Genomic DNA clean-up - MACHEREY
Contents
1. Genomic DNA clean up User manual NucleoSpin gDNA Clean up XS July 2014 Rev 05 MACHEREY NAGEL www mn net com Genomic DNA clean up Protocol at a glance Rev 05 XS NucleoSpin gDNA Clean up XS 1 Prepare sample Adjust up to 400 uL aqueous sample with buffer TE to 800 uL 2 Adjust DNA binding conditions 200 uL NT 3 Bind DNA Load 500 uL diluted sample 11 000 x g 30 s Load remaining sample 11 000 x g 30 s 4 Wash silica membrane Turn spin cup inside the centrifuge by i 180 compared to the loading position CJ 100 uL B5 11 000 x g 2 min 5 Elute DNA 1 6 15 uL BE 11 000 x g 1 min 2 6 15 uL BE 11 000 x g 1 min 6 Removal of residual ethanol and 90 C concentration 8 min 2 x 10 uL elution or 5 min 10 uL 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 Genomic DNA clean up Table of contents 1 Components 4 1 1 Kit contents 4 1 2 Reagents consumables and equipment to be supplied by user 5 1 3 About this user manual 5 2 Product description 6 2 1 Basic principle 6 2 2 Kit specifications 6 2 3 Handling of sample material 8 2 4 Elution procedures 8 2 5 Concentration and removal of residual ethanol 8 3 Storage conditions and preparation of working solutions 10 4 Safety instructions 11 5 NucleoSpin gDNA Clea2. 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 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 16 MACHEREY NAGEL 07 2014 Rev 05 Genomic DNA clean up 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
3. anrufen P 330 Rinse mouth Mund aussp len 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 MACHEREY NAGEL 07 2014 Rev 05 11 NucleoSpin gDNA Clean up XS 5 NucleoSpin gDNA Clean up XS protocol Before starting the preparation Check if Wash Buffer B5 was prepared according to section 3 Prepare buffer TE e g 10 mM Tris HCl pH 7 5 0 1 mM EDTA Prepare sample Note The following dilution step with TE buffer might not be necessary for highly concentrated samples for uncomplicated pre purified DNA or non forensic samples TE For lt 400 pL sample solution dilute with buffer TE not to a final provided to a final volume of 800 uL volume of 800 uL For gt 400 pL sample solution add 1 vol of buffer TE k and increase Buffer NT proportionally in step 2 Mix thoroughly by vortexing and spin down briefly to clear the lid 2 Adjust DNA binding conditions Add 200 pL Buffer NT per 800 pL diluted or undiluted 200 uL NT sample e g add 100 uL Buffer NT to 400 uL sample per 800 uL of sample Mix thoroughly by vortexing Spin down briefly to clear the lid 3 Bind DNA Load 500 uL sample For each sample place one NucleoSpin gDNA Clean 11 000 up XS Column into a Collection Tube 2 mL 30 xg s Add 500 uL of binding mixture to the column Centrifuge for 30 s at 1
4. consequently highly recommended to perform sampling and DNA purification with special care in order to avoid a contamination of the sample or the purified DNA with unwanted DNA containing material e g fingerprints hair particles aerosol dust Moreover a cross contamination between samples has to be excluded The following precautions are recommended Wear personal protection equipment lab coat gloves goggles Use aerosol resistant pipette tips Always change pipette tips between liquid transfers Briefly centrifuge after mixing steps in order to remove droplets from tube lid 2 4 Elution procedures A high DNA concentration in the elution fraction is of importance and desirable for all typical downstream applications This is of particular interest if the total volume of a reaction mixture is limited as this in turn limits the possible amount of DNA that can be added Due to a high default elution volume classical DNA clean up kits often result in weakly concentrated DNA if only small amounts of DNA are processed Such DNA may even require a subsequent concentration before it can be used for typical downstream applications In contrast to classical kits NucleoSpin gDNA Clean up XS allows for efficient elution in avery small volume which results in highly concentrated DNA For forensic samples elution with 2 x 6 uL is recommended to maximize concentration and yield A two fold elution generally yields more DNA than just o
5. 04 50 740904 250 Wash Buffer B5 Concentrate 6 mL 6 mL 6 mL Add 24 mL Add 24 mL Add 24 mL ethanol ethanol ethanol 10 MACHEREY NAGEL 07 2014 Rev 05 Genomic DNA clean up 4 Safety instructions The following components of the NucleoSpin gDNA Clean up XS kits contain hazardous contents Wear gloves and goggles and follow the safety instructions given in this section GHS classification Only harmful features must 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 NT Guanidinium thiocyanate Warning 302 412 260 273 30 60 EUH031 3014312 330 Guanidiniumthiocyanat Achtung 30 60 Hazard phrases H 302 Harmful if swallowed Gesundheitssch dlich bei Verschlucken H 412 Harmful to aquatic life with long lasting effects Sch dlich f r Wasserorganismen mit langfristiger Wirkung EUH031 Contact with acids liberates toxic gas Entwickelt bei Ber hrung mit S ure giftige S ure Precaution phrases P 260 Do not breathe vapours Dampf nicht einatmen P 273 Avoid release to the environment Freisetzung in die Umwelt vermeiden P 301 312 IF SWALLOWED Call a POISON CENTER doctor if you feel unwell BEI VERSCHLUCKEN Bei Unwohlsein GIFTINFORMATIONSZENTRUM Arzt
6. 1 000 x g and discard the flow through Load e5 remaining Add the remaining binding mixture to the column and sample centrifuge for 30 s at 11 000 x g 11 000 x g Place the column into a new Collection Tube 2 mL 30s 12 MACHEREY NAGEL 07 2014 Rev 05 NucleoSpin gDNA Clean up XS Wash silica membrane Turn the NucleoSpin gDNA Clean up XS Column inside the centrifuge by 180 compared to the loading position in order to allow optimal washing efficiency Add 100 uL Buffer B5 Centrifuge for 2 min at 11 000 x g Elute DNA Place the NucleoSpin gDNA Clean up XS Column into a 1 5 mL microcentrifuge tube not supplied Add 6 15 pL Buffer BE directly to the center of the membrane Centrifuge for 1 min at 11 000 x g Add another 6 15 uL Buffer BE directly to the center of the membrane Centrifuge for 1 min at 11 000 x g Note The elution volume can be varied from 5 30 uL The second elution step can be omitted leading to higher concentration but lower yield See section 2 4 for more information Removal of residual ethanol and concentration Incubate eluate with open lid at 90 C for 8 min 2 x 10 uL elution or 5 min 10 uL elution Note For different elution volumes or incubation at 75 C for non denatured DNA see section 2 5 for detailed information 100 pL B5 11 000 x g 2 min 6 15 uL BE 11 000 x g 1 min 6 15 uL BE 11 000 x g 1 min 90 C 8 min 2x 10 p
7. L elution or 90 C 5 min 10 uL elution MACHEREY NAGEL 07 2014 Rev 05 13 Genomic DNA clean up 6 Appendix 6 1 Troubleshooting Problem Possible cause and suggestions Reagents not prepared properly Low DNA yield Add the indicated volume of 96 100 ethanol to the Buffer B5 Concentrate and mix well before use Residual ethanol in eluate No increase of PCR e Please see the detailed description of removal of signal despite an residual traces of ethanol in section 2 5 increased volume of eluate used as Carry over of chaotropic salts template Perform a second washing step with Buffer B5 to remove last traces of Buffer NT Silica abrasion from the membrane Due to the typically low DNA content in very small sam ples and the resulting low total amount of isolated DNA DNA quantification via As absorption measurement is often hampered by the low sensitivity of this method Discrepancy between When performing absorption measurements close to Aas Quantification the detection limit of the photometer the measurement values and PCR may be influenced by minor amounts of silica abrasion quantification values In order to prevent incorrect Az quantification of small DNA amounts centrifuge the eluate for 30s at gt 11 000 x g and take an aliquot for measurement without disturbing any sediment Alternatively use a silica abrasion insensitive DNA quantification method e g PicoGreen fluore
8. 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 MACHEREY NAGEL 07 2014 Rev 05 5 Genomic DNA clean up 2 Product description 2 1 Basic principle The NucleoSpin gDNA Clean up XS kit is designed for a fast and convenient purification of genomic DNA from aqueous samples e g phenol chloroform extracts PCR inhibitors e g indigo are efficiently removed and DNA is concentrated with high recovery Due to its high sensitivity the kit is particularly well suited for trace levels of DNA from forensic samples The optimized protocol allows for up to 400 uL of aqueous sample to be processed without the need for error prone repeated loading steps However multiple loading steps can be used without difficulty to process larger sample volumes The special funnel design of the thrust rings inside the NucleoSpin gDNA Clean up XS Column in combination with the very small membrane allows for high recovery with very small elution volumes 5 30 uL which results in highly concentrated DNA Appropriate conditions under which DNA binds to the silica membrane are created by addition of Binding Buffer NT The mixture is then applied to the NucleoSpin gDNA Clean up XS Column and the DNA binds to a silica membrane A subsequent washing step efficiently removes contaminations and highly pure DNA
9. ell 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 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
10. ics need to be treated carefully to prevent DNA contamination MACHEREY NAGEL therefore has a stringently controlled production process to avoid DNA contamination of consumables Further MACHEREY NAGEL uses ethylene oxide EO treatment to remove amplifiable DNA which might still be introduced during the manufacturing process MACHEREY NAGEL products carrying the forensic quality seal contain plastic materials that are EO treated This means DNA of any kind which might still be introduced into plastic consumables during the production process is inactivated by means of the treatment with ethylene oxide in order to prevent the generation of accidental human profile by PCR amplification Ethylene oxide treatment has been shown to be the method of choice to prevent DNA profiles due to DNA contamination Shaw et al 2008 Figure 1 UV Gamma Electron beam Ethylene oxide 100 30 40 27 4 13 87 N N aS j i l nam 30 70 Full profile Partial profile loadable I Partial profile unloadable BB No profile Figure 1 According to Shaw et al 2008 Comparison of the effects of sterilization techniques on subsequent DNA profiling Int J Legal Med 122 29 33 MACHEREY NAGEL 07 2014 Rev 05 7 Genomic DNA clean up 2 3 Handling of sample material The NucleoSpin gDNA Clean up XS procedure is designed for very small amounts of genomic DNA and the typical downstream applications are thus very sensitive It is
11. is finally eluted with 5 30 uL of a slightly alkaline elution buffer of low ionic strength 5 mM Tris HCI pH 8 5 2 2 Kit specifications The NucleoSpin gDNA Clean up XS kit is recommended for the purification and concentration of genomic DNA from very dilute aqueous samples Typical sample materials comprise for example PCR inhibitor containing solutions Proteinase K reaction mixtures or the aqueous phase of phenol chloroform extractions The robust membrane allows for multiple loading steps to process even large sample volumes The special column design and the very small membrane lead to a significantly reduced dead volume which allows for high recovery of small amounts of DNA with as little as 5 30 uL elution buffer e DNA is ready to use for all common downstream applications like e g real time PCR The preparation time is approximately 20 min for 6 12 samples 6 MACHEREY NAGEL 07 2014 Rev 05 Genomic DNA clean up Table 1 Kit specifications at a glance Parameter NucleoSpin gDNA Clean up XS Sample material lt 400 uL solution containing lt 2 ug DNA Typical recovery 60 70 Fragment size 100 bp approx 50 kbp Aso A280 1 8 1 9 Elution volume 6 15 uL Preparation time 20 min 6 preps exclusive preceding extraction or lysis Format Mini spin column XS design Forensic quality product NucleoSpin gDNA Clean up XS is certified as forensic quality product Consumables used in forens
12. 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 PicoGreen is a registered trademark of Molecular Probes Inc 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 05 17
13. me and the intended final volume Take into consideration that incubation times may vary depending on the heating block or microcentrifuge tubes that are used Shaking the tubes during incubation increases the evaporation rate even more Incubation min 70 C 90 C Figure 2 Concentration and removal of residual ethanol from eluates by heat treatment Eluates of 10 20 and 30 uL were incubated at 75 C non denaturing and 90 C denaturing for 0 40 min without shaking Choose your final volume and read the necessary incubation time from the appropriate curve For other starting volumes just interpolate the array of curves MACHEREY NAGEL 07 2014 Rev 05 9 Genomic DNA clean up 3 Storage conditions and preparation of working solutions Attention Buffer NT contains guanidinium thiocyanate Wear gloves and goggles Storage conditions All kit components can be stored at room temperature 18 25 C and are stable for at least one year Before starting any NucleoSpin gDNA Clean up XS protocol prepare the following g Wash Buffer B5 Add the indicated volume of ethanol 96 100 to Buffer B5 Concentrate Mark the label of the bottle to indicate that ethanol was added Wash Buffer B5 can be stored at room temperature 18 25 C for at least one year NucleoSpin gDNA Clean up XS 10 preps 50 preps 250 preps REF 740904 10 7409
14. n up XS protocol 12 6 Appendix 14 6 1 Troubleshooting 14 6 2 Ordering information 15 6 3 Product use restriction warranty 15 MACHEREY NAGEL 07 2014 Rev 05 3 Genomic DNA clean up 1 Components 1 1 Kit contents NucleoSpin gDNA Clean up XS 10 preps 50 preps 250 preps REF 740904 10 740904 50 740904 250 Binding Buffer NT 25 mL 25 mL 75 mL Wash Buffer B5 Concentrate 6 mL 6 mL 6 mL Elution Buffer BE 13 mL 13 mL 13 mL NucleoSpin gDNA Clean up XS 10 50 250 Columns light green rings Collection Tubes 2 mL 30 3x 50 3 x 250 User manual 1 1 1 For preparation of working solutions and storage conditions see section 3 Composition of Elution Buffer BE 5 mM Tris HCl pH 8 5 4 MACHEREY NAGEL 07 2014 Rev 05 Genomic DNA clean up 1 2 Reagents consumables and equipment to be supplied by user Reagents 96 100 ethanol Buffer TE e g 10 mM Tris HCl pH 7 5 0 1 mM EDTA Consumables 1 5 mL microcentrifuge tubes for sample lysis and DNA elution Disposable pipette tips Equipment Manual pipettors Thermal heating block Centrifuge for microcentrifuge tubes Personal protection equipment lab coat gloves goggles 1 3 About this user manual It is strongly recommended that first time users of the NucleoSpin gDNA Clean up XS 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
15. ne elution with the same total buffer volume Optionally the second elution can be omitted to achieve the highest possible DNA concentration In general larger volumes 10 30 uL increase the overall DNA yield but naturally reduce the final DNA concentration Elution buffer volumes gt 30 uL will only slightly increase total DNA yield 2 5 Concentration and removal of residual ethanol For most applications removal of trace levels of ethanol is not required However if a large volume of eluate has to be used as PCR template a heat incubation of the eluate is recommended An incubation of for example 8 min at 90 C for a 20 uL eluate removes residual ethanol in the eluate and concentrates the DNA to approximately 11 uL resulting in a significantly increased sensitivity in downstream applications 8 MACHEREY NAGEL 07 2014 Rev 05 Genomic DNA clean up The template may then represent up to 40 of the total PCR reaction volume The necessity of this step may be individually tested An incubation at 90 C however will denature DNA If non denatured DNA is required for downstream applications other than PCR e g ligation or cloning we recommend an incubation of 17 min at 75 C to remove ethanol from an eluate of 20 uL Even if ethanol is of no concern for the downstream application the heat incubation is a useful means to concentrate an eluate Use Figure 2 to estimate the necessary incubation time depending on your elution volu
16. 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 MACHEREY NAGEL 07 2014 Rev 05 15 Genomic DNA clean up 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 w
17. scent dye Measurement not in the range of photometer detection limit In order to obtain a significant Axsgo Azgq ratio it is Az6o Azeo ratio too necessary that the initially measured Apso and Apso high or too low values are significantly above the detection limit of the photometer used An Azo value close to the background noise of the photometer will cause unexpected Asago Aogo ratios 14 MACHEREY NAGEL 07 2014 Rev 05 Genomic DNA clean up 6 2 Ordering information Product REF Pack of NucleoSpin gDNA Clean up XS 740904 10 10 preps 740904 50 50 preps 740904 250 250 preps NucleoSpin gDNA Clean up 740230 10 10 preps 740230 50 50 preps 740230 250 250 preps Buffer NT 740614 100 100 mL Buffer B5 Concentrate 740921 20 mL for 100 mL Buffer B5 Buffer BE 740306 100 100 mL Collection Tubes 2 mL 740600 1000 Visit www mn net com for more detailed product information 6 3 Product use restriction warranty NucleoSpin gDNA Clean up XS 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
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