Total RNA and DNA Purification User Manual
Contents
1. MACHEREY NAGEL GmbH amp Co KG Neumann Neander Str 6 8 D 52355 D ren Germany Tel 49 0 24 21 969 270 www mn net com e mail tech bio mn net com Total 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 risk and safety phrases 8 5 Protocol Isolation of RNA and DNA from one undivided sample 9 6 Appendix 11 6 1 Troubleshooting 11 6 2 Ordering information 13 6 3 Product use restriction warranty 13 MACHEREY NAGEL 10 2008 Rev 05 3 Total RNA and DNA Purification 1 Components 1 1 Set contents NucleoSpin RNA DNA Buffer Set 100 preps Cat No 740944 Buffer DNA Wash Concentrate 22 5 ml Buffer DNA Elute 12 0 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 II Cat No 740955 NucleoSpin RNA Plant Cat No 740949 NucleoSpin RNA Protein Cat No 740933 NucleoSpin RNA XS Cat No 740902 Additional collection tubes are required and are not supplied see ordering informa tion 1 3 About this User Manual It is strongly2. MACHEREY NAGEL 10 2008 Rev 05 11 Total RNA and DNA Purification Suboptimal performance of DNA in downstream applications Divalent cations e Eluted DNA contains small amounts of divalent cations If the downstream application comprises e g 50 DNA elu ate of the final reaction volume the divalent cations intro duced 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 Low DNA yield for large sample amounts Sample amount too large e Depending on the type of sample and its DNA content DNA yield may not increase proportional with increased sample amount Sample amounts larger than e g 5 mg tissue or 108 cultured cells may yield less DNA than smaller sample amounts Use smaller sample to ensure good correlation between sample amount and DNA yield 12 MACHEREY NAGEL 10 2008 Rev 05 Total RNA and DNA Purification 6 2 Ordering information Product Cat No Pack of NucleoSpin RNA II 740955 20 50 250 20 50 250 NucleoSpin RNA Plant 740949 10 50 250 10 50 250 NucleoSpin RNA Protein 740933 10 50 250 10 50 250 NucleoSpin RNA XS 740902 10 50 250 10 50 250 NucleoSpin TriPrep 740666 10 50 250 10 50 250 Buffer RA1 740961 50 ml Buffer RA1 740961 500 500 ml Buffer RP1 740934 50 50 ml Buffer RP1 740934 500 500 ml rDNase Set 740963 1 set NucleoSpin Filters 740606 50 NucleoSpin 96 RNA
3. Filter Plate 740711 4 plates Collection Tubes 2 ml 740600 1000 Visit www mn net com for more detailed product information 6 3 Product use restriction warranty NucleoSpin RNA DNA Buffer Set components were developed designed distrib uted and sold FOR RESEARCH PURPOSES ONLY They are suitable FOR IN VITRO USES ONLY No claim or representation is intended for its use to identify any specific organism or for clinical use diagnostic prognostic therapeutic or blood banking It is rather the responsibility of the user to verify the use of the NucleoSpin RNA DNA Buffer Set for a specific application range as the performance characteristic of this kit has not been verified to a specific organism This MACHEREY NAGEL product is shipped with documentation stating specifica tions 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 MACHEREY NAGEL 10 2008 Rev 05 13 Total RNA and DNA Purification 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 an extra copy MACHEREY NAGEL does not warrant against damages or defects arising in shipping and handling transport insurance for customers excluded or out of accid
4. Troubleshooting Problem Possible cause and suggestions DNA is contami nated with RNA Buffer temperature e DNA elution buffer DNA Elute exceeded 30 C during ap plication Use DNA Elute with a temperature preferentially of 18 25 C DNA yield lower than RNA yield Sample material e DNAand RNA yield depend very much on sample material Ratio of RNA yield to DNA yield may vary from approxi mately 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 precau tion keep DNA on ice for short term or at 20 C for long term storage e Some sample materials may contain remaining DNase traces that are not sufficiently washed away by the stan dard 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 yl Buffer RA2 onto the column centrifuge 1 min at 11000 x g and continue with DNA Wash washing steps Low RNA yield or quality See general protocol e See troubleshooting section of individual NucleoSpin protocols Check if Wash Buffer RA3 has been equili brated to room temperature before use Washing at lower temperatures lowers efficiency of salt removal by Wash Buffer RAS
5. 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 fur ther purification DNA eluted with DNA Elute may readily serve as template for PCR is restrictable with restrictions enzymes and is of high molecular weight 220 kb A sd Asso ratios of eluted DNA are within a range from 1 7 2 0 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 nei ther 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 manuals of the NucleoSpin RNA kits MACHEREY NAGEL 10 2008 Rev 05 5 Total RNA and DNA Purification 2 2 Kit specificati
6. 0 98 gt 0 98 gt 0 98 DNA gt 0 99 gt 0 95 gt 0 67 DNA size and quality e Isolated genomic DNA is commonly of high molecular weight gt 20 kb DNA is commonly stable even at 37 C for 2 h with or without addition of a typical restriction enzyme buffer e DNAis digestable with restriction enzymes e DNAis suitable for PCR MACHEREY NAGEL 10 2008 Rev 05 Total RNA and DNA Purification 3 Storage conditions and preparation of working solutions Store solutions at room temperature 20 25 C e The DNA Wash solution is delivered as a concentrate To prepare the final DNA Wash solution add four volumes of ethanol 5096 to the DNA Wash Concentrate add 90 ml 5096 ethanol to 22 5 ml DNA Wash Concentrate e 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 e Further due to its composition DNA Elute does not inhibit microbial growth Therefore make sure not to contaminate DNA Elute with any source of micro bial contaminants NucleoSpin RNA DNA Buffer Set 100 preps Cat No 740944 Buffer DNA Wash Concentrate 22 5 ml add 90 ml ethanol 50 4 Safety instructions risk and safety phrases The NucleoSpin RNA DNA Buffer Set is intended to be used in conjunction with NucleoSpin RNA kits The NucleoSpin RNA DNA Buff
7. Total RNA and DNA Purification User Manual NucleoSpin RNA DNA Buffer Set October 2008 Rev 05 MACHEREY NAGEL MN Total RNA DNA Purification from Tissue Plant Protocol at a glance Rev 05 1 Homogenize NucleoSpin RNA II NucleoSpin RNA Plant NucleoSpin RNA Protein NucleoSpin RNA XS sample C Sample Sample 2 Lyse cells 350 pl RA1 RAP or RP1 100 pl RA1 I 3 5 ul reducing agent 2 ul TCEP Mix Mix 5 yl Carrier RNA 3 Filtrate lysate 1 min 30s 11 000 x g 11 000 x g 4 Adjust RNA 350 yl 70 ethanol 100 ul 70 ethanol binding condi tions Mix Mix 5 Bind RNA DNA Load lysate Load lysate eS 30s 30s 11 000 x g 11 000x g Beast slice 1 wash 500 yl DNA Wash 400 pl DNA Wash membrane 2 wash 500 ul DNA Wash 400 ul DNA Wash e 1 min 1 min 11 000 x g 11 000 x g B Dry membrane 3 min RT 3 min RT C Elute DNA 100 ul DNA Elute 80 ul DNA Elute NucleoSpin RNA DNA Buffer Set ES 1 min 1 min 11 000 x g 11 000 x g 7 Digest DNA 95 yl DNase 25 ul DNase reaction mixture reaction mixture 15 min RT 15 min RT 8 Wash and dry 1 wash 200 pl RA2 100 pl RA2 silica membrane 2m wash 600 yl RA3 400 yl RA3 3 wash 250 ul RAS 200 pl RAS 1s 30 s 30 s and 2 E 11 000 x g 11 000 x g 2 min 2 min rd i 11 000 x g 11 000 x g 9 Elute highly pure RNA 60 yl RNase free H O 1 min 11 000x g 6 10 pl RNase free H O 30s 11 000x g
8. ent or im proper or abnormal use of this product against defects in products or components not manufactured by MACHEREY NAGEL or against 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 spe cial 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 wri
9. er Set does not contain hazardous contents However pay attention to the safety instructions of the individual NucleoSpin RNA kits 8 MACHEREY NAGEL 10 2008 Rev 05 NucleoSpin RNA DNA Buffer Set 5 Protocol Isolation of RNA and DNA from one undivided sample Before starting the procedure e Check if Buffer DNA Wash was prepared according to section 3 e 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 II NucleoSpin RNA Plant NucleoSpin RNA Protein or NucleoSpin RNA XS kit standard protocol Subsequent to binding of nucleic acids onto 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 ul 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 1 min 11 000 x If using NucleoSpin RNA XS add only 400 ul DNA g 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 4 500 pl DNA Wash EITI Add again 500 pl DNA Wash and centrifuge 1 min at oe 11 000 x g Discard Collection Tube with flow through Mg If using NucleoSpin RNA XS add onl
10. ons Typical yields of total RNA and DNA DNA RNA yield ug 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 II 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 10 5 x 105 cells 3 x 10 1 x 108 cells RNA gt 0 98 gt 0 98 DNA gt 0 99 gt 0 95 6 MACHEREY NAGEL 10 2008 Rev 05 Total RNA and DNA Purification 100 o RNA DNA RNA yield ug 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 II 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 sample 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
11. recommended reading the detailed protocol sections of this User Manual if the NucleoSpin RNA DNA Buffer Set is used in combination with NucleoSpin RNA II Cat No 740955 NucleoSpin RNA Plant Cat No 740949 NucleoSpin RNA Protein Cat No 740933 or NucleoSpin RNA XS Cat No 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 refer encing 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 10 2008 Rev 05 Total 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 Il 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 respectively DNA and RNA are immediately ready for downstream applications Samples are lysed in the lysis buf fer supplied
12. tten or oral by MACHEREY NAGEL s employees agent or representatives except written state ments 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 Please contact MACHEREY NAGEL Germany Tel 49 0 24 21 969 270 e mail TECH BIO mn net com Last updated 12 2006 Rev 02 14 MACHEREY NAGEL 10 2008 Rev 05
13. y 400 ul DNA Wash MACHEREY NAGEL 10 2008 Rev 05 9 NucleoSpin RNA DNA Buffer Set 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 Elute DNA Add 100 pl 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 so lution 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 G6 B e 1 min 11 000x g Add 100 pl DNA Elute Proceed with the digestion of residual on column DNA according to the indi vidual NucleoSpin RNA protocols step Digest DNA Add DNase reaction mixture onto the column and perform all subsequent steps as described in the NucleoSpin RNA II NucleoSpin RNA Plant NucleoSpin RNA Protein or NucleoSpin RNA XS protocol 10 MACHEREY NAGEL 10 2008 Rev 05 Total RNA and DNA Purification 6 Appendix 6 1
Download Pdf Manuals
Related Search