RNA clean-up
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1. GmbH amp Co KG Tel 49 24 21 969 270 tech bio mn net com Trademarks LightCycler is a trademark of a member of the Roche Group NucleoSpin is a registered trademark of MACHEREY NAGEL GmbH amp Co KG SYBR is a registered trademark of Molecular Probes Inc All used names and denotations can be brands trademarks or registered labels of their respective owner also if they are not special denotation To mention products and brands is only a kind of information i e it does not offend against trademarks and brands and can not be seen as a kind of recommendation or assessment Regarding these products or services we can not grant any guarantees regarding selection efficiency or operation 20 MACHEREY NAGEL 03 2012 Rev 022. ml certificate of exemption according to 67 548 EEC Art 25 1999 45 EC Art 12 and German GefStoffV 20 3 and TRGS 200 7 1 For further information see Material Safety Data Sheet 10 MACHEREY NAGEL 03 2012 Rev 02 RNA clean up 4 2 GHS classification Only harmful features do not need to be labeled with H and P phrases until 125 mL or 125 g Mindergefahrliche Eigenschaften m ssen bis 125 mL oder 125 g nicht mit H und P S tzen gekennzeichnet werden Component Hazard contents GHS symbol Hazard Precaution phrases phrases Inhalt Gefahrstoff GHS Symbol H S tze P S tze RCU Guanidinium thiocyanate Warning 302 412 260 273 30 60 EUH031 301 312 330 Guanidiniumthiocyanat Achtung 30 60 Hazard phrases H 302 Harmful if swallowed Gesundheitssch dlich bei Verschlucken H 412 Harmful to aquatic life with long lasting effects Sch dlich f r Wasserorganismen mit langfristiger Wirkung EUH031 Contact with acids liberates toxic gas Entwickelt bei Ber hrung mit S ure giftige Gase Precaution phrases P 260 Do not breathe vapours Dampf nicht einatmen P 273 Avoid release to the environment Freisetzung in die Umwelt vermeiden P 301 312 IF SWALLOWED Call a POISON CENTER or doctor physician if you feel unwell BEI VERSCHLUCKEN Bei Unwohlsein GIFTINFORMATIONSZENTRUM oder Arzt anrufen P 330 Rinse mouth Mund aussp len Bei Symptomen der Atemwege GIFTINFORMATIONSZENTRUM oder Arzt anrufe
3. 0 mL ethanol to each bottle MACHEREY NAGEL 03 2012 Rev 02 9 RNA clean up 4 Safety instructions risk and safety phrases The following components of the NucleoSpin RNA Clean up XS kits 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 Sdtze symbol RCU Guanidinium thiocyanate 30 60 x Xn R 20 21 22 S 13 61 Guanidiniumthiocyanat 30 60 32 52 53 Risk phrases 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 32 Contact with acids liberates very toxic gas Entwickelt bei Ber hrung mit S ure sehr giftige Gase 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 Safety phrases S 13 Keep away from food drink and animal foodstuffs Von Nahrungsmitteln Getr nken und Futtermitteln fernhalten S 61 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 Hazard labeling not neccessary if quantity per bottle below 125g or
4. A clean up 3 Storage conditions and preparation of working solutions Attention Buffers RCU contains chaotropic salt Wear gloves and goggles CAUTION Buffer RCU contains guanidinium thiocyanate which can form highly reactive compounds when combined with bleach sodium hypochlorite DO NOT add bleach or acidic solutions directly to the sample preparation waste All 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 96 100 ethanol is available as additional solution in the lab Before starting any NucleoSpin RNA Clean up protocol prepare the following Clean up Buffer RCU Add the indicated volume of 96 100 ethanol to the Clean up Buffer RCU Concentrate See table below or bottle label for necessary volumes Store Buffer RCU at room temperature 18 25 C for up to one year Wash Buffer RA3 Add the indicated volume of 96 100 ethanol see table below to Wash 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 Clean up 10 preps 50 preps 250 preps REF 740903 10 740903 50 740903 250 Clean up Buffer 1 mL 5 mL 25 mL RCU Concentrate Add 3 mL ethanol Add 15 mL ethanol Add 75 mL ethanol Wash Buffer RA3 2 mL 7mL 2x20 mL Concentrate Add 8 mL ethanol Add 28 mL ethanol Add 8
5. 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 IN VITRO diagnostic use Please pay attention to the package of the product IN VITRO diagnostic products are expressly marked as IVD on the packaging 18 MACHEREY NAGEL 03 2012 Rev 02 RNA Clean up 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 specif
6. RNA XS 740902 10 10 preps 740902 50 50 preps 740902 250 250 preps NucleoSpin RNA II 740955 20 20 preps 740955 50 50 preps 740955 250 250 preps NucleoSpin RNA L 740962 20 20 preps NucleoSpin RNA Protein 740933 10 10 preps 740933 50 50 preps 740933 250 250 preps NucleoSpin TriPrep 740966 10 10 preps 740966 50 50 preps 740966 250 250 preps NucleoSpin RNA Clean up 740948 10 10 preps 740948 50 50 preps 740948 250 250 preps NucleoSpin miRNA 740971 10 10 preps 740971 50 50 preps 740971 250 250 preps NucleoSpin RNA Blood 740200 10 10 preps 740200 50 50 preps NucleoSpin RNA Plant 740949 10 10 preps 740949 50 50 preps 740949 250 250 preps NucleoSpin FFPE RNA 740969 10 10 preps 740969 50 50 preps 740969 250 250 preps NucleoSpin RNA DNA Buffer Set 740944 Suitable for 100 preps rDNase Set 740963 1 set DISTRIBUTION AND USE OF NUCLEOSPIN TRIPREP and NUCLEOSPIN RNA DNA BUFFER SET IN THE USA IS PROHIBITED FOR PATENT REASONS MACHEREY NAGEL 03 2012 Rev 02 17 RNA Clean up Product REF Pack of NucleoSpin Filters 740606 50 Collection Tubes 2 mL 740600 1000 6 3 Literature Fleige S Pfaffl MW RNAintegrity and the effect on the real time qRT PCR performance Mol Aspects Med 2006 Apr Jun 27 2 3 126 39 Epub 2006 Feb 15 Review Imbeaud S Graudens E Boulanger V Barlet X Zaborski P Eveno E Mueller O Schroeder A Auffray C Towards standardization of RNA quality assessment using user indepen
7. RNA clean up User manual NucleoSpin RNA Clean up XS March 2012 Rev 02 MACHEREY NAGEL MN RNA clean up 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 Stability of isolated RNA 3 Storage conditions and preparation of working solutions 4 Safety instructions risk and safety phrases 4 1 Risk and safety phrases 4 2 GHS classification 5 Protocols 5 1 RNA clean up and concentration of RNA 5 2 DNA digestion in crude RNA extracts and subsequent clean up 6 Appendix 6 1 Troubleshooting 6 2 Ordering information 6 3 Literature 6 4 Product use restriction warranty an A A N DD DD 10 10 11 12 12 14 15 15 17 18 18 MACHEREY NAGEL 03 2012 Rev 02 RNA clean up 1 Components 1 1 Kit contents NucleoSpin RNA Clean up XS 10 preps 50 preps 250 preps REF 740903 10 740903 50 740903 250 Clean up Butler RCU qim 5 mL 25 mL Concentrate Wash Buffer RA3 2mL 7mL 2x20 mL Concentrate RNase free H O 5mL 15 mL 25 mL NucleoSpin RNA XS Binding 10 50 250 Columns light blue rings plus Collection Tubes Collection Tubes 2 mL 10 50 250 Collection Tubes 1 5 mL 10 50 250 User manual 1 1 1 For preparation of working solu
8. ctivates RNases which are present in virtually all biological materials and creates appropriate binding conditions to allow adsorption of RNA to the silica membrane Two washing steps with a single buffer remove any impurities Pure RNA is finally eluted at low ionic strength conditions with RNase free water supplied in a volume as small as 5 uL The RNA clean up procedure using NucleoSpin RNA Clean up XS kit can be performed at room temperature The eluate should be treated with care because RNA is very sensitive to trace contaminations of RNases often present on general lab ware fingerprints and dust To ensure RNA stability we recommend keeping the RNA solution frozen at 20 C for short term or 70 C for long term storage 2 2 Kit specifications The NucleoSpin RNA Clean up XS kit is recommended for the clean up and concentration of prepurified RNA samples Typical sample material covers nanogramm to microgramm amounts of prepurified RNA e g phenol purified RNA and RNA from reaction mixtures e g DNase treated samples The innovative column design with a funnel shaped thrust ring and a small silica membrane area allows sample volumes of up to 300 uL and elution of RNA in as little as 5 30 uL Thus highly concentrated RNA is eluted and is ready for common downstream applications e g RT PCR RNA enrichment of 20 x up to 50x can be achieved e g input 300 uL sample containing crude RNA 10 ng uL output 5 uL eluate co
9. dent classifiers of microcapillary electrophoresis traces Nucleic Acids Res 2005 Mar 30 33 6 e56 Miller CL Diglisic S Leister F Webster M Yolken RH Evaluating RNA status for RT PCR in extracts of postmortem human brain tissue Biotechniques 2004 Apr 36 4 628 33 Schoor O Weinschenk T Hennenlotter J Corvin S Stenzl A Rammensee HG Stevanovic S Moderate degradation does not preclude microarray analysis of small amounts of RNA Biotechniques 2003 Dec 35 6 1192 6 1198 201 6 4 Product use restriction warranty NucleoSpin RNA Clean up XS 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
10. e 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 Higher RNA yield than theoretically possible Unexpected A go go ratio If performing clean up of samples containing less than approximately 300ng RNA subsequent quantification by Ago Measurement may simulate yields larger than the RNA input This may be due to absorbance of silica abrasion In order to prevent incorrect Ago quantification of small RNA amounts centrifuge the elution tube for 30s at 8 000 11 000 x g and withdraw an aliquot for measurement without disturbing any sediment or use a silica abrasion insensitive RNA quantification method e g RiboGreen fluorescent dye Measurement not in the range of photometer detection limit In order to obtain a significant A o A go ratio it is necessary that the initially measured Ago and Aggo Values are significantly above the detection limit of the photometer used An A go value close to the background noise of the photometer will cause unexpected A go ggo ratios 16 MACHEREY NAGEL 03 2012 Rev 02 RNA clean up 6 2 Ordering information Product REF Pack of NucleoSpin RNA Clean up XS 740903 10 10 preps 740903 50 50 preps 740903 250 250 preps NucleoSpin
11. e may be varied in the range of 5 30 uL 11 000 x g 30 s For further details on alternative elution procedures see section 2 4 MACHEREY NAGEL 03 2012 Rev 02 13 NucleoSpin RNA Clean up XS 5 2 DNA digestion in crude RNA extracts and subsequent clean up Several commonly used RNA purification methods co purify DNA to a considerable extent e g phenol based RNA purification This often requires a subsequent removal of contaminating DNA and clean up of the RNA from the reaction mixture 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 MACHEREY NAGEL rDNase Set to be ordered separately see ordering information contains high quality recombinant RNase free DNase rDNase and reaction buffer It is optimized for a highly efficient digestion in order to remove even traces of contaminating DNA 1 Digest DNA reaction setup Prepare enzyme buffer premix Add 1 uL rDNase to 10 pL Reaction Buffer for rDNase Add 1 10 volume of enzyme buffer premix to the crude RNA extract e g to 10 uL RNA extract add 1 uL of the premix comprising buffer and enzyme Gently swirl the tube in order to mix the solutions Spin down gently approx 1 s at 1 000 x g to collect every droplet of the solution at the bottom of the tube Note Dissolve lyophiliz
12. ed rDNase rDNase Set see ordering information in 540 uL RNase free H O as described in the corresponding user manual 2 Incubate sample Incubate for 10 min at 37 C 3 Repurify RNA Repurify RNA with the NucleoSpin RNA Clean up XS kit according to section 5 1 14 MACHEREY NAGEL 03 2012 Rev 02 NucleoSpin RNA Clean up XS 6 Appendix 6 1 Troubleshooting Problem Possible cause and suggestions RNA is degraded no RNA obtained Poor RNA quality or yield 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 Reagents not applied or restored properly Sample and reagents have not been mixed completely Always vortex vigorously after each reagent has been added No ethanol has been added to Clean up Buffer RCU Binding of RNA to the silica membrane is only effective in the presence of ethanol Adjust binding conditions by adding ethanol to Clean up Buffer RCU Concentrate as described in section 3 Kit storage Store kit components at room temperature Storage at low temperatures may cause salt precipitation Keep bottles tightly closed in order to prevent evaporation or contamination lonic strength and pH influ
13. ence Aggo absorption as well as ratio Azeo A280 For adsorption measurement use 5mM Tris pH 8 5 as diluent Please see also Manchester K L 1995 Value of A go go 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 Sample material Sample material not stored properly Keep thawed samples on ice before addition of Buffer RCU MACHEREY NAGEL 03 2012 Rev 02 15 RNA Clean up Problem Possible cause and suggestions Contamination Sample material already contaminated with DNA of RNA with Digest contaminating DNA in an RNA sample according to genomic DNA section 5 2 Carry over of ethanol or salt Do not let the flow through touch the column outlet after the second wash using Wash Buffer RA3 Be sure to centrifuge at the corresponding speed for the respective time in order to remove ethanolic Wash Buffer RA3 completely Check if Wash Buffer RA3 has been equilibrated to room Suboptimal temperature before use Washing at lower temperatures p lowers efficiency of salt removal by Wash Buffer RAS performance of RNA in Depending on the robustness of the used RT PCR system downstream RT PCR might be inhibited if complete eluates are used as experiments template for RT PCR Use less eluate as template Stor
14. ications 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 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
15. in connection with the sale or the failure of MACHEREY NAGEL products to perform in accordance with the stated specifications This warranty is exclusive and MACHEREY NAGEL makes no other warranty expressed or implied The warranty provided herein and the data specifications and descriptions of this MACHEREY NAGEL product appearing in MACHEREY NAGEL published catalogues and product literature are MACHEREY NAGEL s sole representations concerning the product and warranty No other statements or representations written or oral by MACHEREY NAGEL s employees agent or representatives except written statements MACHEREY NAGEL 03 2012 Rev 02 19 RNA clean up 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 warraniy 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
16. n For further information please see Material Safety Data Sheets www mn net com Weiterf hrende Informationen finden Sie in den Sicherheitsdatenbl ttern www mn net com MACHEREY NAGEL 03 2012 Rev 02 11 NucleoSpin RNA Clean up XS 5 Protocols 5 1 RNA clean up and concentration of RNA Before starting the preparation Check if Buffer RCU and Buffer RA3 were prepared according to section 3 1 Sample preparation Provide up to 300 uL sample containing up to 90 ug RNA such as prepurified RNA e g phenol purified or RNA from reaction mixtures e g labelling reactions in a microcentrifuge tube not provided For appropriate sample amounts see section 2 3 Note Fill up RNA samples smaller than 100 L L with RNase free water to 100 uL RNA samples from 100 200 uL should be filled up with RNase free water to 200 uL 2 Adjust RNA binding conditions 1 vol RCU Add one volume of Buffer RCU to the sample e g 100 uL RCU to 100 uL sample and mix 2 x 5 s If Mix necessary spin down gently approx 1 s at 1 000 x g 2x 5s to clean the lid 3 Bind RNA Take one NucleoSpin RNA XS Column light blue ring placed in a Collection Tube for each preparation Load up to 300 pL sample mix to the column Centrifuge for 30 s at 11 000 x g es volumes exceeding 300 HL load the sample mix in Ss Load sample wo subsequent centrifugation steps onto the column H mi Place the column in a new Collecti
17. ntaining pure RNA 510 ng uL enrichment of factor 51 MACHEREY NAGEL in house data The RNA recovery rate is typically 85 95 High quality RNA RNA Integrity Number RIN gt 9 according to Agilent 2100 Bioanylzer assays can be obtained from high quality RNA samples The RIN of the processed sample is typically equal 0 3 to the RIN of the input sample RNA quality always depends on the sample quality see section 6 3 for further aspects The NucleoSpin RNA Clean up XS kit allows clean up and concentration of RNA with an A go ggo ratio generally exceeding 1 9 measured in TE buffer pH 7 5 Due to the high RNA purity large amounts of eluates can be used as template in RT PCR without inhibition e g 8 uL of 10 uL eluates as template in a 20 uL qRT PCR setup generating stronger signal compared to reactions 6 MACHEREY NAGEL 03 2012 Rev 02 RNA clean up with less template in a LightCycler PCR with the Sigma SYBR Green Quantitative RT PCR Kit Table 1 Kit specifications at a glance Parameter NucleoSpin RNA Clean up XS Technology Silica membrane technology Format Mini spin columns XS design Sample material lt 300 uL RNA solution containing lt 90 ug RNA Fragment size gt 200 nt Typical recovery 85 95 Asso Aaso 1 9 2 1 Elution volume 5 30 uL Preparation time Approx 20 min 6 preps Binding capacity 110 ug 2 3 Handling preparation and storage of starting materials RNA intended
18. on Tube 2 mL Maximal loading capacity of NucleoSpin RNA XS Columns cS n N 9 is 600 uL However for maximum performance loading at most 300 uL onto the column for one centrifugation step is recommended For larger volumes load the sample mix in two or more if necessary successive centrifugation steps Repeat the procedure if larger volumes are to be processed For high demanding applications the recovery rate can further be increased as follows Centrifuge 30 s at 2 000 x g prior to centrifugation for 30 s at 11 000 x g 12 MACHEREY NAGEL 03 2012 Rev 02 NucleoSpin RNA Clean up XS 4 Wash and dry silica membrane 1 wash 400 pL RA3 Add 400 uL Buffer RA3 to the NucleoSpin RNA XS 11 000 x g Column Centrifuge for 30 s at 11 000 x g Discard flow 30s through and place the column back into the Collection Tube Add 200 pL Buffer RA3 to the NucleoSpin RNA XS e gt Column Centrifuge for 2 min at 11 000 x g to dry the 200 uL RAS membrane Place the column into a nuclease free Collection Tube 1 5 mL supplied gaa 9 min If for any reason the liquid level in the Collection Tube has reached the NucleoSpin RNA XS Column after centrifugation discard flow through and centrifuge again 5 Elute RNA Elute the RNA in 10 pL RNase free H O supplied and 10 pL centrifuge at 11 000 x g for 30 s RNase free If higher RNA concentrations or higher elution volumes H20 are desired elution volum
19. tions and storage conditions see section 3 4 MACHEREY NAGEL 03 2012 Rev 02 RNA clean up 1 2 Reagents consumables and equipment to be supplied by user Reagents 96 100 ethanol to prepare Wash Buffer RA3 and to adjust RNA binding conditions Consumables 1 5 mL microcentrifuge tubes Sterile RNase free tips Equipment Manual pipettors Vortex mixer Centrifuge for microcentrifuge tubes Personal protection equipment e g 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 Clean up XS 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 03 2012 Rev 02 5 RNA clean up 2 Product description 2 1 The basic principle A major aspect of RNA clean up is preventing degradation of the RNA during the clean up procedure The NucleoSpin RNA Clean up XS method achieves this by mixing the crude RNA extract with a binding buffer containing chaotropic ions and ethanol This buffer immediately ina
20. to be used as sample for the NucleoSpin RNA Clean up XS procedure should be handled with the same care as any RNA sample The stability of prepurified RNA samples e g RNA isolated with phenol based protocols depends very much on the performed procedure Wear gloves at all times during the preparation Change gloves frequently MACHEREY NAGEL 03 2012 Rev 02 T RNA clean up 2 4 Elution procedures A high RNA concentration in the elution fraction is desirable for all typical downstream applications In particular with regard to limited volumes of reaction mixes high RNA concentration can be a crucial criterion Due to a high default elution volume standard kits often result in low concentrated RNA if only small samples are processed Such RNA often even requires a subsequent concentration to be suitable for the desired application In contrast to standard kits NucleoSpin RNA Clean up XS allows an efficient elution in avery small volume resulting in highly concentrated RNA Elution volumes in the range of 5 30 uL are recommended the default volume is 10 uL 2 5 Stability of isolated RNA Eluted RNA should immediately be put and always kept on ice during work for optimal stability Contamination with almost omnipresent RNases general lab ware fingerprints dust may be a risk for isolated RNA For short term storage freeze at 20 C for long term storage freeze at 70 C 8 MACHEREY NAGEL 03 2012 Rev 02 RN
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