and use NucleoSpin Filters for
24. ree H O RNase free H O to each vial MACHEREY NAGEL 02 2011 Rev 06 11 Total RNA isolation from plant 4 Safety instructions risk and safety phrases The following components of the NucleoSpin RNA Plant 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 Inhalt Gefahrstoff Gefahrstoffsymbol R S tze S S tze rDNase rDNase x May cause sensitization by R 42 43 S 22 24 RNase free lyophilized Xn inhalation and skin contact rDNase Sensibilisierung durch Einatmen lyophilisiert und Hautkontakt m glich RA1 Guanidinium x Harmful by inhalation in R 20 21 22 S 13 thiocyanate Xn contact with the skin and if swallowed Guanidinium Gesundheitssch dlich beim thiocyanat Einatmen Verschlucken und Ber hrung mit der Haut RA2 Guanidinium x Flammable Harmful by in R 10 S 7 13 16 thiocyanate Xn halation in contact with skin 20 21 22 and if swallowed Guanidinium Entz ndlich Gesundheitssch d thiocyanat lich beim Einatmen Verschlucken und Ber hrung mit der Haut RAP Guanidine x Harmful if swallowed R 22 hydrochloride Xn Irritating to eyes and skin 36 38 Guanidin Gesundheitssch dlich beim hydrochlorid Verschlucken Reizt die Augen und die Haut MDB Guanidinium Flammable R 10 S 7 16 thiocyanate 1096 ethanol 1096 Guanidinium Entz ndlich thiocyanat 1096
25. to replacement of products free of charge in the event products fail to perform 24 MACHEREY NAGEL 02 2011 Rev 06 Total RNA isolation from plant 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 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 con
26. up tissue must then be homogenized with a NucleoSpin Filter or by passing gt 5 through a 0 9 mm syringe needle Thawing of undisrupted plant tissue should only be done in the presence of Buffer RA1 during simultaneous mechanical disruption e g with a rotor stator homogenizer This ensures that the RNA is not degraded by RNases before the preparation has started The spinning rotor disrupts and simultaneously homogenizes the sample by mechanical shearing of DNA within seconds up to minutes homogenization time depends on sample Take care to keep the rotor tip submerged in order to avoid excess foaming Select a suitably sized homogenizer 5 7 mm diameter rotors can be used for homogenization in microcentrifuge tubes 8 MACHEREY NAGEL 02 2011 Rev 06 Total RNA isolation from plant 2 4 Elution procedures It is possible to adapt elution method and volume of water used for the subsequent application of interest In addition to the standard method described in the individual protocols recovery rate about 70 90 there are several modifications possible High yield Perform two elution steps with the volume indicated in the individual protocol About 90 100 of bound nucleic acid will be eluted High yield and high concentration Elute with the standard elution volume and apply the eluate once more onto the column for reelution Eluted RNA should immediately be put on ice and always kept on ice for optimal stability becaus
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