NucleoSpin® gDNA Clean-up - MACHEREY
Contents
1. Genomic DNA clean up User manual NucleoSpin gDNA Clean up July 2014 Rev 02 MACHEREY NAGEL www mn net com gDNA clean up Protocol at a glance Rev 02 1 Adjust DNA binding conditions NucleoSpin gDNA Clean up 150 uL sample 450 uL DB Vortex 5 s For smaller sample volumes adjust to 150 uL with water for larger sample volumes increase binding buffer proportionally 2 Bind DNA Load sample on NucleoSpin gDNA Clean up Column 11 000 x g 30s 3 Wash silica membrane El 700 uL DW Vortex 2 s 11 000 x g 30s Ej 700 uL DW Vortex 2 s 11 000 x g 30s 4 Dry silica membrane 11 000 x g 1 min 5 Elute DNA 50 uL DE RT 1 min 11 000 x g 30s Qptional Repeat elution with first eluate or another 50 uL of fresh Buffer DE Heating elution buffer to 70 C might further promote elution 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 gDNA clean up Table of contents 1 Components 4 1 1 Kit contents 4 1 2 Reagents consumables and equipment to be supplied by user 4 1 3 About this user manual 5 2 Product description 6 2 1 Basic principle 6 2 2 Kit specifications 6 2 3 Removal of RNA 7 2 4 How to interpret yield and purity from UV VIS 7 3 Storage conditions and preparation of working solutions 9 4 S2. To facilitate the decision whether the yield as determined from A readings can be trusted or not the ratio of the absorption at 260 nm and 230 nm can be used The ratio Azeo Az30 Should be higher than 2 0 for pure DNA and is acceptable down to ratios of about 1 5 Smaller values around or even below 1 0 indicate significant amounts of impurities and the real DNA concentration is far below its calculated value Purity ratio Azgo Azgo MACHEREY NAGEL 07 2014 Rev 02 7 gDNA clean up Another indicator of DNA purity is the ratio Aago Aago which should be between 1 8 and 1 9 Values below 1 8 indicate protein contamination whereas higher values indicate RNA contamination However this ratio should be treated with caution since contamination with protein and RNA at the same time can compensate each other and result in a perfect Aggo Aaso Agarose gel electrophoresis As a consequence the DNA should always be run on an agarose gel to evaluate the DNA quality in terms of size distribution and to verify the UV VIS quantification especially if Azgo Ao3q and Azgo Azgo are beyond the acceptable range 8 MACHEREY NAGEL 07 2014 Rev 02 gDNA clean up 3 Storage conditions and preparation of working solutions Attention Buffer DB contains guanidine hydrochloride Wear gloves and goggles Storage conditions All kit components should be stored at room temperature 18 25 C and are stable for at least one year Storage
3. gDNA clean up 6 Appendix 6 1 Troubleshooting Problem Possible cause and suggestions Reagents not applied or restored properly Poor or no DNA yield Always dispense exactly the buffer volumes given in the protocol Always follow the given instructions closely with regard to order and mode of mixing shaking vortexing etc Add the indicated volume of ethanol 96 100 to Wash Buffer DW Concentrate and mix thoroughly see section 5 for more information Keep bottles tightly closed in order to prevent evapora tion or contamination Carry over of ethanol or salt Suboptimal performance of DNA in downstream experiments e Make sure to dry the silica membrane and the NucleoSpin gDNA Clean up Column completely before elution to avoid carry over of ethanolic Wash Buffer DW Check if Buffer DW has been equilibrated to room temperature 18 25 C before use Washing at lower temperatures decreases the efficiency of salt removal 6 2 Ordering information Product REF Pack of NucleoSpin gDNA Clean up NucleoSpin gDNA Clean up RNase A lyophilized Collection Tubes 2 mL 740230 10 50 250 10 50 250 preps XS 740904 10 50 250 10 50 250 preps 740505 50 50 mg 740505 100 mg 740600 1000 Visit www mn net com for more detailed product information MACHEREY NAGEL 07 2014 Rev 02 13 gDNA clean up 6 3 Product use restriction warranty NucleoSpin gDNA Clean up kit co
4. the purification procedure All technical literature is available on the internet at www mn net com MACHEREY NAGEL 07 2014 Rev 02 5 gDNA clean up 2 2 1 Product description Basic principle Prepurified and especially high molecular weight genomic DNA dissolved in water elution buffer or any reaction buffer is mixed with Binding Buffer DB and loaded onto a NucleoSpin gDNA Clean up Column All kinds of contaminants are removed by two washing steps with Wash Buffer DB After a drying step pure and concentrated DNA can be eluted with Elution Buffer DE 5 mM Tris HCl pH 8 5 2 2 Kit specifications The NucleoSpin gDNA Clean up kit is designed for the rapid purification of previously isolated small and especially high molecular weight genomic DNA It is used to clean up and concentrate genomic DNA after crude extraction methods for example using Trizol or after enzymatic or chemical reactions No need for organic denaturants or chloroform extractions Any impurities like phenol enzymes salts dyes labels nucleotides small oligonucleotides and even up to 5 detergents e g SDS Triton Tween Lauroylsarcosin are removed completely Binding Buffer DB and Wash Buffer DW are specifically developed to allow a very gentle binding and washing to ensure the highest possible DNA recovery for high molecular weight DNA as well as for DNA fragments down to 100 bp The eluted DNA is ready to use for all s
5. x g 30 5 Discard flow through and place the column back into the collection tube 3 Wash silica membrane 1 wash 700 pL DW Add 700 pL Buffer DW to the NucleoSpin gDNA Clean up Column Vortex 2s Close the lid vortex for 2 s and centrifuge for 30s at ED 11 000 x g 11 000 x g 30s Discard flow through and place the column back into the collection tube MACHEREY NAGEL 07 2014 Rev 02 11 NucleoSpin gDNA Clean up Add 700 pL Buffer DW to the NucleoSpin gDNA Clean 700 pL DW up Column Vortex 2s Close the lid vortex for 2 s and centrifuge for 30 s at 11 000 x g 11 000 x g 30s Discard flow through and place the column back into the collection tube Dry silica membrane Centrifuge for 1 min at 11 000 x g and discard the 11 000 x g collection tube 1 min Note Residual ethanolic wash buffer might inhibit enzymatic reactions Elute DNA Place the NucleoSpin gDNA Clean up Column into a new 1 5 mL microcentrifuge tube not provided Add 50 uL Buffer DE to the column 50 pL DE Do not close the lid and incubate for 1 min at room RT temperature 18 25 C 1 min Close the lid and centrifuge for 30 s at 11 000 x g es 11 000 x g 1 min Note DNA yield can be increased by eluting a second time Either re apply the first eluate to the column or use 50 uL of fresh Elution Buffer DE Heating the elution buffer to 70 C can further increase the elution efficiency MACHEREY NAGEL 07 2014 Rev 02
6. 014 Rev 02 gDNA clean up 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 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 r
7. afety instructions 10 5 NucleoSpin gDNA Clean up protocol 11 6 Appendix 13 6 1 Troubleshooting 13 6 2 Ordering information 13 6 3 Product use restriction warranty 14 MACHEREY NAGEL 07 2014 Rev 02 3 gDNA clean up 1 Components 1 1 Kit contents NucleoSpin gDNA Clean up 10 preps 50 preps 250 preps REF 740230 10 740230 50 740230 250 Binding Buffer DB 25 mL 25 mL 125 mL Wash Buffer DW Concentrate 6 mL 25 mL 3 x 50 mL Elution Buffer DE 13 mL 13 mL 30 mL NucleoSpin gDNA Clean up 10 50 250 Columns light green rings Collection Tubes 2 mL 10 50 250 User manual 1 1 1 1 2 Reagents consumables and equipment to be supplied by user Reagents 96 100 ethanol Consumables 1 5 mL microcentrifuge tubes Disposable pipette tips Equipment Manual pipettors Centrifuge for microcentrifuge tubes Personal protection equipment e g lab coat gloves goggles For preparation of working solutions and storage conditions see section 3 Composition of Elution Buffer DE 5 mM Tris HCl pH 8 5 4 MACHEREY NAGEL 07 2014 Rev 02 gDNA clean up 1 3 About this user manual It is strongly recommended that first time users of the NucleoSpin gDNA Clean up kit read the detailed protocol sections of this user manual 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
8. at lower temperatures may cause precipitation of salts If precipitation occurs incubate the bottle for several minutes at about 30 40 C and mix well until the precipitate is dissolved Before starting any NucleoSpin gDNA Clean up protocol prepare the following g any Wash Buffer DW Add the indicated volume of ethanol 96 100 to Buffer DW Concentrate Mark the label of the bottle to indicate that ethanol has been added Buffer DW is stable at room temperature 18 25 C for at least one year NucleoSpin gDNA Clean up 10 preps 50 preps 250 preps REF 740230 10 740230 50 740230 250 Wash Buffer DW 6 mL 25 mL 3 x 50 mL Concentrate Add 14 mL ethanol Add 60 mL ethanol Add 110 mL ethanol to each bottle MACHEREY NAGEL 07 2014 Rev 02 9 gDNA clean up 4 Safety instructions The following component of the NucleoSpin gDNA Clean up kits contain hazardous contents Wear gloves and goggles and follow the safety instructions given in this section GHS classification Only harmful features need not 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 Component Hazard contents GHS symbol Hazard Precaution phrases phrases Inhalt Gefahrstoff GHS Symbol H S tze P S tze DB Guanidine hydrochloride Danger H 225 P 210 233 1 10 ethanol 55 75 403 235 Guanidinhydrochlorid 1 10 Gefahr Et
9. epresentatives 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 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 NucleoSpin is a trademark of MACHEREY NAGEL GmbH amp Co KG 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 MACHEREY NAGEL 07 2014 Rev 02 15
10. hanol 55 75 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 Hazard phrases H 225 Highly flammable liquid and vapour Fl ssigkeit und Dampf leicht entz ndbar Precaution phrases P 210 Keep away from heat hot surfaces sparks open flames and other ignition sources No smoking Von Hitze hei en Oberfl chen Funken offenen Flammen sowie anderen Z ndquellenarten fernhalten Nicht rauchen P 233 Keep container tightly closed Beh lter dicht verschlossen halten P 403 235 Store in a well ventilated place Keep cool K hl an einem gut bel fteten Ort aufbewahren 10 MACHEREY NAGEL 07 2014 Rev 02 NucleoSpin gDNA Clean up 5 NucleoSpin gDNA Clean up protocol Before starting the preparation Check if Wash Buffer DW was prepared according to section 3 Adjust DNA binding conditions Add 450 uL Binding Buffer DB to 150 pL DNA solution Vortex for 5 s Note If sample volume is less than 150 uL fill up with water to 150 uL If more than 150 uL of sample has to be processed increase Binding Buffer DB proportionally Multiple loading steps might be necessary in step 2 j 150 uL sample 450 pL DB Vortex 5 2 BindDNA Place a NucleoSpin gDNA Clean up Column in a Load up to 700 pL sample solution onto the column 11 000 x g Centrifuge for 30 s at 11 000
11. mponents 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 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 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 o
12. r 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 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 14 MACHEREY NAGEL 07 2
13. tandard downstream applications such as PCR endonuclease restriction Southern Blotting and labeling MACHEREY NAGEL 07 2014 Rev 02 gDNA clean up Table 1 Kit specifications at a glance Parameter NucleoSpin gDNA Clean up Typical sample size 150 uL DNA solution Typical amount of DNA lt 25 ug Typical recovery 80 90 Fragment size 100 bp approx 50 kbp Binding capacity 50 ug Elution volume 50 100 uL Preparation time lt 15 min 10 preps Format Mini spin column 2 3 Removal of RNA Nucleotides and small oligonucleotides are removed by the gentle binding conditions and the stringent washing steps To remove contamination of RNA completely it is recommend to add 1 ug of RNase A see ordering information to 150 uL of sample and to incubate at room temperature 18 25 C for 5 15 min 2 4 How to interpret yield and purity from UV VIS The most common method to determine the DNA yield is UV VIS spectroscopy The DNA concentration in the final eluate can be calculated from its absorption maximum at 260 nm A50 based on the fact that an absorption of Aago 1 corresponds to 50 ug mL double stranded DNA However this calculation assumes the absence of any other compound that absorbs UV light at 260 nm Any contamination with phenol RNA protein or detergents etc significantly contributes to the total absorption at 260 nm thus leading to an overestimation of the real DNA concentration Purity ratio Azgo Az30
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