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1. RNA isolation from plant User manual NucleoSpin RNA Plant February 2013 Rev 08 MACHEREY NAGEL MN www mn net com RNA isolation from plant Protocol at a glance Rev 08 1 Homogenize sample cs 2 Lyse cells 350 uL RA1 35 pL B mercaptoethanol or 350 uL RAP 35 uL B mercaptoethanol Mix 3 Filtrate lysate ze 4 Adjust RNA binding 350 pL 70 ethanol conditions Mix Bind RNA NucleoSpin RNA Plant Desalt sillca membrane 350 uL MDB 11 000 x g 1 min Digest DNA ges 95 pL DNase reaction mixture RT 15 min Wash and dry silica 200 pL RAW2 membrane 600 uL RA3 250 uL RA3 11 000 xg 30s 11 000 xg 2 min 60 uL RNase free H O 11000xg 1 min MACHEREY NAGEL GmbH amp Co KG Neumann Neander Str 6 8 52355 Daren Germany el 49 24 21 969 270 ax 49 24 21 969 199 tech bio mn net com www mn net com 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 samples 3 Storage conditions and preparation of working solutions 4 Safety instructions 4 1 Risk and safety phrases 4 2 GHS classification 5 Protocols 5 1 RNAisolation from plant tissue or filamentous fungi 5 2 rDNase digestion in solution 6 Appe2. 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 MACHEREY NAGEL 02 2013 Rev 08 350 pL y 7096 ethanol Mix Load lysate 11 000 x g c5 c F 350 uL MDB 11 000 x g 1 min NucleoSpin RNA Plant 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 RAW2 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 RAW2 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 Note Make sure that residual buffer from the previous steps is washed away with Buffer RA3 especially if the lysate has been in contact with the inner rim of the column during loading of the lysate onto the column For efficient washing of the inner rim flush it with Buffer RAS Add 250 uL Buffer RA3 to the NucleoSpin RNA Plant Column Centrifuge for 2
3. 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 concentration Centrifuge for 10 min at maximum speed Wash RNA pellet with 70 ethanol Dry RNA pellet and resuspend RNA in RNase free H O 20 MACHEREY NAGEL 02 2013 Rev 08 RNA isolation from plant 6 Appendix 6 1 Troubleshooting Problem Possible cause and suggestions RNA is degraded no RNA obtained RNase contamination 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 Poor RNA quality or yield Reagents not applied or restored properly 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 Sa
4. Reizt die Augen und die Haut R 42 43 May cause sensitization by inhalation and skin contact Sensibilisierung durch Einatmen und Hautkontakt m glich R 52 53 Harmful to aquatic organisms may cause long term adverse effects in the aquatic environment Sch dlich f r Wasserorganismen kann in Gew ssern l ngerfristig sch dliche Wirkungen haben MACHEREY NAGEL 02 2013 Rev 08 11 RNA isolation from plant Safety phrases 13 Keep away from food drink and animal foodstuffs Von Nahrungsmitteln Getr nken und Futtermitteln fernhalten S 16 Keep away from sources of ignition No smoking Von Z ndquellen fernhalten Nicht rauchen 22 Do not breathe dust Staub nicht einatmen S24 Avoid contact with the skin Ber hrung mit der Haut vermeiden S 26 n case of contact with eyes rinse immediately with plenty of water and seek medical advice Bei Ber hrung mit den Augen gr ndlich mit Wasser absp len und Arzt konsultieren S 37 39 Wear suitable protective clothing and gloves Bei der Arbeit geeignete Schutzhandschuhe und Schutzkleidung tragen S61 Avoid release to the environment Refer to special instructions safety data sheet Freisetzung in die Umwelt vermeiden Besondere Anweisungen einholen Sicherheitsdaten bl tter zu Rate ziehen 4 2 GHS classification Only harmful features do not need to be labeled with H and P phrases until 125 mL or 125g Mindergef hrliche Eigenschaften m ssen bis 125 mL oder 125 g nicht mit H und P S tzen gekenn
5. 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 MACHEREY NAGEL 02 2013 Rev 08 9 RNA isolation from plant 3 Storage conditions and preparation of working solutions Attention Buffers RA1 RAW2 RAP and MDB contain chaotropic salt Wear gloves and goggles CAUTION Buffers RA1 and MDB contain guanidinium thiocyanate and Buffer RAW2 and Buffer RAP contain guanidine hydrochloride which can form highly reactive compounds when combined with bleach sodium hypochlorite DO NOT add bleach or acidic solutions directly to the sample preparation
6. 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 centrifu gation discard flow through and centrifuge again 95 uL rDNase reaction mixture RT 15 min 200 uL RAW2 11 000 x g 30s F 4 600 uL RA3 11 000 x g e 30s 250 uL RA3 11 000 x g 2 min MACHEREY NAGEL 02 2013 Rev 08 17 NucleoSpin RNA Plant 18 Elute RNA Elute the RNA in 60 pL RNase free H O supplied and centrifuge at 11 000 x g for 1 min If higher RNA concentrations are desired elution can be done with 40 uL Overall yield however will decrease when using smaller elution volumes C For alternative elution procedures see section 2 4 60 pL RNase free H O 11 000 x g 1 min MACHEREY NAGEL 02 2013 Rev 08 NucleoSpin RNA Plant 5 2 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
7. 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 Il 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 D STR BUT ON AND USE OF THE NUCLEOSP N RNA DNA BUFFER SET N THE USA S PROH B TED FOR PATENT REASONS 6 MACHEREY NAGEL 02 2013 Rev 08 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 suitable for applications like reverse transcriptase PCR RT PCR Northern blotting primer extension or RNase protection assays rDNase is sup
8. 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 level 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 MACHEREY NAGEL 02 2013 Rev 08 19 NucleoSpin RNA Plant C Repurify RNA Repurify RNA with a suitable RNA cleanup procedure for example by use of the
9. aterial Ensure thorough sample disruption and use NucleoSpin Filters for easy homogenization of disrupted starting material LOW A As ratio Carry over of guanidinium thiocyanate Carefully load the lysate to the NucleoSpin RNA Il Column and try to avoid a contamination of the upper part of the column and the column lid Make sure that a sufficient amount concentration of RNA is used for quantification so that the A value is significantly higher than the background level Clogged NucleoSpin Column Contamination Sample material Too much starting material used Overloading may lead to decreased overall yield Reduce amount of sample material or use larger volume of Buffer RA1 Insufficient disruption and or homogenization of starting material Ensure thorough sample disruption and use NucleoSpin Filters for easy homogenization of disrupted starting material rDNase not active Reconstitute and store lyophilized rDNase according to instructions given in section 3 rDNase solution not properly applied OFRNA with Pipette rDN lution directly onto the center of the sili genomic DNA ipette rDNase solution directly onto the center of the silica membrane Too much cell material used Reduce quantity of cells or tissue used 22 MACHEREY NAGEL 02 2013 Rev 08 RNA isolation from plant DNA detection system too sensitive The amount of DNA contamination is effectively reduced during the on col
10. bar Harmful if swallowed Gesundheitssch dlich bei Verschlucken Causes skin irritation Verursacht Hautreizungen May cause an allergic skin reaction Kann allergische Hautreaktionen verursachen Causes serious eye irritation Verursacht schwere Augenreizung May cause allergy or asthma symptoms or breathing difficulties if inhaled Kann bei Einatmen Allergie asthmaartige Symptome oder Atembeschwerden verursa chen Harmful to aquatic life with long lasting effects Sch dlich f r Wasserorganismen mit langfristiger Wirkung Contact with acids liberates toxic gas Entwickelt bei Ber hrung mit S ure giftige Gase Precaution phrases P 210 P 233 P 260 P 261 P 273 P 280 P 3014312 P 3024352 P 3044341 Keep away from heat sparks open flames hot surfaces No smoking Von Hitze Funken offener Flamme heiBen Oberfl chen fernhalten Keep container tightly closed Beh lter dicht verschlossen halten Do not breathe vapours Dampf nicht einatmen Avoid breathing dust Einatmen von Staub vermeiden Avoid release to the environment Freisetzung in die Umwelt vermeiden Wear protective gloves eye protection Schutzhandschuhe Augenschutz tragen F SWALLOWED Call a PO SON CENTER or doctor physician if you feel unwell BEI VERSCHLUCKEN Bei Unwohlsein GIFTINFORMATIONSZENTRUM oder Arzt anrufen F ON SK N Wash with plenty of soap and water BEI KONTAKT MIT DER HAUT Mit viel Wasser und Seife waschen F NHALED f breath
11. designed and sold FOR RESEARCH PURPOSES ONLY except however any other function of the product being expressly described in original MACHEREY NAGEL product leaflets MACHEREY NAGEL products are intended for GENERAL LABORATORY USE ONLY MACHEREY NAGEL products are suited for QUALIFIED PERSONNEL ONLY MACHEREY NAGEL products shall in any event only be used wearing adequate PROTECTIVE CLOTHING For detailed information please refer to the respective Material Safety Data Sheet of the product MACHEREY NAGEL products shall exclusively be used in an ADEQUATE TEST ENVIRONMENT MACHEREY NAGEL does not assume any responsibility for damages due to improper application of our products in other fields of application Application on the human body is STRICTLY FORBIDDEN The respective user is liable for any and all damages resulting from such application DNA RNA PROTEIN purification products of MACHEREY NAGEL are suitable for 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 f
12. e Material Safety Data Sheets www mn net com Weiterf hrende Informationen finden Sie in den Sicherheitsdatenbl ttern www mn net com 14 MACHEREY NAGEL 02 2013 Rev 08 NucleoSpin RNA Plant 5 5 1 Protocols RNA isolation from plant tissue or filamentous fungi Before starting 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 pL Buffer RA1 and 3 5 pL 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 a new 1 5 mL microcentrifuge tube not 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 su
13. enaturing 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 up tissue must then be homogenized with a NucleoSpin Filter or by passing 5 through a 0 9 mm syringe needle Thawing of undisrupted plant tiss
14. ing is difficult remove to fresh air and keep at rest ina position comfortable for breathing BEI EINATMEN Bei Atembeschwerden an die frische Luft bringen und in einer Position ruhigstellen die das Atmen erleichtert MACHEREY NAGEL 02 2013 Rev 08 13 RNA isolation from plant Precaution phrases P 30543514338 F N EYES Rinse continuously with water for several minutes Remove P 330 P 332 313 P 333 313 P 337 313 P 3424311 P 4034235 P 363 contact lenses if present and easy to do continue rinsing BEI KONTAKT MIT DEN AUGEN Einige Minuten lang behutsam mit Wasser sp len Vorhandene Kontaktlinsen nach M glichkeit entfernen Weiter sp len Rinse mouth Mund aussp len F skin irritation occurs Get medical advice attention Bei Hautreizung Arztlichen Rat einholen rztliche Hilfe hinzuziehen F skin irritation or a rash occurs Get medical advice attention Bei Hautreizung oder ausschlag Arztlichen Rat einholen rztliche Hilfe hinzuziehen Get medical advice attention Bei anhaltender Augenreizung Arztlichen Rat einholen rztliche Hilfe hinzuziehen f experiencing respiratory symptoms Call a PO SON CENTER or doctor physician Bei Symptomen der Atemwege GIFTINFORMATIONSZENTRUM oder Arzt anrufen Store in a well ventilated place Keep cool K hl an einem gut bel fteten Ort augbewahren Wash contaminated clothing before reuse Kontaminierte Kleidung vor erneutem Tragen waschen For further information please se
15. lized Add 120 uL Add 550 uL Add 550 uL RNase free H O RNase free H O RNase free H O to each vial 10 MACHEREY NAGEL 02 2013 Rev 08 RNA isolation from plant 4 Safety instructions The following components of the NucleoSpin RNA Plant kit contain hazardous contents Wear gloves and goggles and follow the safety instructions given in this section 4 1 Risk and safety phrases Component Hazard contents Hazard Risk Safety symbol phrases phrases Inhalt Gefahrstoff Gefahrstoff R S tze S S tze symbol rDNase rDNase lyophilized x Xn R 42 43 S 22 24 RNase free rDNase lyophilisiert RA1 Guanidinium thiocyanate 30 60 x Xn R 20 21 22 813 61 Guanidiniumthiocyanat 30 60 32 52 53 RAW2 Guanidine hydrochloride 24 36 x Xn R 10 22 S16 ethanol 20 35 Guanidinhydrochlorid 24 36 Ethanol 20 35 RAP Guanidine hydrochloride 50 66 x Xn R 22 36 38 S 26 37 39 Guanidin hydrochlorid 50 66 MDB Guanidinium thiocyanate 1 15 id R 10 ethanol 5 20 Guanidiniumthiocyanat 1 1596 Ethanol 5 2096 Risk phrases R 10 Flammable Entz ndlich R 20 21 22 Harmful by inhalation in contact with skin and if swallowed Gesundheitssch dlich beim Einatmen Verschlucken und Ber hrung mit der Haut R 22 Harmful if swallowed Gesundheitssch dlich beim Verschlucken R 32 Contact with acids liberates very toxic gas Entwickelt bei Ber hrung mit S ure sehr giftige Gase R 36 38 rritating to eyes and skin
16. ls 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 HCl 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 sensitive to trace contaminations of RNases often found on general lab ware fingerprints and dust To ensure RNA stability keep RNA frozen at
17. m plant components not manufactured by MACHEREY NAGEL or damages resulting from such non MACHEREY NAGEL components or products MACHEREY NAGEL makes no other warranty of any kind whatsoever and SPECIFICALLY DISCLAIMS AND EXCLUDES ALL OTHER WARRANTIES OF ANY KIND OR NATURE WHATSOEVER DIRECTLY OR INDIRECTLY EXPRESS OR IMPLIED INCLUDING WITHOUT LIMITATION AS TO THE SUITABILITY REPRODUCTIVITY DURABILITY FITNESS FOR A PARTICULAR PURPOSE OR USE MERCHANTABILITY CONDITION OR ANY OTHER MATTER WITH RESPECT TO MACHEREY NAGEL PRODUCTS In no event shall MACHEREY NAGEL be liable for claims for any other damages whether direct indirect incidental compensatory foreseeable consequential or special including but not limited to loss of use revenue or profit whether based upon warranty contract tort including negligence or strict liability arising in connection with the sale or the failure of MACHEREY NAGEL products to perform in accordance with the stated specifications This warranty is exclusive and MACHEREY NAGEL makes no other warranty expressed or implied The warranty provided herein and the data 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 excep
18. mple and reagents have not been mixed completely Always vortex vigorously after each reagent has been added No ethanol has been added after Iysis Binding of RNA to the silica membrane is only effective in the presence of ethanol Kit storage lonic strength and pH influence A A oso A go 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 order to prevent evaporation or contamination 2s absorption as well as ratio For adsorption measurement use 5mM Tris pH8 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 of nucleic acid purity Biotechniques 22 474 481 MACHEREY NAGEL 02 2013 Rev 08 21 RNA isolation from plant Poor RNA 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 m
19. ndix 6 1 Troubleshooting 6 2 Ordering information 6 3 References 6 4 Product use restriction warranty ana A A o o ON O O 10 11 11 12 15 15 19 21 21 24 24 25 MACHEREY NAGEL 02 2013 Rev 08 RNA isolation from plant 1 Components 1 1 Kit contents NucleoSpin RNA Plant 10 preps 50 preps 250 preps REF 740949 10 740949 50 740949 250 Lysis Buffer RA1 10 mL 25 mL 125 mL Lysis Buffer RAP 10 mL 25 mL 125 mL Wash Buffer RAW2 15 mL 15 mL 80 mL Mash Eller PAS 6mL 12 5 mL 3 x 25 mL Concentrate Membrane Desalting 10 mL 25 mL 125 mL Buffer MDB Reaction Buffer for rDNase 3mL 7 mL 30 mL rDNase RNase free 1 vial 1 vial 5 vials lyophilized size D size F size F RNase free H O 15 mL 15 mL 125 mL NucleoSpin Filters 10 50 250 violet rings NucleoSpin RNA Plant 10 50 250 Columns light blue rings plus Collection Tubes Collection Tubes 2 mL 30 150 750 Collection Tubes 1 5 mL 10 50 250 User manual 1 1 1 For prepara ion of working solu ions and s orage condi ions see sec ion 3 4 MACHEREY NAGEL 02 2013 Rev 08 RNA isolation from plant 1 2 Reagents consumables and equipment to be supplied by user 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 Ly
20. or its use to identify any specific organism or for clinical use included but not limited to diagnostic prognostic therapeutic or blood banking It is rather in the responsibility of the user or in any case of resale of the products in the responsibility of the reseller to inspect and assure the use of the DNA RNA protein purification products of MACHEREY NAGEL for a well defined and specific application MACHEREY NAGEL shall only be responsible for the product specifications and the performance range of MN products according to the specifications of in house quality control product documentation and marketing material This MACHEREY NAGEL product is shipped with documentation stating specifications and other technical information MACHEREY NAGEL warrants to meet the stated specifications MACHEREY NAGEL s sole obligation and the customer s sole remedy is limited to replacement of products free of charge in the event products fail to perform as warranted Supplementary reference is made to the general business terms and conditions of MACHEREY NAGEL which are printed on the price list Please contact us if you wish to 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 MACHEREY NAGEL 02 2013 Rev 08 25 RNA isolation fro
21. pernatant without any formed pellet to a new 1 5 mL microcentrifuge tube not provided MACHEREY NAGEL 02 2013 Rev 08 Grind sample 350 pL RA1 3 5 uL B ME or 350 pL RAP 3 5 uL B ME 15 NucleoSpin RNA Plant Adjust RNA binding conditions Discard the NucleoSpin Filter and add 350 pL ethanol 70 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 pL ethanol 70 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 pL 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
22. plied 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 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 100 mg tissue Fragment size gt 200 nt Typical yield 3 70 ug from 100 mg plant material A seo Peso 1 9 2 1 Elution volume 60 uL Preparation time 30 min 6 preps Binding capacity 200 ug MACHEREY NAGEL 02 2013 Rev 08 7 RNA isolation from plant 2 3 Handling preparation and storage of starting materials RNA is not protected against digestion until the sample material is flash frozen or disrupted in the presence of RNase inhibiting or d
23. rly 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 MACHEREY NAGEL 02 2013 Rev 08 23 RNA isolation from plant 6 2 Ordering information Product REF Pack of NucleoSpin RNA Plant 740949 10 50 250 10 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 Buffer Set 740944 Suitable for 100 preps NucleoSpin TriPrep 740966 10 50 250 10 50 250 740961 50 mL Butfer RAT 740961500 500 mL 740936 50 50 mL BUDSDPAE 740936 500 500 mL 740385 50 50 mL Buffer RLI 740385 125 125 mL rDNase Set 740963 1 set NucleoSpin Filters 740606 50 Collection Tubes 2 mL 740600 1000 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 D STR BUT ON AND USE OF NUCLEOSP N RNA DNA BUFFER SET and NUCLEOSP N TR PREP N THE USA S PROH B TED FOR PATENT REASONS 24 MACHEREY NAGEL 02 2013 Rev 08 RNA isolation from plant 6 4 Product use restriction warranty NucleoSpin RNA Plant kit components are intended developed
24. sis 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 Please contact Technical Service regarding information about changes of the current user manual compared to previous revisions MACHEREY NAGEL 02 2013 Rev 08 5 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 large amounts of chaotropic ions This lysis buffer immediately inactivates RNases which are present in virtually all biological materia
25. t 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 mn net com 26 MACHEREY NAGEL 02 2013 Rev 08
26. ue 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 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 96 there are several modifications possible High yield Perform two elution steps with the volume indicated in the individual protocol About 90 100 96 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 because 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 8 MACHEREY NAGEL 02 2013 Rev 08 RNA isolation
27. umn digestion with rDNase Anyhow it can not be guaranteed that the purified RNA is 100 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 Contamination of a 1 kbp fragment in a 30 cycle reaction Generally no PCR of RNA with product is obtained while skipping the DNase digest usually genomic DNA leads to positive PCR results 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 spanning 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 prope
28. waste Store lyophilized rDNase RNase free at 4 C on arrival stable for at least 1 year All other kit components should be stored at room temperature 18 25 C and are stable up for at least 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 Wash Buffer RA3 can be stored at room temperature 18 25 C for at least one year NucleoSpin RNA Plant 10 preps 50 preps 250 preps REF 740949 10 740949 50 740949 250 Wash Buffer RA3 6 mL 12 5 mL 3 x 25 mL Concentrate Add 24 mL ethanol Add 50 mL ethanol Add 100 mL ethanol to each vial rDNase RNase 1 vial size D 1 vial size F 5 vials size F free lyophi
29. zeichnet werden Component Hazard contents GHS symbol Hazard Precaution phrases phrases Inhalt Gefahrstoff GHS Symbol H S tze P S tze rDNase rDNase lyophilized Danger 317 334 261 280 RNase free rDNase Iyophilisiert Gefahr 302 352 3044341 3334313 3424311 363 RA1 Guanidinium thiocyanate Warning 302 412 260 273 30 60 O EUHO31 301 312 330 Guanidiniumthiocyanat Achtung 30 60 RAW2 Guanidine hydrochloride Warning 226 302 210 233 24 36 ethanol 20 35 d O 301 312 330 Guanidinhydrochlorid 24 36 Achtung 403 235 Ethanol 20 35 RAP Guanidine hydrochloride Warning 302 315 280 301 312 50 66 319 302 352 idi j Achtung 305 351 338 Guanidin hydrochlorid 50 66 330 3324313 337 313 Hazard labeling no necessary if quaniy per bo le below 125g or mL cerificae of exemp ion according o 67 548 EEC Ar 25 1999 45 EC Ar 12 and German GefS offV 20 3 and TRGS 2007 1 For fur her informa ion see Ma erial Safe y Da a Shee 12 MACHEREY NAGEL 02 2013 Rev 08 RNA isolation from plant Component Hazard contents GHS symbol Hazard Precaution phrases phrases Inhalt Gefahrstoff GHS Symbol H S tze P S tze MDB Guanidinium thiocyanate Warning 226 210 233 1 15 ethanol 5 20 d 4034235 Guanidiniumthiocyanat 1 1596 Achtung Ethanol 5 20 Hazard phrases H 226 H 302 H 315 H 317 H 319 H 334 H412 EUHOS1 Flammable liquid and vapour Fl ssigkeit und Dampf entz nd
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