NucleoSpin® 96 Soil - MACHEREY
Contents
1. Genomic DNA from soil User manual NucleoSpin 96 Soil July 2014 Rev 02 MACHEREY NAGEL www mn net com Genomic DNA from soil 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 The basic principle 6 2 2 Kit specifications 6 2 3 Automated processing on robotic platforms 7 2 4 Relevance of humic substances as PCR inhibitors 7 2 5 Amount of starting material 8 2 6 Choice of lysis buffer 8 2 7 Mechanical sample lysis 10 2 8 Repeated extraction 10 2 9 How to interpret DNA yield and purity from UV VIS 10 3 Storage conditions and preparation of working solutions 13 4 Safety instructions 14 5 NucleoSpin 96 Soil 16 5 1 Purification of DNA from soil and sediment vacuum processing 16 5 2 Purification of DNA from soil and sediment centrifuge processing 23 6 Appendix 29 6 1 Troubleshooting 29 6 2 Ordering information 31 6 3 Product use restriction warranty 32 MACHEREY NAGEL 07 2014 Rev 02 3 Genomic DNA from soil 1 Components 1 1 Kit contents NucleoSpin 96 Soil 2x96 preps 4x 96 preps REF 740787 2 740787 4 Lysis Buffer SL1 150 mL 2x 150 mL Lysis Buffer SL2 150 mL 2x 150 mL Lysis Buffer SL3 50 mL 2x50 mL Enhancer SX 50 mL 2x50 mL Binding Buffer SB 250 mL 500 mL Wash Buffer SW1 125 mL 300 mL Wash Buffer SW2 Concentrate 100 mL 2x 100 mL Elution Buffer SE 602. 000 x g 2 min 24 MACHEREY NAGEL 07 2014 Rev 02 NucleoSpin 96 Soil centrifuge processing Detailed protocol Before starting the preparation check Lysis Buffer SL1 or SL2 for precipitated SDS Dissolve any precipitate by incubating the buffer at 30 40 C for 10 min and shaking the bottle every 2 min 1 Prepare sample See section 2 5 and 2 6 for more information on the amount of starting material and the choice of lysis buffer See section 2 8 for the repeated extraction of a sample to improve DNA yield Transfer 250 500 mg fresh sample material to a NucleoSpin Bead Tube containing the ceramic beads Important Do not fill the tube higher than the 1 mL mark Add 700 uL Buffer SL1 or Buffer SL2 Note for very dry material If the sample material soaks up too much lysis buffer fill the NucleoSpin Bead Tube up to the 1 5 mL mark with fresh lysis buffer Note for very wet material Remove excess liquid before addition of lysis buffer if necessary after spinning down the sample 2 Adjust lysis conditions Add 150 uL Enhancer SX and close the cap Note Enhancer SX ensures the highest possible DNA yield It can however also promote the release of humic acids See section 2 6 on how to lower the volume or omit the buffer entirely in order to increase DNA purity 3 Lyse sample See section 2 7 for more information on homogenization methods e g FastPrep 24 instrument Vortex adapte
3. 280 P 301 312 P 305 351 338 P 330 P 337 313 P 403 235 Wear protective gloves eye protection Schutzhandschuhe Augenschutz tragen IF SWALLOWED Call a POISON CENTER or doctor physician if you feel unwell BEI VERSCHLUCKEN Bei Unwohlsein GIFTINFORMATIONSZENTRUM oder Arzt anrufen IF IN EYES Rinse continuously with water for several minutes Remove contact lenses if present and easy to do continue rinsing BEI KONTAKT MIT DEN AUGEN Einige Minuten lang behutsam mit Wasser sp len Vorhandene Kontaktlinsen nach M glichkeit entfernen Weiter aussp len Rinse mouth Mund aussp len Get medical advice attention Bei anhaltender Augenreizung Arztlichen Rat einholen rztliche Hilfe hinzuziehen Store in a well ventilated place Keep container tightly closed K hl an einem gut bel fteten Ort aufbewahren 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 IN The symbol shown on labels refers to the precaution phrases of this section Das auf Etiketten dargestellte Symbol weist auf die P S tzen dieses Kapitels hin MACHEREY NAGEL 07 2014 Rev 02 15 NucleoSpin 96 Soil vacuum processing 5 NucleoSpin 96 Soil 5 1 Purification of DNA from soil and sediment vacuum processing The NucleoSpin 96 Soil kit can be used with the NucleoVac 96 Vacuum Manifold see order
4. amount of starting material see section 2 5 for more information Enhancer SX can facilitate the release of humic substances Reduce Enhancer SX to 10 uL or omit the buffer entirely see section 2 6 for more information Make sure to carefully follow the washing instructions e Dilute DNA 1 10 to reduce concentration of inhibitors 30 MACHEREY NAGEL 07 2014 Rev 02 Genomic DNA from soil 6 2 Ordering information Product REF Pack of NucleoSpin 96 Soil 740787 2 2x 96 preps 740787 4 4x96 preps Buffer SB 740785 50 50 mL Buffer SL1 740781 30 30 mL Buffer SL2 740782 30 30 mL Buffer SL3 740783 30 30 mL Enhancer SX 740784 50 50 mL NucleoSpin Bead Tubes 740786 50 50 740476 4 sets Block MN Square well Blog 740476 24 24 sets 740479 4 sets MN Wash Plat en 740479 24 24 sets A ee a 12 strips with 8 al a i ips wi tubes each and 12 Cap Strips 740471 24 24 sets 740478 48 sets t Gap Sinps 740478 24 288 sets Self adhering PE Foil 740676 50 NucleoVac 96 Vacuum Manifold 740681 1 NucleoVac Vacuum Regulator 740641 1 Visit www mn net com for more detailed product information MACHEREY NAGEL 07 2014 Rev 02 31 Genomic DNA from soil 6 3 Product use restriction warranty NucleoSpin 96 Soil 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 leafl
5. into vacuum manifold base Insert spacers labeled MTP MULTI 96 PLATE with the notched side up Place the MN Wash Plate onto the spacers Close the manifold with the manifold lid and place the NucleoSpin Soil Binding Plate on top of the lid see Binding setup page 19 Load binding mixtures from step 6 onto the binding plate Apply vacuum 0 2 to 0 6 bar until all liquid has passed the plate Flow through rate should be about 1 2 drops per second Adjust vacuum strength accordingly Wash and dry silica membrane Load 500 uL Buffer SB Apply vacuum 0 2 to 0 6 bar until all liquid has passed the plate Load 550 uL Buffer SW1 Apply vacuum 0 2 to 0 6 bar until all liquid has passed the plate Load 700 uL Buffer SW2 Apply vacuum 0 2 to 0 6 bar until all liquid has passed the plate Load 700 uL Buffer SW2 Apply vacuum 0 2 to 0 6 bar until all liquid has passed the plate MACHEREY NAGEL 07 2014 Rev 02 21 NucleoSpin 96 Soil vacuum processing Dry silica membrane Assemble drying setup Put NucleoSpin Soil Binding Plate on a clean paper towel to remove residual wash buffer from plate outlets Discard MN Wash Plate and remove waste container from vacuum manifold Close the manifold with the manifold lid and place the NucleoSpin Soil Binding Plate back on top of the lid see Drying setup page 19 Apply maximum vacuum for 15 min to dry membrane and to eliminate last trace
6. mL 125 mL NucleoSpin Bead Tubes 2x96 4x96 NucleoSpin Inhibitor Removal Plate 2 4 light gray rings NucleoSpin Soil Binding Plate 2 4 light green rings MN Wash Plate 2 4 MN Square well Block 2 4 Rack of Tube Strips 2 4 User manual 1 1 1 For preparation of working solutions and storage conditions see section 3 2 Composition of Elution Buffer SE 5 mM Tris HCl pH 8 5 For use with vacuum only Set of 1 rack 12 strips with 8 tubes each Cap Strips included 4 MACHEREY NAGEL 07 2014 Rev 02 Genomic DNA from soil 1 2 Reagents consumables and equipment to be supplied by user Reagents 96 100 ethanol For more detailed information regarding special hardware required for centrifuge or vacuum processing see section 5 or contact Technical Service tech bio mn net com 1 3 About this user manual It is strongly recommended that first time users of the NucleoSpin 96 Soil 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 the purification procedure All technical literature is available on the internet at www mn net com Please contact Technical Service regarding information about changes of the current user manual compared to previous revisions MACHEREY NAGEL 07 2014 Rev 02 5 Genomic DN
7. material and the choice of lysis buffer See section 2 8 for the repeated extraction of a sample to improve DNA yield Transfer 250 500 mg fresh sample material to a NucleoSpin Bead Tube containing the ceramic beads Important Do not fill the tube higher than the 1 mL mark Add 700 uL Buffer SL1 or Buffer SL2 Note for very dry material If the sample material soaks up too much lysis buffer fill the NucleoSpin Bead Tube up to the 1 5 mL mark with fresh lysis buffer Note for very wet material Remove excess liquid before addition of lysis buffer if necessary after spinning down the sample 2 Adjust lysis conditions Add 150 uL Enhancer SX and close the cap Note Enhancer SX ensures the highest possible DNA yield It can however also promote the release of humic acids See section 2 6 on how to lower the volume or omit the buffer entirely in order to increase DNA purity 3 Lyse sample See section 2 7 for more information on homogenization methods e g FastPrep 24 instrument Vortex adapter Attach the NucleoSpin Bead Tubes horizontally to a vortexer for example by taping or using a special adapter Vortex the samples at full speed and room temperature 18 25 C for 5 min MACHEREY NAGEL 07 2014 Rev 02 19 NucleoSpin 96 Soil vacuum processing Precipitate contaminants Centrifuge for 2 min at 11 000 x gto eliminate the foam caused by the detergent Note The clear supe
8. A from soil 2 Product description 2 1 The basic principle The sample material is resuspended in Lysis Buffer SL1 or SL2 supplemented with the Enhancer SX and mechanically disrupted using ceramic beads Proteins and PCR inhibitors are precipitated with Lysis Buffer SL3 and subsequently pelleted by centrifugation together with the ceramic beads and undissolved sample material The supernatant is taken off and cleared by passing it through a NucleoSpin Inhibitor Removal Plate DNA binding conditions are then adjusted by addition of Binding Buffer SB to the flow through and the lysate is loaded onto a NucleoSpin Soil Binding Plate Residual humic substances especially humic acids and other PCR inhibitors are removed by efficient washing with Binding Buffer SB and Wash Buffers SW1 SW2 After a drying step ready to use DNA can be eluted with Elution Buffer SE 5 mM Tris HCI pH 8 5 2 2 Kit specifications The NucleoSpin 96 Soil kit is designed for the isolation of high molecular weight genomic DNA from microorganisms like Gram positive and Gram negative bacteria archaea fungi and algae in soil sludge and sediment samples Suitable for soils from forest bog farmland grassland etc Suitable for stool samples The kit offers two special lysis buffers Buffer SL1 and Buffer SL2 which can be combined with the chemical additive Enhancer SX to guarantee highest possible yields with excellent purity for all types o
9. Prep 24 instrument MP Biomedicals set instrument to 5 m s for 30 s or an adapter for Vortex Genie 2 MO BIO In most cases however this kind of equipment is not necessary The same result can be achieved by taping the lysis tubes horizontally to a standard vortexer The lysis time should be as short as necessary to avoid shearing of DNA and to minimize the release of humic acids Depending on the sample however it might be advantageous to increase the lysis time to 10 20 or 30 min Homogenization and cell disruption should be performed at room temperature 18 25 C to avoid SDS precipitation in the lysis buffers Overheating the sample for example by prolonged bead beating in a bead mill or the FastPrep 24 instrument should be avoided to minimize liberation of humic acids 2 8 Repeated extraction For sample materials containing a high amount of microorganisms a single extraction step might not be sufficient to disrupt every cell and to release all DNA Extracting the sample twice may help to increase DNA yield significantly Therefore follow the protocol until the first centrifugation in step 4 But instead of adding Lysis Buffer SL3 directly to the NucleoSpin Bead Tube transfer the supernatant to a new collection tube not provided and complete step 4 with this supernatant Then repeat steps 1 4 with the same soil sample in the NucleoSpin Bead Tube Filter both final supernatants of step 4 through a NucleoSpin Inhibi
10. ccelerations of 5 600 6 000 x g To collect flow throughs in step 7 and 8 additional MN Square well or Round well Blocks are required see ordering information They can be recycled by washing with detergent and hot water Incubate for 5 min in 0 4 M HCI rinse with water again and autoclave before next use Protocol at a glance For detailed information on each step see page 26 Before starting the preparation check Lysis Buffer SL1 or SL2 for precipitated SDS Dissolve any precipitate by incubating the buffer at 30 40 C for 10 min and shaking the bottle every 2 min 1 Prepare sample 250 500 mg sample to NucleoSpin Bead Tube 700 pL SL1 or SL2 2 Adjust lysis conditions 150 pL Enhancer SX 3 Lyse sample Mechanically homogenize 4 Precipitate contaminants 11 000 x g 2 min 150 pL SL3 Vortex 5 s 0 4 C 5 min 11 000 x g 1 min 5 Filter lysate Assemble filtration setup Load samples 5 600 6 000 x g 5 min MACHEREY NAGEL 07 2014 Rev 02 23 NucleoSpin 96 Soil centrifuge processing Adjust binding conditions 250 pL SB Mix Bind DNA Load samples 5 600 6 000 x g 5 min Wash silica membrane 500 pL SB 5 600 6 000 x g 5 min 550 pL SW1 5 600 6 000 x g 2 min 700 pL SW2 5 600 6 000 x g 2 min 700 pL SW2 5 600 6 000 x g 5 min Dry silica membrane 5 600 6 000 x g 15 min or 37 C 20 min 10 Elute DNA 100 200 pL SE 1 min 5 600 6
11. consecutive elution steps e g 2 x 100 uL yield more DNA than one elution step with 200 uL Check DNA yield and quality by UV VIS and agarose gel see section 2 9 for more details 28 MACHEREY NAGEL 07 2014 Rev 02 Genomic DNA from soil 6 Appendix 6 1 Troubleshooting Problem Possible cause and suggestions Poor or no DNA yield Suboptimal lysis conditions Too much sample material was filled into the NucleoSpin Bead Tube Too little head space does not allow the necessary motion of the beads to disrupt the sample Use less sample material see section 2 5 for more information Compare the yields obtained with Lysis Buffer SL1 and SL2 in parallel purifications each with and without addition of Enhancer SX to find the optimal lysis buffer conditions see section 2 6 for more information Insufficient disruption and or homogenization of starting material Shaking of the NucleoSpin Bead Tube was too weak or not long enough Increase shaking time and velocity or use another shaking device see section 2 7 for more information Make sure that the NucleoSpin Bead Tube is fixed horizontally on the vortexer Reagents not applied or restored properly Always dispense exactly the buffer volumes given in the protocol Always follow closely the given instructions with regard to order and mode of mixing shaking vortexing etc Add the indicated volume of ethanol 96 100 to Wash Buffer SW2 Conc
12. ct 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 FastPrep is a registered trademark of MP Biomedicals LLC NucleoSpin is a registered trademark of MACHEREY NAGEL GmbH amp Co KG Vortex Genie is a registered trademark of Scientific Industries 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 02 33
13. emble binding setup Load samples 0 2 to 0 6 bar 8 Wash silica membrane 500 uL SB 0 2 to 0 6 bar 550 pL SW1 0 2 to 0 6 bar 700 pL SW2 0 2 to 0 6 bar 700 pL SW2 0 2 to 0 6 bar 9 Dry silica membrane Assemble drying setup Full vacuum 15 min or 37 C 20 min 10 Elute DNA Assemble elution setup 100 200 pL SE 1 min 0 2 to 0 4 bar Setup of vacuum manifold MACHEREY NAGEL 07 2014 Rev 02 17 NucleoSpin 96 Soil vacuum processing Filtration Binding Drying Elution setup setup setup setup gt SS SS NucleoSpin Inhibitor Removal Plate NucleoSpin Soil Binding Plate NucleoSpin Soil Binding Plate NucleoSpin Soil Binding Plate light gray rings light green rings light green rings light green rings MN Wash Plate MN Square well Block Waste Container Manifold base with spacers Manifold base with spacers Manifold base without spacers Manifold base with spacers for SQUARE WELL BLOCK inserted MTP MULTI 96 PLATE inserted Microtube Rack inserted 18 MACHEREY NAGEL 07 2014 Rev 02 NucleoSpin 96 Soil vacuum processing Detailed protocol Before starting the preparation check Lysis Buffer SL1 or SL2 for precipitated SDS Dissolve any precipitate by incubating the buffer at 30 40 C for 10 min and shaking the bottle every 2 min 1 Prepare sample See section 2 5 and 2 6 for more information on the amount of starting
14. entrate and mix thoroughly see section 3 for more information Store kit components at room temperature 18 25 C Storage at lower temperatures may cause salt precipitation Check Lysis Buffer SL1 and SL2 for white precipitate If precipitation occurred incubate the bottle for 10 min at 30 40 C and shake every 2 minutes until all precipitate is dissolved see section 3 for more information Keep bottles tightly closed in order to prevent evaporation or contamination Sample material not stored properly Whenever possible use fresh material MACHEREY NAGEL 07 2014 Rev 02 29 Genomic DNA from soil Problem Possible cause and suggestions Too harsh mechanical sample disruption DNA i Reduce intensity or incubation time of mechanical sample lysis is degraded DNA is degraded by DNases Add at least 10 15 uL Enhancer SX to the lysate DNA yield was overestimated If DNA eluates are not completely free of contaminants e g RNA protein humic substances UV VIS quantification based on Aso is not reliable due to the contribution of the contaminants to the absorption at 260 nm Carry over of ethanol or salt Suboptimal Make sure to dry the silica membrane and the NucleoSpin Soil i P Binding Plate completely before elution to avoid carry over of per ormance ethanolic Wash Buffer SW2 of DNA in downstream experiments Contamination with PCR inhibitors The DNA purity can be increased by lowering the
15. eoSpin Soil Binding Plate outlets and application of the MN Wash Plate protecting the bottom and the binding plate outlets from lysate and wash buffer spray The MN Frame see ordering information can be used to position the disposable MN Wash Plate inside the vacuum chamber Visit MN online at www mn net com or contact your local MACHEREY NAGEL distributor for technical support regarding hardware software setup instructions and selection of the protocol 2 4 Relevance of humic substances as PCR inhibitors Humic substances are produced by bacteria fungi and protozoa in soil sediments and waters during the degradation of plant or other organic matter They consist of very high molecular weight compounds with undefined structures Building blocks are mainly heterocyclic aromatic compounds that are linked by ether or ethoxy groups and which carry hydroxyl methoxy carbonyl or carboxyl groups MACHEREY NAGEL 07 2014 Rev 02 7 Genomic DNA from soil According to their solubility in water they are divided into humin humic acids and fulvic acids The completely insoluble and black humin has an average molecular weight of around 300 000 g mol The dark brown to grey colored humic acids are slightly smaller They carry a lot of hydroxyl and carboxyl groups and are therefore mainly soluble at neutral or alkaline pH The only slightly yellow to light brown colored fulvic acids with an average molecular weight of 2 000 g mol are so
16. ets 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 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 t
17. f sample material Efficient mechanical lysis of the sample material is achieved by bead beating using the ceramic NucleoSpin Beads The optimized buffer chemistry and the NucleoSpin Inhibitor Removal Plate completely remove humic substances and other PCR inhibitors typically present in soil and sediment samples The eluted DNA is ready to use for all standard downstream applications In most cases the concentrated DNA can be used as PCR template without further dilution for highest sensitivity The kit can be processed with vacuum or centrifugation manually or semi automated 6 MACHEREY NAGEL 07 2014 Rev 02 Genomic DNA from soil Table 1 Kit specifications at a glance Parameter NucleoSpin 96 Soil Technology Silica membrane technology Format 96 well plates Processing vacuum or centrifugation Sample material lt 500 mg soil or sediment Typical yield 2 10 ug Elution volume 100 200 uL Preparation time 150 min plate Binding capacity 50 ug 2 3 Automated processing on robotic platforms NucleoSpin 96 Soil can be processed semi automated on many common laboratory workstations The bead beating lysis still has to be processed manually but the DNA clean up can be automated using vacuum for loading washing and elution Please contact MN for scripts and general considerations on a specific robot The risk of cross contamination is reduced by optimized vacuum settings an improved shape of the Nucl
18. he 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 32 MACHEREY NAGEL 07 2014 Rev 02 Genomic DNA from soil components not manufactured by MACHEREY NAGEL or damages resulting from such non MACHEREY NAGEL components or products MACHEREY NAGEL makes no other war
19. ing information When processing less than 96 samples Self adhering PE Foil see ordering information should be used to close and protect unused wells to ensure proper vacuum Establish a reliable vacuum source for the NucleoVac 96 Vacuum Manifold It can be used in combination with a vacuum pump house vacuum or a water aspirator We recommend a vacuum of 0 2 to 0 4 bar reduction of atmospheric pressure that can be adjusted by the NucleoVac Vacuum Regulator see ordering information Alternatively adjust the vacuum so that sample and buffers run through the plate at a rate of 1 2 drops per second Protocol at a glance For detailed information on vacuum manifold setups see page 19 For detailed information on each step see page 20 Before starting the preparation check Lysis Buffer SL1 or SL2 for precipitated SDS Dissolve any precipitate by incubating the buffer at 30 40 C for 10 min and shaking the bottle every 2 min 1 Prepare sample 250 500 mg sample to NucleoSpin Bead Tube 700 pL SL1 or SL2 2 Adjust lysis conditions 150 pL Enhancer SX 3 Lyse sample Mechanically homogenize 4 Precipitate contaminants 11 000 x g 2 min 150 pL SL3 Vortex 5 s 0 4 C 5 min 11 000x g 1 min 16 MACHEREY NAGEL 07 2014 Rev 02 NucleoSpin 96 Soil vacuum processing 5 Filter lysate Assemble filtration setup Load samples 0 7 bar 6 Adjust binding conditions 250 uL SB Mix 7 Bind DNA Ass
20. like leaves stones or twigs e g by sieving as well as excess of water e g by discarding the supernatant after spinning down sediment samples 2 6 Choice of lysis buffer Due to the highly varying composition of different soils organic matter inorganic matter humic substances metal ions polysaccharides pH etc it is impossible to obtain best results in DNA yield and purity for all sample types with only one single lysis buffer system There are several parameters that can be adjusted in a way that lysis works perfect for one sample but fails with another Therefore the NucleoSpin 96 Soil kit is equipped with two lysis buffers SL1 and SL2 and Enhancer SX 8 MACHEREY NAGEL 07 2014 Rev 02 Genomic DNA from soil Those three components allow a perfect fine tuning for every type of soil sample for maximum yield and purity Unfortunately for the reasons given above there is no way to predict the best choice of Iysis buffer for a specific sample This can only be determined experimentally Therefore both Iysis buffers should be tested in parallel for each new sample material After mixing the sample with Iysis buffer inthe NucleoSpin Bead Tube the Enhancer SX is added routinely to the sample prior to the mechanical homogenization This buffer ensures the highest possible DNA yield with most sample materials However in case of a very high humic acid content in the sample material the Enhancer SX might also reduce
21. luble under alkaline as well as under acidic conditions Due to the high molecular weight and the mainly polyanionic nature of humic substances most purification methods do not distinguish between these molecules and DNA For the same reason they act as extremely potent PCR inhibitors Even smallest amounts of humic substances can inhibit for example DNA polymerases or restriction enzymes and result in a complete failure of enzymatic downstream applications Frequently the problem is circumvented by dilution of the isolated DNA prior to PCR analysis However this results in a significantly reduced sensitivity because low abundance DNA may be lost completely Thus highest DNA yields with as little PCR inhibitor contaminations as possible are of utmost importance for any DNA analysis of soil samples 2 5 Amount of starting material NucleoSpin 96 Soil is suitable for processing 250 500 mg of sample material However do not fill the NucleoSpin Bead Tube higher than the 1 mL mark including the ceramic beads to ensure sufficient head space for an efficient mechanical disruption Usually a reduction of starting material also helps to improve the lysis efficiency and to increase the purity of the DNA Very dry material can soak up large volumes of lysis buffer In this case either reduce the amount of sample material or add additional lysis buffer up to the 1 5 mL mark of the NucleoSpin Bead Tube If possible remove foreign material
22. mum at 230 nm and the absorption sores up below 230 nm In this case only a small part of the absorbance at 260 nm is caused by DNA most of it is just the tailing absorption of the humic acid contamination However the calculated DNA yield seems to be higher in the contaminated sample Thus DNA yield determined by UV VIS might be distorted by co purifying contaminants and we recommend to check the DNA yield also by agarose gel electrophoresis 1 4 1 2 1 0 0 8 0 6 0 4 0 2 0 0 T T T T T T T T 210 220 230 240 250 260 270 280 290 300 Wave length nm Absorption Figure 2 UV VIS quantification of A pure DNA and B contaminated DNA A 7 7 ug in 100 UL 1 84 Asgo Azgo 1 71 A260 A230 B 9 3 ug in 100 UL 1 35 A260 A280 0 27 A260 A230 Purity ratio Az go Az39 To facilitate the decision whether the yield as determined from Azgq readings can be trusted or not the ratio of the absorption at 260 nm and 230 nm can be used The ratio Aggo As39 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 as shown in Figure 2 indicate significant amounts of impurities and the real DNA concentration is far below its calculated value Additionally not only humic acids but also proteins saccharides and other contaminants can be detected by a low Asgo Aas0 ratio Purity ratio A260 A280 Another indicator of DNA purity is the ratio Aago Aaso Which should be bet
23. onent Hazard contents GHS symbol Hazard Precaution phrases phrases Inhalt Gefahrstoff GHS Symbol H S tze P S tze SB Guanidinium thiocyanate Warning 302 412 260 273 30 60 EUH 031 301 312 330 Guanidiniumthiocyanat Achtung 30 60 sWw1 Guanidine hydrochloride Warning 226 302 210 233 280 36 50 isopropanol 319 336 301 312 20 50 305 351 338 Guanidinhydrochlorid 36 50 Achtung 330 337 313 Isopropanol 20 50 403 235 Hazard phrases H 226 Flammable liquid and vapour Fl ssigkeit und Dampf entz ndbar H 302 Harmful if swallowed Gesundheitssch dlich bei Verschlucken H 319 Causes serious eye irritation Verursacht schwere Augenreizung H 412 Harmful to aquatic life with long lasting effects Sch dlich f r Wasserorganismen mit langfristiger Wirkung EUH 031 Contact with acids liberates toxic gas Entwickelt bei Ber hrung mit S ure giftige Gase May cause drowsiness or dizziness Kann Schl frigkeit und Benommenheit verursachen H 336 Precaution phrases P 210 Keep away from heat sparks open flames hot surfaces No smoking Von Hitze Funken offener Flamme hei en Oberfl chen fernhalten Nicht rauchen P 233 Keep container tightly closed Beh lter dicht verschlossen halten P 260 Do not breathe vapours Dampf nicht einatmen P 273 Avoid release to the environment Freisetzung in die Umwelt vermeiden 14 MACHEREY NAGEL 07 2014 Rev 02 Genomic DNA from soil P
24. r Attach the NucleoSpin Bead Tubes horizontally to a vortexer for example by taping or using a special adapter Vortex the samples at full speed and room temperature 18 25 C for 5 min MACHEREY NAGEL 07 2014 Rev 02 25 NucleoSpin 96 Soil centrifuge processing Precipitate contaminants Centrifuge for 2 min at 11 000 x gto eliminate the foam caused by the detergent Note The clear supernatant can be transferred to a new collection tube not provided prior to the following precipitation This might result in more consistent yields from prep to prep and is highly recommended for carbonate containing samples See also section 2 8 for repeated extraction of a sample to improve DNA yield Add 150 uL Buffer SL3 and vortex for 5 s Incubate for 5 min at 0 4 C Centrifuge for 1 min at 11 000 x g Filter lysate Place the NucleoSpin Inhibitor Removal Plate onto the MN Square well Block Load up to 1 mL clear supernatant from step 4 to each well of the plate Centrifuge at 5 600 6 000 x g for 5 min Repeat loading step to process more than 1 mL of lysate see section 2 8 for repeated extraction Discard NucleoSpin Inhibitor Removal Plate Adjust binding conditions Add 250 uL Buffer SB to the flow through in each well of the MN Square well Block Mix by pipetting up and down Bind DNA Place the NucleoSpin Soil Binding Plate on top of a new square well or round well block not pro
25. ranty 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 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 contra
26. reactive compounds when combined with bleach sodium hypochlorite DO NOT add bleach or acidic solutions directly to the sample preparation waste Storage conditions e All kit components should be stored at room temperature 18 25 C and are stable for at least one year Storage 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 the first NucleoSpin 96 Soil procedure prepare the following Wash Buffer SW2 Add the indicated volume of ethanol 96 100 to Buffer SW2 Concentrate Mark the label of the bottle to indicate that ethanol was added Buffer SW2 is stable at room temperature 18 25 C for at least one year NucleoSpin 96 Soil 2x96 preps 4x 96 preps REF 740787 2 740787 4 Wash Buffer SW2 Concentrate 100 mL 2x 100 mL Add 400 mL ethanol Add 400 mL ethanol to each bottle MACHEREY NAGEL 07 2014 Rev 02 13 Genomic DNA from soil 4 Safety instructions The following components of the NucleoSpin 96 Soil kits contain hazardous contents Wear gloves and goggles and follow the safety instructions given in this section GHS classification Only harmful features do not need to 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 Comp
27. rnatant can be transferred to a new collection tube not provided prior to the following precipitation This might result in more consistent yields from prep to prep and is highly recommended for carbonate containing samples See also section 2 8 for repeated extraction of a sample to improve DNA yield Add 150 uL Buffer SL3 and vortex for 5 s Incubate for 5 min at 0 4 C Centrifuge for 1 min at 11 000 x g Filter lysate Assemble filtration setup Insert spacers labeled SQUARE WELL BLOCK with the notched side up Place a MN Square well Block onto the spacers Close the manifold with the manifold lid and place the NucleoSpin Inhibitor Removal Plate on top of the lid see Filtration setup page 19 Load up to 1 mL of clear supernatant from step 4 into each well of the plate Apply vacuum 0 7 bar until all liquid has passed the plate Repeat loading step to process more than 1 mL of lysate see section 2 8 for repeated extraction Note Use centrifuge in case of clogging minimal bucket height has to be 85 mm to accommodate entire square well block filter plate sandwich Discard NucleoSpin Inhibitor Removal Plate Adjust binding conditions Add 250 uL Buffer SB to the flow through in each well of the MN Square well Block Mix by pipetting up and down MACHEREY NAGEL 07 2014 Rev 02 NucleoSpin 96 Soil vacuum processing 7 Bind DNA Assemble binding setup Place the waste container
28. s of ethanol Note Ethanol in Buffer SW2 inhibits enzymatic reactions and has to be removed completely before eluting the DNA An additional 10 min incubation of the binding plate at 37 C can further improve ethanol removal 10 Elute DNA Assemble elution setup Insert spacers MICROTUBE RACK into the vacuum manifold base Place the Rack of Tube Strips onto the spacers Close the manifold with the manifold lid and place the NucleoSpin Soil Binding Plate back on top of the lid see Elution Setup on page 19 Load 100 200 uL Buffer SE directly into the center of the silica membrane of each well Incubate for 1 min Apply vacuum 0 2 to 0 4 bar until all liquid has passed the plate Note Preheating Buffer SE to 70 C or and incubating the entire plate at elevated temperature for 5 min can increase final yield significantly Furthermore two consecutive elution steps e g 2 x 100 uL yield more DNA than one elution step with 200 uL Check DNA yield and quality by UV VIS and agarose gel see section 2 9 for more details 22 MACHEREY NAGEL 07 2014 Rev 02 NucleoSpin 96 Soil centrifuge processing 5 2 Purification of DNA from soil and sediment centrifuge processing For this protocol a microtiter plate centrifuge is required that is able to accommodate the NucleoSpin Soil Binding Plate stacked on the MN Square well Block minimum bucket height 85 mm The centrifuge should be able to reach a
29. the purity of the DNA by facilitating the release of humic acids into the lysate Therefore the volume of added Enhancer SX can be lowered from 150 uL to for example 10 uL or the buffer can be entirely omitted This usually increases the purity A260 A230 Of the sample significantly Table 2 however lower the DNA yield Figure 1 Ideally for a new sample material both lysis buffers Buffer SL1 and SL2 should be tested with and without adding Enhancer SX These initial four preparations will help you to find the ideal lysis condition for your special soil composition 1 E n hn Figure 1 Total DNA purified from wheat field soil with four different lysis buffer combinations 20 of 100 uL eluate were analyzed on a 1 TAE agarose gel Lane 1 Marker X Hindlll Lane 2 Lysis Buffer SL1 Lane 3 Lysis Buffer SL1 Enhancer SX Lane 4 Lysis Buffer SL2 Lane 5 Lysis Buffer SL2 Enhancer SX Table 2 Yields and purity ratios of DNA purified from wheat field soil Buffer SL1 SL2 Enhancer SX z Yield 2 3 ug 2 3 ug 1 4 ug 3 1 ug Apso A280 1 69 1 60 1 76 1 72 Apso A230 1 85 0 96 1 78 0 99 MACHEREY NAGEL 07 2014 Rev 02 9 Genomic DNA from soil 2 7 Mechanical sample lysis A thorough mechanical lysis step is essential to break up the soil crumbs to free the cells within the soil and to break up cells and spores Ceramic beads have proven to be most effective in combination with a bead mill a Fast
30. tor Removal Plate as described in step 5 Add Binding Buffer SB to both filtrates according to step 6 and finally load both samples on one NucleoSpin Soil Binding Plate according to step 7 in multiple loading steps Note that the supplied buffer volumes are calculated for only one extraction per well The excess of Enhancer SX and Binding Buffer SB might not be sufficient to allow two extraction steps 2 9 How to interpret DNA 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 A250 based on the fact that an absorption of A2s 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 for example RNA protein or especially humic substances significantly contributes to the total absorption at 260 nm and therefore leads to an overestimation of the real DNA concentration 10 MACHEREY NAGEL 07 2014 Rev 02 Genomic DNA from soil Figure 2 shows a typical UV absorbance spectrum of pure DNA solid line exhibiting a peak at 260 nm a decrease of absorption with a minimum at 230 nm and only a moderate increase in absorption below 230 nm In comparison the spectrum of a sample that is contaminated with humic acids demonstrates only a small shoulder at 260 nm it lacks the mini
31. vided Load binding mixtures from step 6 onto the binding plate Centrifuge at 5 600 6 000 x g for 5 min Discard flow through MACHEREY NAGEL 07 2014 Rev 02 NucleoSpin 96 Soil centrifuge processing 8 Wash and dry silica membrane Load 500 uL Buffer SB Centrifuge at 5 600 6 000 x g for 2 min Discard flow through Load 550 uL Buffer SW1 Centrifuge at 5 600 6 000 x g for 2 min Discard flow through Load 700 uL Buffer SW2 Centrifuge at 5 600 6 000 x g for 2 min Discard flow through Load 700 uL Buffer SW2 Centrifuge at 5 600 6 000 x g for 5 min Discard flow through 9 Dry silica membrane Centrifuge at 5 600 6 000 x g for 15 min or incubate the plate for 20 min at 37 C Note Ethanol in Buffer SW2 inhibits enzymatic reactions and has to be removed completely before eluting the DNA Incubation at 37 C is more effective to remove traces of wash buffer and should be preferred if possible MACHEREY NAGEL 07 2014 Rev 02 27 NucleoSpin 96 Soil centrifuge processing 10 Elute DNA Place the NucleoSpin Soil Binding Plate on an opened Rack of Tube Strips Load 100 200 uL Buffer SE directly into the center of the silica membrane of each well Incubate for 1 min Centrifuge at 5 600 6 000 x g for 2 min Note Preheating Buffer SE to 70 C or and incubating the entire plate at elevated temperature for 5 min can increase final yield significantly Furthermore two
32. ween 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 MACHEREY NAGEL 07 2014 Rev 02 11 Genomic DNA from soil contamination with protein and RNA at the same time can compensate each other and result in a perfect Azgo Azgo Agarose gel electrophoresis As a consequence the DNA should always be run on an agarose gel to verify the UV VIS quantification especially if Azgo Az39 and Azgo Azgo are beyond the acceptable range Figure 3 demonstrates that the contaminated sample B of Figure 2 actually contains much less DNA than the pure sample A in contrast to the UV VIS results which can easily be misinterpreted Figure 3 Gel analysis of A pure and B contaminated genomic DNA from soil 10 uLofeach sample were run on a 1 TAE agarose gel 1h 100 V The larger gel band of pure DNAA proves a higher yield and concentration compared to the contaminated DNA sample which is in contrast to the UV VIS quantification A 7 7 ug 100 uL B 9 3 ug 100 uL 12 MACHEREY NAGEL 07 2014 Rev 02 Genomic DNA from soil 3 Storage conditions and preparation of working solutions Attention Buffers SB and SW1 contain guanidinium thiocyanate and guanidine hydrochloride respectively Wear gloves and goggles CAUTION Buffers SB1 and SW1 contain guanidinium thiocyanate and guanidine hydrochloride which can form highly
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