NucleoZOL - MACHEREY
Contents
1. For samples containing 100 mg tissue mL NucleoZOL 15 min incubation at room temperature is recommended Centrifuge samples for 15 min at 12 000 x g Centrifugation can be performed at 4 28 C A semi solid pellet containing DNA proteins and polysaccharides forms at the bottom of the tube The RNA is still solubilized in the supernatant Precipitate large RNA Pipette 1 mL of the supernatant to a new tube Leave a layer of the supernatant on top of the precipitate Add 400 pL 75 ethanol to 1 mL supernatant for precipitation of the RNA Incubate samples at room temperature for 10 min Centrifuge the samples for 8 min at 12 000 x g A white pellet containing the RNA will be formed at the bottom of the tube Transfer the supernatant containing the small RNA to a new tube and store it at 4 C or at 20 C The small RNA containing supernatant can be stored at 20 C for one year Precipitate small RNA Add 0 8 volumes of isopropanol to the supernatant 1 4 mL obtained after precipitation of RNA step 3 Incubate the samples for 30 min at 4 C Centrifuge the sample for 15 min at 12 000 x g Centrifugation can be performed at 4 28 C Precipitated RNA will form a white pellet at the bottom of the tube MACHEREY NAGEL 07 2015 Rev 01 13 NucleoZOL Wash RNA Add 400 600 uL 75 ethanol when working with 1 5 mL tubes For larger tubes add 500 uL 75 ethanol per 1 mL supernatant used for precipitat2. RNA isolation User manual NucleoZOL July 2015 Rev 01 MACHEREY NAGEL www mn net com NucleoZOL Table of contents 1 Components 4 1 1 Kit contents 4 1 2 Reagents consumables and equipment to be supplied by the user 4 1 3 RNase free work environment 5 1 4 About this user manual 5 2 Product description 6 2 1 The basic principle 6 2 2 Product specifications 7 2 3 Handling preparation and storage of starting materials 8 2 4 RNA reconstitution 8 Storage conditions and preparation of working solutions 9 4 Safety instructions 10 5 NucleoZOL protocols 12 5 1 Isolation of small and large RNA in two separate fractions 12 5 2 Isolation of total RNA 15 6 Appendix 18 6 1 Digestion of residual DNA solution 18 6 2 Troubleshooting 19 6 3 Ordering information 20 6 4 Product use restriction warranty 21 MACHEREY NAGEL 07 2015 Rev 01 3 NucleoZOL 1 Components 1 1 Kit contents 740404 200 NucleoZOL reagent 1 2 Reagents consumables and equipment to be supplied by the user Reagents RNase free water 75 ethanol 70 isopropanol 100 isopropanol 4 bromoanisole optional see p 16 Consumables 1 5 mL 2 0 mLor 15 mL centrifuge tubes depending on the amount of sample to be processed per preparation Sterile RNase free tips Equipment Manual pipettors Vortex mixer Centrifuge for microcentrifuge tubes Equipment for sample disruption and homogenization Personal protecti
3. C or 70 C for at least one year for later use Cells grown in monolayer Remove cell culture medium and lyse cells by addition of at least 1 mL NucleoZOL to the culture disk diameter 3 5 cm 10 cm Ensure complete lysis by repeated pipetting Calculate the amount of reagent based on culture dish area not on cell number An insufficient volume of the reagent will lead to DNA contamination of the isolated RNA Cells grown in suspension Sediment cells and lyse directly by the addition of NucleoZOL Add at least 1 mL NucleoZOL per 10 cells and lyse cells by pipetting up and down several times Do not wash the cells before addition of NucleoZOL Washing of cells might contribute to RNA degradation Liquid samples Add 1 mL NucleoZOL per 400 uL liquid sample for homogenization and lysis For processing sample volumes smaller than 400 uL add 1 mL of NucleoZOL and add RNase free water to a final volume of 1 4 mL Fatty samples Homogenize lipid rich samples as described above Centrifuge the samples for 5 min at 12 000 x g After centrifugation a fat layer appears on top of the sample Pierce the upper layer with a syringe pipette tip and transfer the supernatant into a new tube 12 MACHEREY NAGEL 07 2015 Rev 01 NucleoZOL Precipitate contaminants Add 400 pL RNase free water per 1 mL NucleoZOL to the lysate Shake the sample vigorously for 15 s Incubate at room temperature 18 25 C for 5 15 min
4. 12 000 x g 4 25 C Residual DNA proteins and polysaccharides accumulate in the organic phase at the bottom of the tube RNA is still solubilized in the supernatant 4 Precipitate total RNA Pipette RNA containing supernatant from step 2 or 3 into a fresh tube Add 1 mL of isopropanol per 1 mL supernatant in order to precipitate RNA Incubate samples at room temperature for 10 min Centrifuge samples for 10 min at 12 000 x g Typically RNA is obtained as a white pellet at the bottom of the tube For spleen samples RNA forms a gel like membrane on the bottom of the tube Upon washing with ethanol the membrane becomes more visible 16 MACHEREY NAGEL 07 2015 Rev 01 NucleoZOL Wash RNA Use 400 600 uL 75 ethanol when precipitating in 1 5 mL tubes For larger tubes add 500 pL 75 ethanol per 1 mL supernatant Centrifuge the pellets for 1 3 min at 4 000 8 000 x g Remove ethanol from the pellet by pipetting Repeat the ethanol washing step Do not dry the RNA pellet between wash steps Drying the RNA pellet may lead to a decrease in solubility Reconstitute RNA Dissolve the RNA pellet in RNase free water to obtain an RNA concentration of 1 2 ug uL Vortex the sample 2 5 min at room temperature for efficient solubilization For accurate determination of RNA concentration by OD measurement with a cuvette dilute RNA in RNase free water with a slightly alkaline pH 1 mM NaOH or buffer with a pH gt 8 e
5. fraction or in separate fractions from a variety of sample materials such as cells tissue and liquids from human or animal origin plants yeast bacteria viral materials and other sources One of the most important factors during the isolation of RNA is to prevent degradation First cells and tissues are lysed and homogenized in NucleoZOL reagent based on guanidinium thiocyanate and phenol Contaminating molecules such as DNA polysaccharides and proteins are precipitated by the addition of water and removed by centrifugation The NucleoZOL procedure allows the separate isolation of small and large RNA by adding ethanol and isopropanol respectively RNA can be reconstituted by RNase free water A chloroform induced phase separation is not necessary for high quality RNA isolation The RNA is ready for use in qRT PCR microarrays RNase protection assays poly A isolation blotting and other applications 6 MACHEREY NAGEL 07 2015 Rev 01 NucleoZOL 2 2 Product specifications Table 1 Product specifications at a glance Technology One phase extraction Sample material lt 1x 107 cultured cells bacteria and yeast por mE NucleoZoL lt 100 mg human animal plant tissue lt 0 4 mL viral fluids Fragment size Small RNA 10 200 nt large RNA gt 200 nt Typical yield total RNA Liver 6 8 ug mg tissue Kidney spleen 3 4 ug mg tissue Muscle brain lung 0 5 1 5 ug mg Cultured cells 4 10 ug 10 cells Typical
6. g Elution Buffer AE see ordering information section 6 3 Distilled water typically has a pH lt 7 MACHEREY NAGEL 07 2015 Rev 01 17 NucleoZOL 6 Appendix 6 1 Digestion of residual DNA solution NucleoZOL efficiently removes DNA when processing samples according to the standard protocol resulting in minimal residual DNA in the purified RNA Residual DNA will not be detectable in most downstream applications If large samples or samples with high levels of DNA are processed it may be difficult to remove all traces of DNA The amount of residual DNA depends on the sample type amount DNA content and the detection sensitivity of the method used to analyze residual DNA A typical example is an RT PCR reaction in which the primer molecules do not differentiate between cDNA derived from RNA and contaminating genomic DNA The effect is prominent 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 lt 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 High quality RNase free recombinant rDNase REF 740963 see ordering information 6 3 facilitates such a digestion in solution in order
7. 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 Trademarks patents disclaimer NucleoSpin is a registered trademark of MACHEREY NAGEL GmbH amp Co KG RNA ater is a trademark of Ambion Inc NucleoZOL reagent US Patents 7 794 932 and 8 367 817 B2 and international patents 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 22 MACHEREY NAGEL 07 2015 Rev 01
8. to remove traces of contaminating DNA 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 Gently swirl the tube in order to mix the solution Spin down gently approx 1s at 1 000 x g to collect every droplet of the solution at the bottom of the tube B Incubate sample Incubate for 10 min at 37 C C Repurify RNA Repurify RNA with a suitable RNA cleanup procedure NucleoSpin RNA Clean up NucleoSpin RNA Clean up XS kits see ordering information 6 3 or by ethanol precipitation Ethanol precipitation exemplary Add 0 1 volume of 3M 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 if the sample contains 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 water 18 MACHEREY NAGEL 07 2015 Rev 01 NucleoZOL 6 2 Troubleshooting Problem Insufficient yield Possible cause and suggestions Homogenization or sample Iysis is incomplete Improve homogenization by testing more stringent conditions Solubilization
9. able consequential or special including but not limited to loss of use revenue or profit whether based upon warranty contract tort including negligence or strict liability arising in connection with the sale or the failure of MACHEREY NAGEL products to perform in accordance with the stated specifications This warranty is exclusive and MACHEREY NAGEL makes no other warranty expressed or implied The warranty provided herein and the data specifications and descriptions of this MACHEREY NAGEL product appearing in MACHEREY NAGEL published catalogues and product literature are MACHEREY NAGEL s sole representations concerning the product and warranty No other statements or representations written or oral by MACHEREY NAGEL s employees agent or representatives except written statements signed by a duly authorized officer of MACHEREY NAGEL are authorized they should 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
10. ake off immediately all contaminated clothing and wash it before reuse Alle kontaminierten Kleidungsst cke sofort ausziehen und vor erneutem Tragen waschen Store locked up Unter Verschluss aufbewahren Dispose of contents container to Inhalt Beh lter zuf hren 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 A The symbol shown on labels refers to further safety information in this section Das auf Etiketten dargestellte Symbol weist auf weitere Sicherheitsinformationen dieses Kapitels hin MACHEREY NAGEL 07 2015 Rev 01 11 NucleoZOL 5 5 1 NucleoZOL protocols Isolation of small and large RNA in two separate fractions Please note that RNA is separated in small RNA 10 200 nt and large RNA gt 200 nt in two fractions following this protocol 1 Homogenize and lyse sample Homogenize tissue samples with a rotor stator homogenizer or another mechanical disruption device using up to 100mg of tissue per 1mL NucleoZOL For tissues with high DNA content e g spleen it is recommended to use 50 mg of tissue mL reagent For simplicity this protocol describes RNA isolation using 1 mL of NucleoZOL For processing of samples in 1 5 mL or 2 mL microcentrifuge tubes use an 880 uL aliquot of the homogenate 80 mg tissue 800 uL NucleoZOL Residual homogenate can be stored at 20
11. dering information section 6 3 to approach an RNA concentration of approximately 1 2 ug uL for the large RNA fraction and approximately 0 1 ug uL for the small RNA fraction MACHEREY NAGEL 07 2015 Rev 01 NucleoZOL 3 Storage conditions and preparation of working solutions Attention NucleoZOL contains phenol corrosive liquid poison and guanidium thiocyanate irritant Wear gloves and eye protection CAUTION Read the warning note on the container and MSDS NucleoZOL contains phenol and guanidinium thiocyanate which CAUSES BURNS and can be fatal When working with NucleoZOL use gloves and eye protection face shield safety goggles Do not get the reagent on skin or clothing Avoid breathing fumes In case of contact Immediately flush eyes or skin with a large amount of water for at least 15 minutes and if necessary seek medical attention NucleoZOL can be stored at room temperature 18 25 C and is stable for at least one year MACHEREY NAGEL 07 2015 Rev 01 9 NucleoZOL 4 Safety instructions NucleoZOL contains hazardous contents Wear gloves and goggles and follow the safety instructions given in this section GHS classification Only harmful features do not need to be labeled with H and P phrases up to 125 mL or 125 g Mindergef hrliche Eigenschaften m ssen bis 125 mL oder 125 g nicht mit H und P S tzen gekennzeichnet werden GHS Hazard Precaution Component Hazard contents symbol phrase
12. einholen Do not handle until all safety precautions have been read and understood Vor Gebrauch alle Sicherheitshinweise lesen und verstehen Do not breathe vapors Dampf nicht einatmen Avoid release to the environment Freisetzung in die Umwelt vermeiden Wear protective gloves eye protection Schutzhandschuhe Augenschutz tragen IF SWALLOWED Immediately call a POISON CENTER doctor BEI VERSCHLUCKEN Sofort GIFTINFORMATIONSZENTRUM Arzt anrufen IF SWALLOWED Rinse mouth Do NOT induce vomiting BEI VERSCHLUCKEN Mund aussp len KEIN Erbrechen herbeif hren IF ON SKIN Wash with plenty of water BEI BERUHRUNG MIT DER HAUT Mit viel Wasser waschen IF ON SKIN or hair Take off immediately all contaminated clothing Rinse skin with water shower BEI BER HRUNG MIT DER HAUT oder dem Haar Alle kontaminierten Kleidungsst cke sofort ausziehen Haut mit Wasser abwaschen duschen IF INHALED Remove person to fresh air and keep comfortable for breathing BEI EINATMEN Die Person an die frische Luft bringen und f r ungehinderte Atmung sorgen IF IN EYES Rinse cautiously with water for several minutes BEI BER HRUNG MIT DEN AUGEN Einige Minuten lang behutsam mit Wasser sp len IF exposed or concerned Get medical advice attention BEI Exposition oder falls betroffen Arztlichen Rat einholen rztliche Hilfe hinzuziehen Call a POISON CENTER doctor GIFTINFORMATIONSZENTRUM Arzt anrufen T
13. ion Centrifuge for 1 3 min at 4 000 8 000 x g Remove ethanol using a micropipette Repeat washing step Do not dry the pellet Add 400 600 pL 70 isopropanol when working with 1 5 mL tubes For larger tubes add 500 pL 70 isopropanol per 1 mL supernatant used for precipitation Centrifuge for 1 3 min at 4 000 8 000 x g Remove ethanol using a micropipette Repeat washing step Do not dry the pellet Drying the RNA pellet may lead to a decrease in solubility Reconstitute RNA Dissolve the RNA pellet in RNase free water to obtain an RNA concentration of 1 2 ug uL for the large RNA fraction and about 0 1 ug L for the small RNA fraction Vortex the sample for 2 5 min at room temperature for efficient solubilization For accurate determination of RNA concentration by OD measurement with a cuvette dilute RNA in RNase free water with a slightly alkaline pH 1 mM NaOH or buffer with a pH gt 8 e g Elution Buffer AE see ordering information 6 3 Distilled water typically has a pH lt 7 Note The large RNA fraction contains RNA gt 200 nt and contains 80 85 of cellular RNA 14 MACHEREY NAGEL 07 2015 Rev 01 NucleoZOL 5 2 Isolation of total RNA Total RNA including small RNA e g miRNA is isolated with the following protocol 1 Homogenization Homogenize tissue samples with a rotor stator homogenizer or another mechanical disruption device using up to 100 mg of tissue per 1 mL NucleoZOL Fo
14. of the RNA pellet is incomplete Increase volume of RNase free water for dissolving of RNA and prolong mixing for solubilization Ratio A260 280 lt 1 6 Volume of NucleoZOL used for homogenization was too low Increase volume of NucleoZOL Low pH during spectrophotometric quantification Use Buffer AE 5 mM Tris pH 8 5 for sample dilution for spectrophotometric quantification RNA pellet was only partly solubilized Increase volume of RNase free water for dissolving of RNA and prolong mixing for solubilization Contamination of polysaccharide or proteoglycan Perform phase separation as described in section 5 2 3 Degraded RNA Inadequate tissue sampling e Make sure to use fresh tissue or flash freeze tissue immediately upon harvest Inappropriate storage conditions Store samples at 70 C Cell dissolution during trypsinization Make sure cells stay intact during trypsinization RNase contaminated solutions or tubes Make sure to work in an RNase free environment MACHEREY NAGEL 07 2015 Rev 01 19 NucleoZOL Problem Contamination with DNA Possible cause and suggestions Volume of NucleoZOL used for homogenization was too low Increase volume of NucleoZOL Sample material contains strong buffers organic solvents alkaline solution or salt The precipitation of DNA step 2 can be improved by the following modification Increase incubation time to 15 min after addition of wa
15. on equipment e g lab coat gloves goggles Well ventilated working environment RNase free working environment 4 MACHEREY NAGEL 07 2015 Rev 01 NucleoZOL 1 3 RNase free work environment The reagent has been tested for functionality However an RNase free working environment is also a critical factor for performing successful RNA isolation and handling Therefore general recommendations to avoid RNase contamination should be followed Maintain a separate area dedicated pipettors and materials when working with RNA Wear gloves when handling RNA and reagents to avoid contact with skin which is a source of RNases Change gloves frequently Use sterile RNase free plastic tubes Tubes for lysate preparation and RNA precipitation have to be supplied by the user Keep all kit components sealed when not in use and all tubes tightly closed when possible 1 4 About this user manual Please read the detailed protocol if using NucleoZOL for the first time Experienced users may refer to the short instruction manual All technical literature is available on the Internet at www mn net com Please contact Technical Service regarding information about changes of to the current user manual compared to the previous revisions MACHEREY NAGEL 07 2015 Rev 01 5 NucleoZOL 2 Product description 2 1 The basic principle NucleoZOL is designed for the isolation of total RNA small and large RNA in a single
16. pped 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 07 2015 Rev 01 21 NucleoZOL 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 foresee
17. r 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 IN 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 IF THERE IS NO IVD SIGN THE PRODUCT SHALL NOT BE SUITABLE FOR IN 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 shi
18. r tissues with high DNA content e g spleen it is recommended to use 50 mg of tissue mL reagent For simplicity this protocol describes RNA isolation using 1 mL of NucleoZOL For processing the sample in 1 5 mL or 2 mL microcentrifuge tubes use an 880 uL aliquot of the homogenate 80 mg tissue 800 uL NucleoZOL Residual homogenate can be stored at 20 C or 70 C for at least one year for later use Cells grown in monolayer Remove cell culture medium and lyse cells by addition of at least 1 mL of NucleoZOL to the culture disk diameter 3 5 cm 10 cm Ensure complete lysis by repeated pipetting Calculate the amount of reagent based on culture dish area not on cell number An insufficient volume of the reagent will lead to DNA contamination of the isolated RNA Cells grown in suspension Sediment cells and lyse directly by the addition of NuceoZOL Add at least 1 mL NucleoZOL per 10 cells and lyse cells by pipetting up and down several times Do not wash the cells before addition of NucleoZOL Washing of cells might contribute to RNA degradation Liquid samples Add 1 mL NucleoZOL per 400 uL liquid sample for homogenization and lysis For processing sample volumes smaller than 400 uL add 1 mL of NucleoZOL and add water to a final volume of 1 4 mL Fatty samples Homogenize lipid rich samples as described above Centrifuge the samples for 5 min at 12 000 x g After centrifugation a fat layer appears on top of the
19. s phrases Inhalt Gefahrstoff GHS Symbol H S tze P S tze NucleoZOL Phenol 30 60 and 301 311 201 202 260 guanidinium thiocyanate amp 314 331 273 280 30 60 OD 341 373 301 310 Phenol 30 60 und 412 301 330 331 Guanidinthiocyanat 30 60 EUHO31 302 352 DANGER 303 361 353 CAS 108 95 2 593 84 0 GEFAHR 3044340 305 351 338 3084313 311 361 364 405 501 Hazard phrases H301 Toxic if swallowed Giftig bei Verschlucken H311 Toxic in contact with skin Giftig bei Hautkontakt H314 Causes severe skin burns and eye damage Verursacht schwere Ver tzungen der Haut und schwere Augensch den H331 Toxic if inhaled Giftig bei Einatmen H341 Suspected of causing genetic defects Kann vermutlich genetische Defekte verursachen H373 May cause damage to organs through prolonged or repeated exposure Kann die Organe sch digen bei l ngerer oder wiederholter Exposition H 412 Harmful to aquatic life with long lasting effects Sch dlich f r Wasserorganismen mit langfristiger Wirkung EUHO31 Contact with acids liberates toxic gas Entwickelt bei Ber hrung mit S ure giftige Gase 10 MACHEREY NAGEL 07 2015 Rev 01 NucleoZOL Precaution phrases P201 P202 P260 P273 P280 P301 310 P301 330 331 P302 352 P303 361 353 P304 340 P305 351 338 P308 313 P311 P361 364 P405 P501 Obtain special instructions before use Vor Gebrauch besondere Anweisungen
20. sample Pierce the upper layer with a syringe pipette tip and transfer the supernatant into a new tube MACHEREY NAGEL 07 2015 Rev 01 15 NucleoZOL 2 Precipitate contaminants Add 400 uL RNase free water per 1 mL NucleoZOL to the Iysate Shake the sample vigorously for 15 s Incubate at room temperature for 5 15 min For samples containing 100 mg tissue mL NucleoZOL 15 min incubation at room temperature is recommended Centrifuge samples for 15 min at 12 000 x g Centrifugation can be performed at 4 28 C A semi solid pellet containing DNA proteins and polysaccharides forms at the bottom of the tube The RNA is still solubilized in the supernatant Transfer 1 mL supernatant 75 of total supernatant volume to a fresh tube Leave a layer of the supernatant above the DNA protein pellet The pellet containing DNA protein and polysaccharides comprises approximately 10 in volume of the total homogenate water mix e g about 8 pellet for 80 mg tissue lysed in 1 mL reagent 3 Phase separation optional The basic protocol for total RNA isolation can be complemented by an optional phase separation This is useful for samples with high DNA content and or extracellular material Add 5 uL 0 5 of supernatant volume 4 bromoanisole to 1 mL transferred supernatant Mix well for 15 s and incubate at room temperature for 3 5 min Do not substitute 4 bromoanisole with bromchloropropane or chloroform Centrifuge for 10 min at
21. ter step 2 Centrifuge at 16 000 x g Use support protocol 6 1 for subsequent rDNase digestion in solution Contamination with proteo glycan fat or polysaccharide Inefficient precipitation of contaminants Centrifuge the initial crude homogenate step 1 for phase separation in an additional step for 10 min at 12 000 x g 6 3 Ordering information Product REF Pack of NucleoZOL 740404 200 200 mL NucleoSpin RNA Clean up XS 740903 10 50 250 10 50 250 preps NucleoSpin RNA Clean up 740948 10 50 250 10 50 250 preps RNase free Water 740378 1000 1000 mL Elution Buffer AE 740917 1 1000 mL rDNase Set 740963 one set 20 MACHEREY NAGEL 07 2015 Rev 01 NucleoZOL 6 4 Product use restriction warranty NucleoZOL 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 ou
22. yield large RNA Liver 5 7 ug mg tissue Kidney spleen 3 4 ug mg tissue Muscle brain lung 0 5 1 5 ug mg Cultured cells 3 8 ug 10 cells A260 280 total RNA 1 8 2 1 Typical RIN RNA integrity number gt 9 Elution volume flexible MACHEREY NAGEL 07 2015 Rev 01 7 NucleoZOL 2 3 Handling preparation and storage of starting materials Sample harvest and RNase inhibition RNA is not protected against digestion until the sample material is flash frozen or disrupted in the presence of RNase inhibiting or denaturing agents Sample harvest methods 2 4 Use freshly harvested sample for immediate lysis and RNA purification Samples can be stored in NucleoZOL after disruption at 20 C to 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 in NucleoZOL should be thawed slowly before starting with the isolation of RNA Flash freeze sample in liquid N immediately upon harvest and store at 70 C Frozen samples are stable up to 6 months Mortar and pestle can be used to pulverize the sample in a frozen state Make sure that the sample does not thaw prior to contact with the reagent Samples can be submerged and stored in RNA stabilizing reagents such as RNAlater Remove excess RNAlater solution from the tissue before processing the sample RNA reconstitution The precipitated RNA can be dissolved in variable volumes of RNase free water see or
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