Total RNA and DNA Purification
Contents
1. Total RNA and DNA Purification User manual NucleoSpin RNA DNA buffer set April 2005 Rev 02 MACHEREY NAGEL MN Protocol at a glance Rev 02 Total RNA DNA Purification from Tissue Plant 1 Homogenization of sample NucleoSpin RNA II NucleoSpin RNA Plant 30 mg 100 mg 2 Cell Lysis 7 350 pl RA1 3 5 yl B mercaptoethanol or 350 pl RAP 3 5 yl B mercaptoethanol Mix 3 Filtration of lysate 1 min S 11 000 xg 4 Adjust RNA binding 350 pl 70 ethanol conditions 5 Bind RNA DNA 30 sec 8 000 x g 6 Column wash 1 wash 200 ul DNA wash 3 2 wash 600 ul DNA wash 5 5 os 1 min co 11 000 x g 2 lt 7 Dry membrane 3 min 2 RT O a 8 DNA elution 100 pl DNA elute 2 o A i E a gt 1 min 2 11 000 x g 9 Digest DNA 95 ul DNase reaction mixture RT 15 min 10 kk ons ed qa Mua 1 wash 200 pl RA2 2 wash 600 ul RA3 3 wash 250 ul RA3 1 and2t 30 see 8 000 x g grd f 2 min 11 000 x g 11 Elute highly pure RNA y 60 ul H20 RNase free 1 min 11 000 x g Total RNA and DNA purification Table of contents 1 Set contents 4 2 Product description 5 2 1 The basic principle 5 2 2 About this user manual 5 3 Storage conditions an preparation of working solution 6 4 Safety Instructions risk and safety phrases 6 5 Protocols 7 5 1 Isolation of RNA and DNA from one undivided sample 7 6 Appendix 9 6 1 Troubleshooting 9 6 2 Ordering informa2. NucleoSpin RNA Plant 740949 50 50 NucleoSpin RNA Plant 740949 250 250 NucleoSpin 8 RNA 740698 12x8 NucleoSpin 8 RNA 740698 5 60 x8 NucleoSpin 96 RNA 740709 2 2 x 96 NucleoSpin 96 RNA 740709 4 4 x 96 NucleoSpin 96 RNA 740709 24 24 x 96 Lysis buffer RA1 740961 50 ml Lysis buffer RA1 740961 500 500 ml DNase set 740963 1 set NucleoSpin Filter 740606 50 NucleoSpin collection tubes 740600 1000 NucleoSpin 96 RNA Filter Plate 740711 4 plates 10 MACHEREY NAGEL 04 2005 Rev 02 Total RNA and DNA purification 6 3 Product use restriction warranty The NucleoSpin RNA DNA buffer set components were developed designed 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 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 Supplementar
3. d 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 MACHEREY NAGEL 04 2005 Rev 02 11 Total RNA and DNA purification 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 2421 969 270 and 275 e mail TECH BlIO mn net com 12 MACHEREY NAGEL 04 2005 Rev 02
4. ials may contain recalcitrant DNase 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 RA2 onto the column centrifuge 1 min at 11000 x g and continue with DNA wash washing steps See general protocol e See troubleshooting section of individual NucleoSpin protocols FOW RNA Check if Wash buffer RA3 has been equilibrated to room SS LEDE temperature before use Washing at lower temperatures lowers efficiency of salt removal by Wash buffer RAS Divalent cations Suboptimal e Eluted DNA contains small amounts of divalent cations If the performance downstream application comprises e g 50 DNA eluate of the of DNA in final reaction volume the divalent cations introduced into the downstream reaction by the DNA eluate may alter the performance application Decrease the divalent cation concentration of the reaction by 1 5 mM for compensation MACHEREY NAGEL 04 2005 Rev 02 9 Total RNA and DNA purification 6 2 Ordering information Product Cat No Pack of NucleoSpin RNA II 740955 20 20 NucleoSpin RNA II 740955 50 50 NucleoSpin RNA II 740955 250 250 NucleoSpin RNA Clean up 740948 10 10 NucleoSpin RNA Clean up 740948 50 50 NucleoSpin RNA Clean up 740948 250 250 NucleoSpin RNA L 740962 20 20 NucleoSpin RNA Plant 740949 20 20
5. id binding conditions and binding of nucleic acids to the NucleoSpin RNA binding column according to the NucleoSpin RNA II or NucleoSpin RNA Plant kit standard protocol steps 1 5 Subsequent to binding of nucleic acids onto the column continue as follows A Column wash 1 add 500 ul Add 500 ul DNA wash to the column and centrifuge for DNA wash 1 min at 11 000 x g Discard flow through and reuse collecting tube The DNA wash solution is used instead Of the MDB ade 1 min membrane desalting buffer from the NucleoSpin RNA II or 11 000 NucleoSpin RNA Plant kit MDB will not be used in this 4 xg procedure here B Column wash 2 add 500 ul DNA wash Add again 500 pl DNA wash and centrifuge 1 min at 11 000 x g Discard collecting tube with flow through 1 min 11 000 x g C Dry membrane Insert the NucleoSpin RNA binding column into a fresh 1 5 ml elution tube Open the lid of the spin column and let it stand for 3 minutes incubate for 3 min The procedure ensures complete removal of ethanol from the column MACHEREY NAGEL 04 2005 Rev 02 7 NucleoSpin RNA DNA buffer set DNA elution 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 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 u
6. ow 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 A260 280 ratios of eluted DNA are within a range from 1 70 2 00 After DNA elution residual on column DNA is digested on the NucleoSpin column as described in the 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 RNA 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 4 1 allows the clean up of up to 100 ug of RNA per NucleoSpin RNA Binding Column or the isolation of total RNA from up to 1 x 10 cultured cells section 4 2 2 2 About this user manual Experienced users who are performing the isolation of RNA and DNA using the NucleoSpin RNA DNA buffer set in combination with NucleoSpin RNA II cat no 740955 or NucleoSpin RNA Plant kit cat no 740949 may refer to the Protocol at a glance instead of this user manual The Protocol at a glance is designed
7. p 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 Digestion of residual on column DNA washing and elution of RNA Prepare and apply DNase reaction mixture according to the NucleoSpin RNA protocol step 7 Digest DNA Add DNase reaction mixture onto the column and proceed with all subsequent steps as described in the NucleoSpin RNAII or NucleoSpin RNA Plant protocol steps 8 9 MACHEREY NAGEL 04 2005 Rev 02 add 100 ul DNA elute 1 min 11 000 x g 95 ul DNase reaction mixture RT 15 min Total RNA and DNA purification 6 Appendix 6 1 Troubleshooting Problem Possible cause and suggestions DNA is buffer temperature contaminated e DNA elution buffer DNA elute exceeded 30 C during application with RNA Use DNA elute with a temperature preferentially of 18 25 C Sample material vw e DNA and RNA yield depend very much on sample material RNA yield Ratio of RNA yield to DNA yield may vary from approximately 1 20 DNase contamination e 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 DNA degrades short term or at 20 C for long term storage upon storage Some sample mater
8. tion 10 6 3 Product use restriction warranty 11 MACHEREY NAGEL 04 2005 Rev 02 3 Total RNA and DNA purification 1 Set contents 100 preps Cat No 740944 Buffer DNA wash concentrate 22 5 ml Buffer DNA elute 12 0 ml Protocol 1 The content of this set is sufficient for 100 DNA isolations in combination with NucleoSpin RNA II cat no 740955 or NucleoSpin RNA Plant kit cat no 740949 Additional collecting tubes and DNA elution tubes are required and are not supplied For preparation of working solutions and storage conditions see section 3 4 MACHEREY NAGEL 04 2005 Rev 02 Total RNA and DNA purification 2 Product description 2 1 The basic principle The Support Set for RNA and DNA isolation with NucleoSpin kits is intended to be used in conjunction with the NucleoSpin RNA II or the NucleoSpin RNA Plant kit for isolation of RNA and DNA from one undivided sample with one single NucleoSpin column This patent t pending technology enables successive elution of DNA and RNA from a NucleoSpin column with low salt buffer and water respectively DNA and RNA are then immediately ready for downstream applications According to the NucleoSpin RNA II or NucleoSpin RNA Plant protocol samples are lysed in lysis buffer RA1 or RAP 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 l
9. to be used only as a supplemental tool for quick referencing while performing the purification procedure First time users are strongly advised to read this user manual MACHEREY NAGEL 04 2005 Rev 02 5 Total RNA and DNA purification 3 Storage conditions an preparation of working solution 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 98 to the DNA wash concentrate add 90 ml ethanol to 22 5 ml DNA wash concentrate e Due to its composition the DNA elute solution DNA elution buffer does not inhibit DNases i e DNA elute does not contain substances e g EDTA to complex divalent cations Therefore be aware not to contaminate DNA elute with DNases 100 preps Cat No 740944 Buffer DNA wash 22 5 ml concentrate add 90 ml ethanol 98 4 Safety Instructions risk and safety phrases The NucleoSpin RNA DNA buffer set for the isolation with NucleoSpin RNA Kits does not contain hazardous contents However pay attention to the safety instructions of the individual NucleoSpin RNA kits 6 MACHEREY NAGEL 04 2005 Rev 02 NucleoSpin RNA DNA buffer set 5 Protocols 5 1 Isolation of RNA and DNA from one undivided sample Before starting the procedure prepare wash buffer DNA wash according to section 3 Perform sample homogenization cell lysis lysate filtration adjusting of nucleic ac
10. y 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 accident or improper 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 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 expresse
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