NucleoSpin® RNA/DNA Buffer Set - MACHEREY
Contents
1. RNA and DNA purification User manual NucleoSpin RNA DNA Buffer Set May 2014 Rev 08 MACHEREY NAGEL MN www mn net com RNA and DNA purification Protocol at a glance Rev 08 1 Homogenize NucleoSpin RNA miRNA RNA Blood RNA Plant NucleoSpin RNA Protein NucleoSpin RNA XS sample So Sample Sample 2 Lyse cells 350 uL RA1 RAP or RP1 100 uL RA1 y 3 5 uL reducing agent 2 uL TCEP Mix Mix 5 uL Carrier RNA 3 Filtrate lysate cS 11 000 x g 11 000 x g 1 min 30s 4 Adjust RNA 350 uL 70 ethanol 100 uL 70 ethanol binding condi tions Mix Mix 5 Bind RNA DNA Load lysate Load lysate i cS 11 000 x g 11 000 x g 30s 30s A p Wash silica 48 wash 500 pL DNA Wash 400 uL DNA Wash membrane 2 4 wash 500 uL DNA Wash 400 uL DNA Wash 11 000 x g 11 000 x g 1 min 1 min B Dry membrane RT 3 min RT 3 min C Elute DNA 100 uL DNA Elute 80 uL DNA Elute NucleoSpin RNA DNA Buffer Set MACHEREY NAGEL GmbH amp Co KG Neumann Neander Str 6 8 52355 D ren Germany Tel 49 24 21 969 270 Fax 49 24 21 969 199 tech bio mn net com www mn net com 11 000 x g 1 min es 11 000 x g 11 000 x g 1 min 1 min 7 Digest DNA 95 uL DNase 25 uL DNase reaction mixture reaction mixture RT 15 min RT 15 min 8 Wash and dry 1 wash 200 uL RA2 100 uL RA2 silica membrane 2 wash 600 pL RA3 400 pL RA3 3 wash 250 uL RA3 200 uL RA3 1 11 000 x2. It is strongly recommended reading the detailed protocol sections of this user manual if the NucleoSpin RNA DNA Buffer Set is used in combination with NucleoSpin RNA REF 740955 NucleoSpin miRNA REF 740971 NucleoSpin RNA Blood REF 740200 NucleoSpin RNA Plant REF 740949 NucleoSpin RNA Protein REF 740933 or NucleoSpin RNA XS REF 740902 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 For preparation of working solutions and storage conditions see section 3 4 MACHEREY NAGEL 05 2014 Rev 08 RNA and DNA purification 2 Product description 2 1 The basic principle The NucleoSpin RNA DNA Buffer Set is intended to be used with one of the following RNA purification kits NucleoSpin RNA NucleoSpin miRNA NucleoSpin RNA Blood REF 740200 NucleoSpin RNA Plant NucleoSpin RNA Protein or NucleoSpin RNA XS The combination the NucleoSpin RNA DNA Buffer Set with either of the RNA purification kits enables the isolation of RNA and DNA from one undivided sample with one single NucleoSpin RNA Binding Column This patented technology enables successive elution of DNA and RNA from a NucleoSpin Column with low salt buffer and water res
3. protocols Check if Wash BufferRA3 has been equilibrated to room temperature before use Washing at lower temperatures lowers efficiency of salt removal by Wash Buffer RA3 Suboptimal performance of DNA in downstream applications Divalent cations Eluted DNA contains small amounts of divalent cations If the downstream application comprises for example 50 DNA eluate of the final reaction volume the divalent cations introduced into the reaction by the DNA eluate may alter the performance Decrease the divalent cation concentration of the reaction by 1 5 mM for compensation MACHEREY NAGEL 05 2014 Rev 08 11 RNA and DNA purification Sample amount too large Depending on the type of sample and its DNA content Low DNA yield for large sample DNA yield may not increase proportional with increased sample amount Sample amounts larger than for example amounts 5 mg tissue or 10 cultured cells may yield less DNA than smaller sample amounts Use smaller sample to ensure good correlation between sample amount and DNA yield 6 2 Ordering information Product REF Pack of NucleoSpin RNA DNA Buffer Set NucleoSpin RNA NucleoSpin miRNA NucleoSpin RNA Blood NucleoSpin RNA Plant NucleoSpin RNA Protein NucleoSpin RNA XS NucleoSpin TriPrep Buffer RA1 Buffer RA1 Buffer RP1 Buffer RP1 rDNase Set NucleoSpin Filters NucleoSpin 96 RNA Filter Plate Collection Tubes 2
4. NucleoSpin RNA Blood NucleoSpin RNA Plant NucleoSpin RNA Protein or NucleoSpin RNA XS protocol 10 MACHEREY NAGEL 05 2014 Rev 08 RNA and DNA purification 6 Appendix 6 1 Troubleshooting Problem Possible cause and suggestions DNA is Buffer temperature contaminated DNA elution buffer DNA Elute exceeded 30 C during with RNA application Use DNA Elute with a temperature preferentially of 18 25 C DNA yield lower than RNA yield Sample material DNA and RNA yield depend very much on sample material Ratio of RNA yield to DNA yield may vary from approximately 1 20 DNA degrades upon storage DNase contamination DNA elution buffer DNA Elute does not contain divalent cations complexing substances e g EDTA Therefore DNA is not protected against DNases Keep DNA Elute solution clean and avoid any contamination As a precaution keep DNA on ice for short term or at 20 C for long term storage Some sample materials may contain remaining DNase traces that are not sufficiently washed away by the standard procedure Perform a wash step of the column with Buffer RA2 after loading the lysate onto the column and before starting the washing steps with DNA Wash solution Add 500 uL Buffer RA2 onto the column centrifuge 1 min at 11 000 x g and continue with DNA Wash washing steps Low RNA yield or quality See general protocol See troubleshooting section of individual NucleoSpin
5. Wash EIS Add again 500 pL DNA Wash and centrifuge 1 min at 11 000 x g 11 000 x g Discard Collection Tube with flow through 1 min If using NucleoSpin RNA XS add only 400 uL DNA Wash MACHEREY NAGEL 05 2014 Rev 08 9 NucleoSpin RNA DNA Buffer Set B Dry membrane Insert the NucleoSpin RNA Binding Column into a new 1 5 mL microcentrifuge tube not supplied Open the lid of the NucleoSpin RNA Binding Column and let it stand for 3 minutes The procedure ensures complete removal of ethanol from the column C Elute DNA Add 100 uL DNA Elute DNA elution buffer directly onto the membrane and incubate 1 min Elute the DNA by centrifuging for 1 min at 11 000 x g If using NucleoSpin RNA XS add only 80 uL DNA Elute for elution The temperature of the DNA Elute solution shall not exceed 30 C otherwise RNA will partly elute with the DNA Elute solution DNA Elute solution may stay for 1 min up to 15 min on the column before DNA is eluted A 1 5 min incubation time is recommended Eluted DNA is immediately ready for downstream applications without further purification Incubate for 3 min 11 000 x g El Add 100 pL DNA Elute lt 1 min Proceed with the digestion of residual on column DNA according to the individual NucleoSpin RNA protocols step Digest DNA Add DNase reaction mixture onto the column and perform all subsequent steps as described in the NucleoSpin RNA NucleoSpin miRNA
6. g 11 000 x g and 2 e gt 30 s 30 s 11 000 x g 11 000 x g rd 3 2 min 2 min 9 Elute highly B pen z 60 uL RNase free HO 10 uL RNase free H O o 6 11 000 x g 30 s RNA and DNA purification Table of contents 1 Components 4 1 1 Set contents 4 1 2 Consumables and equipment to be supplied by user 4 1 3 About this user manual 4 2 Product description 5 2 1 The basic principle 5 2 2 Kit specifications 3 Storage conditions and preparation of working solutions 8 4 Safety instructions 8 5 Protocol isolation of RNA and DNA from one undivided sample 9 6 Appendix 11 6 1 Troubleshooting 11 6 2 Ordering information 12 6 3 Product use restriction warranty 13 MACHEREY NAGEL 05 2014 Rev 08 3 RNA and DNA purification 1 Components 1 1 Set contents NucleoSpin RNA DNA Buffer Set 100 preps REF 740944 Buffer DNA Wash Concentrate 2x12 mL Buffer DNA Elute 12 mL User manual 1 1 2 Consumables and equipment to be supplied by user The content of this set is sufficient for 100 DNA isolations in combination with RNA isolations performed with the following kits NucleoSpin RNA REF 740955 NucleoSpin miRNA REF 740971 NucleoSpin RNA Blood REF 740200 NucleoSpin RNA Plant REF 740949 NucleoSpin RNA Protein REF 740933 NucleoSpin RNA XS REF 740902 Additional collection tubes are required and are not supplied see ordering information 1 3 About this user manual
7. GEL 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 MACHEREY NAGEL 05 2014 Rev 08 13 RNA and DNA purification 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
8. L products MACHEREY NAGEL does not warrant the correctness of any of those applications Please contact MACHEREY NAGEL GmbH amp Co KG Tel 49 24 21 969 270 tech bio mn net com 14 MACHEREY NAGEL 05 2014 Rev 08
9. e amount and nucleic acid yield 0 08 1 25 mg 0 08 2 5 mg 0 08 5 mg mouse liver mouse liver mouse liver RNA gt 0 98 gt 0 98 gt 0 98 DNA gt 0 99 gt 0 95 gt 0 67 DNA size and quality Isolated genomic DNA is commonly of high molecular weight gt 20 kb DNA is commonly stable even at 37 C for 2h with or without addition of a typical restriction enzyme buffer DNA is digestable with restriction enzymes DNA is suitable for PCR MACHEREY NAGEL 05 2014 Rev 08 RNA and DNA purification 3 Storage conditions and preparation of working solutions Store solutions at room temperature 18 25 C The DNA Wash solution is delivered as a concentrate To prepare the final DNA Wash solution add four volumes of ethanol 50 to the DNA Wash Concentrate add 90 mL 50 ethanol to 22 5 mL DNA Wash Concentrate Due to its composition DNA Elute DNA elution buffer does not inhibit DNases i e DNA Elute does not contain substances e g EDTA to complex divalent cations Therefore make sure not to contaminate DNA Elute with DNases Further due to its composition DNA Elute does not inhibit microbial growth Therefore make sure not to contaminate DNA Elute with any source of microbial contaminants NucleoSpin RNA DNA Buffer Set 100 preps REF 740944 2x12 mL Bufer ONA Wash Concentrate Add 48 mL ethanol 50 to each bottle 4 Safety instructions The NucleoSpin RNA DNA Buf
10. fer Set is intended to be used in conjunction with NucleoSpin RNA kits The NucleoSpin RNA DNA Buffer Set does not contain hazardous contents However pay attention to the safety instructions of the individual NucleoSpin RNA kits 8 MACHEREY NAGEL 05 2014 Rev 08 NucleoSpin RNA DNA Buffer Set 5 Protocol isolation of RNA and DNA from one undivided sample Before starting the procedure Check if Buffer DNA Wash was prepared according to section 3 Perform sample homogenization cell lysis lysate filtration adjusting of nucleic acid binding conditions and binding of nucleic acids to the NucleoSpin RNA Binding Column according to the NucleoSpin RNA NucleoSpin miRNA NucleoSpin RNA Blood REF 740200 NucleoSpin RNA Plant NucleoSpin RNA Protein or NucleoSpin RNA XS kit standard protocol Subsequent to binding of nucleic acids to the column continue as follows with step A the membrane desalting step of the individual NucleoSpin RNA protocols is replaced by steps A C A Wash silica membrane 500 pL Add 500 pL DNA Wash to the NucleoSpin RNA Binding DNA Wash Column and centrifuge for 1 min at 11 000 x g Discard flow through and reuse Collection Tube 11 000 x g 1 min If using NucleoSpin RNA XS add only 400 uL DNA Wash The DNA Wash solution is used instead of MDB Membrane Desalting Buffer from the NucleoSpin RNA kits MDB will not be used in this procedure 500 pL DNA
11. 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 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 NAGE
12. mL DISTRIBUTION AND USE OF NUCLEOSPIN RNA DNA BUFFER SET and NUCLEOSPIN TRIPREP IN THE USA IS PROHIBITED FOR PATENT REASONS 740944 740955 10 50 250 740971 10 50 250 740200 10 50 740949 10 50 250 740933 10 50 250 740902 10 50 250 740666 10 50 250 740961 740961 500 740934 50 740934 500 740963 740606 740711 740600 100 preps 20 50 250 preps 10 50 250 preps 10 50 preps 10 50 250 preps 10 50 250 preps 10 50 250 preps 10 50 250 preps 50 mL 500 mL 50 mL 500 mL 1 set 50 4 plates 1000 12 MACHEREY NAGEL 05 2014 Rev 08 RNA and DNA purification Visit www mn net com for more detailed product information 6 3 Product use restriction warranty NucleoSpin RNA DNA Buffer Set 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 impr
13. oper 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 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 NA
14. pectively DNA and RNA are immediately ready for downstream applications Samples are lysed in the lysis buffer supplied in the NucleoSpin RNA kits Lysis Buffer RA1 RAP or RP1 Ethanol is added to facilitate conditions for binding of nucleic acids to the NucleoSpin RNA Binding Column After wash steps DNA and RNA are eluted sequentially DNA is eluted with a low salt solution DNA Elute which selectively elutes DNA and keeps RNA on the column Eluted DNA is immediately ready for downstream applications without further purification DNA eluted with DNA Elute may readily serve as template for PCR is restrictable with restrictions enzymes and is of high molecular weight gt 20 kb A A ratios of eluted DNA are within a range from 1 7 2 0 280 After DNA elution residual on column DNA is digested on the NucleoSpin Column as described in the relating NucleoSpin RNA protocol After additional washing steps pure RNA is eluted with RNase free water DNA elution prior to RNA elution does neither compromise RNA quality nor quantity Sequential DNA and RNA isolation from one sample with this support set and NucleoSpin RNA kits has been successfully performed with various sample materials e g HeLa cells pig liver kidney and spleen parsley leaf maize leaf and root The standard protocol section 5 allows the purification of DNA and RNA from a variety of sample types Suitable sample types are described in the respective user manual
15. s of the NucleoSpin RNA kits MACHEREY NAGEL 05 2014 Rev 08 5 RNA and DNA purification 2 2 Kit specifications Typical yields of RNA and DNA 100 RNA DNA RNA yield ug DNA 0 1 1 ros osr 1 je ee er E E 10 000 100 000 1 000 000 HeLa cell number Figure 1 DNA and RNA yield from different amounts of HeLa cells Different amounts of HeLa cells were used as sample material DNA and RNA were isolated with the NucleoSpin RNA DNA Buffer Set in combination with the NucleoSpin RNA kit DNA and RNA were isolated as described in Figure 1 Obtained correlation coefficients between sample amount and RNA and DNA yield are shown in Table 1 Table 1 Correlation between sample amount and nucleic acid yield 3 x 104 5 x 10 cells 3 x 104 1 x 10 cells RNA gt 0 98 gt 0 98 DNA gt 0 99 gt 0 95 6 MACHEREY NAGEL 05 2014 Rev 08 RNA and DNA purification RNA DNA RNA yield yg DNA 0 01 0 1 1 10 Mouse liver mg Figure 2 DNA and RNA yield from different amounts of mouse liver tissue Different amounts of mouse liver tissue were used as sample material DNA and RNA were isolated with the NucleoSpin RNA DNA Buffer Set in combination with the NucleoSpin RNA kit DNA and RNA were isolated as described in Figure 2 Obtained correlation coefficients between sample amount and RNA and DNA yield are shown in Table 2 Table 2 Correlation between sampl
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