Genomic DNA from soil
Contents
1. Genomic DNA from soil User manual NucleoSpin Soil November 2011 Rev 03 MACHEREY NAGEL MN Genomic DNA from soil Protocol at a glance Rev 03 1 Prepare sample NucleoSpin Soil NucleoSpin Bead Tube 250 500 mg sample material 700 uL SL1 or SL2 2 Adjust lysis 150 uL Enhancer SX contaminants T conditions 3 Sample lysis Horizontally vortex 5 min at RT or use other homogenizers according to manufacturers protocol 4 Precipitate 11 000 x g 2 min 150 uL SL3 Vortex 5 s 0 4 C 5 min 11 000 x g 1 min 5 Filter lysate Load supernatant on NucleoSpin Inhibitor Removal Column red ring 11 000 x g 1 min 6 Adjust binding cam m de K conditions 250 uL SB Vortex 5 s 7 Bind DNA Load 550 uL sample on NucleoSpin Soil Column green ring 11 000 x g 1 min Load remaining sample 11 000 x g 1 min 8 Wash silica i E 00 ul sB 11 000 x g 30 s membrane EJ 550 u swt 11 000 x g 30s 3 E 700uL sw2 vortex2s 11 000 x g 30 s a 700 uL SW2 Vortex2s 11 000 x g 30 s 9 Dry silica membrans F eS 11 000 x g 2 min 10 Elute DNA 30 100 uL SE Ee Cc gt RT 1 min 2 11 000 x g 30s 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 from soil Table of contents 1 Components 4 1 1 Kit contents2. 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 Concentrate 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 11 2011 Rev 03 21 Genomic DNA from soil Problem Possible cause and suggestions Too harsh mechanical sample disruption Reduce intensity or incubation time of mechanical sample DNA is lysis 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 lt Ma
3. 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 Relevance of humic substances as PCR inhibitors 7 2 4 Amount of starting material 8 2 5 Choice of lysis buffer 8 2 6 Mechanical sample lysis 10 2 7 Repeated extraction 10 2 8 Elution procedures 10 2 9 How to interpret DNA yield and purity from UV VIS 11 3 Storage conditions and preparation of working solutions 13 4 Safety instructions 14 4 1 Risk and safety phrases 14 4 2 GHS classification 15 5 Protocol purification of DNA from soil and sediment 17 6 Appendix 21 6 1 Troubleshooting 21 6 2 Ordering information 23 6 3 Product use restriction warranty 23 MACHEREY NAGEL 11 2011 Rev 03 3 Genomic DNA from soil 1 Components 1 1 Kit contents NucleoSpin Soil 10 preps 50 preps 250 preps REF 740780 10 740780 50 740780 250 Lysis Buffer SL1 30 mL 2x 30 mL 250 mL Lysis Buffer SL2 30 mL 2x30 mL 250 mL Lysis Buffer SL3 5 mL 15 mL 50 mL Enhancer SX 3 mL 10 mL 50 mL Binding Buffer SB 10 mL 2x25 mL 2x125 mL Wash Buffer SW1 6 mL 30 mL 2x75 mL Wash Buffer SW2 Concentrate 6 mL 20 mL 2x 50 mL Elution Buffer SE 5 mL 15 mL 30 mL NucleoSpin Bead Tubes 10 50 250 NucleoSpin Inhibitor Removal 10 50 250 Columns red rings NucleoSpin Soil Columns 10 50 250 green rings Collection Tubes 2 mL 10 50 250 Collection Tubes 2 mL lid 10 50 250 User
4. additional lysis buffer up to the 1 5 mL mark of the NucleoSpin Bead Tube If possible remove foreign material 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 5 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 Soil kit is equipped with two lysis buffers SL1 and SL2 and an Enhancer SX 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 lysis buffer for a specific sample This can only be determined experimentally Therefore both lysis buffers should be tested in parallel for each new sample material After mixing the sample with lysis buffer in the 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 sa
5. 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 pL 12 MACHEREY NAGEL 11 2011 Rev 03 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 Storage conditions 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 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 Soil 10 preps 50 preps 250 preps REF 740780 10 740780 50 740780 250 Wash Buffer SW2 6 mL 20 mL 2x50 mL Concentrate Add 24 mL ethanol Add 80 mL ethanol Add 200 mL ethanol to each bottle MACHEREY NAGEL 11 2011 Rev 03 13 Genomic DNA from soil 4 Safety instructions The following components of the Nu
6. 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 IN VITRO USES ONLY MACHEREY NAGEL 11 2011 Rev 03 23 Genomic DNA from soil 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 clini
7. the same soil sample in the NucleoSpin Bead Tube Filter both final supernatants of step 4 through a NucleoSpin Inhibitor Removal Column 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 Column according to step 7 in multiple loading steps Note that the supplied buffer volumes are calculated for only one extraction The excess of Enhancer SX and Bindung Buffer SB might not be sufficient to allow two extraction steps for all 10 50 or 250 preps of the kit 2 8 Elution procedures It is possible to adapt the elution method temperature and volume of elution buffer used for the subsequent application of interest In addition to the standard method where an increase of DNA concentration can be achieved by reducing the elution volume from 100 to 30 uL there are two options to increase the DNA yield Heat the elution buffer to 80 C Perform two subsequent elution steps with fresh elution buffer 10 MACHEREY NAGEL 11 2011 Rev 03 Genomic DNA from soil 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 Azggo based on the fact that an absorption of Apso 1 corresponds to 50 ug mL double stranded DNA However this calculation assumes the absence of any other compound
8. 1 Flammable liquid and vapour Fl ssigkeit und Dampf entz ndbar Harmful if swallowed Gesundheitssch dlich bei Verschlucken Causes serious eye irritation Verursacht schwere Augenreizung Harmful to aquatic life with long lasting effects Sch dlich f r Wasserorganismen mit langfristiger Wirkung Contact with acids liberates toxic gas Entwickelt bei er hrung mit S ure giftige Gase MACHEREY NAGEL 11 2011 Rev 03 15 Genomic DNA from soil Precaution phrases P 210 P 233 P 260 P 273 P 280 P 301 312 P 305 351 338 P 330 P 3374313 P 403 235 Keep away from heat sparks open flames hot surfaces No smoking Von Hitze Funken offener Flamme heiBen Oberfl chen fernhalten Nicht rauchen Keep container tightly closed Beh lter dicht verschlossen halten Do not breathe vapours Dampf nicht einatmen Avoid release to the environment Freisetzung in die Umwelt vermeiden 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 l
9. Manual 1 1 1 For preparation of working solutions and storage conditions see section 3 Composition of Elution Buffer SE 5 mM Tris HCl pH 8 5 4 MACHEREY NAGEL 11 2011 Rev 03 Genomic DNA from soil 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 Equipment for sample disruption and homogenization see section 2 6 Personal protection equipment e g lab coat gloves goggles 1 3 About this user manual It is strongly recommended that first time users of the NucleoSpin 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 11 2011 Rev 03 5 Genomic DNA 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 inhibit
10. atant of step 4 onto the filter Centrifuge for 1 min at 11 000 x g Note With very wet samples e g sediments the volume of clear supernatant of step 4 can exceed 700 uL significantly In this case transfer the NucleoSpin Inhibitor Removal Column to a new collection tube not provided and load the remaining supernatant Centrifuge for 1 min at 11 000 x g Combine the flow throughs Discard the NucleoSpin Inhibitor Removal Column If a pellet is visible in the flow through transfer the clear supernatant to a new collection tube not provided Vortex RT 5 min 11 000 x g 2 min 150 pL SL3 Vortex 5s 0 4 C 5 min 11 000 x g 1 min Load supernatant 11 000 x g 1 min 18 MACHEREY NAGEL 11 2011 Rev 03 NucleoSpin Soil Adjust binding conditions Add 250 uL Buffer SB and close the lid Vortex for 5 s Bind DNA Place a NucleoSpin Soil Column green ring in a Collection Tube 2 mL Load 550 uL sample onto the column Centrifuge for 1 min at 11 000 x g Discard flow through and place the column back into the collection tube Load the remaining sample onto the column Centrifuge for 1 min at 11 000 x g Discard flow through and place the column back into the collection tube Wash and dry silica membrane Add 500 uL Buffer SB to the NucleoSpin Soil Column Centrifuge for 30 s at 11 000 x g Discard flow through and place the column back into the collec
11. ation MACHEREY NAGEL 11 2011 Rev 03 25
12. c DNA from soil Safety phrases 13 16 S 26 S 39 61 Keep away from food drink and animal foodstuffs Von Nahrungsmitteln Getr nken und Futtermitteln fernhalten Keep away from sources of ignition No smoking Von Z ndquellen fernhalten Nicht rauchen In case of contact with eyes rinse immediately with plenty of water and seek medical advice Bei Ber hrung mit den Augen gr ndlich mit Wasser absp len und Arzt konsultieren Wear eye face protection Schutzbrille Gesichtsschutz tragen Avoid release to the environment Refer to special instructions safety data sheet Bei Ber hrung mit den Augen sofort gr ndlich mit Wasser absp len und Arzt konsultieren 4 2 GHS classification Only harmful features do not need to be labeled with H and P phrases until 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 SB Guanidine 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 isolpropanol 319 301 312 20 50 305 351 338 Guanidinhydrochlorid 36 50 Achtung 330 337 313 Isopropanol 20 50 403 235 Hazard phrases H 226 H 302 H 319 H 412 EUH 03
13. cal 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 components not manufactured by MACHEREY NAGEL or damages resulting from such non MACHEREY NAGEL components or prod
14. cleoSpin Soil kits contain hazardous contents Wear gloves and goggles and follow the safety instructions given in this section 4 1 Risk and safety phrases Component Hazard contents Hazard Risk Safety symbol phrases phrases Inhalt Gefahrstoff Gefahrstoff R S tze S S tze symbol SB Guanidine thiocyanate 30 60 x Xn R 20 21 22 S 13 61 Guanidiniumthiocyanat 30 60 32 52 53 swi Guanidine hydrochloride x Xn R 10 22 36 S 16 26 36 50 isolpropanol 20 50 39 Guanidinhydrochlorid 36 50 Isopropanol 20 50 Risk phrases R10 Flammable Entztindlich R 20 21 22 Harmful by inhalation in contact with the skin and if swallowed Gesundheitssch dlich beim Einatmen Verschlucken und Bwr hrung mit der Haut R22 Harmful if swallowed Gesundheitssch dlich beim Verschlucken R 32 Contact with acids liberates very toxic gas Entwickelt bei Ber hrung mit S ure sehr giftige Gase R 36 Irritating to eyes Reizt die Augen R 52 53 Harmful to aquatic organisms may cause long term adverse effects in the aquatic environment Sch dlich f r Wasserorganismen kann in Gew ssern l mgerfristig sch dliche Wirkungen haben Hazard labeling not necessary if quantity per bottle below 125g or mL certificate of exemption according to 67 548 EEC Art 25 1999 45 EC Art 12 and German GefStoffV 20 3 and TRGS 200 7 1 For further information see Material Safety Data Sheet 14 MACHEREY NAGEL 11 2011 Rev 03 Genomi
15. en 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 16 MACHEREY NAGEL 11 2011 Rev 03 NucleoSpin Soil 5 Protocol purification of DNA from soil and sediment 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 4 and 2 5 for more information on the amount of starting material and the choice of lysis buffer See section 2 7 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 250 500 mg sample Important Do not fill the tube higher than the 1mL mark 700 pL Add 700 uL Buffer SL1 or Buffer SL2 Siler SEa 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 buf
16. f 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 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 oper
17. fer if necessary after spinning down the sample 2 Adjust lysis conditions Add 150 uL Enhancer SX and close the cap 150 pL SX Note Enhancer SX ensures the highest possible DNA yield It can however also promote the release of humic acids See section 2 5 on how to lower the volume or omit the buffer entirely in order to increase DNA purity MACHEREY NAGEL 11 2011 Rev 03 17 NucleoSpin Soil Sample lysis See section 2 6 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 Precipitate contaminants Centrifuge for 2 min at 11 000 x g to 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 7 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 a NucleoSpin Inhibitor Removal Column red ring in a Collection Tube 2 mL lid Load up to 700 uL clear supern
18. h an average molecular weight of 2 000 g mol are soluble 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 MACHEREY NAGEL 11 2011 Rev 03 T Genomic DNA from soil 2 4 Amount of starting material NucleoSpin 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
19. h yield Buffer SE to the column Do not close the lid and incubate for 1 min at room temperature 18 25 C Close the lid and centrifuge for 30 s at 11 000 x g Note Quantify DNA not only by UV VIS but also run an agarose gel to verify yield and DNA quality see section 2 9 for more information 700 pL SW2 Vortex 2s 11 000 x g 30s 700 pL SW2 Vortex 2s 11 000 x g 30s 11 000 x g 2 min G i 30 100 pL SE 2 RT 1 min 11 000 x g 30s 20 MACHEREY NAGEL 11 2011 Rev 03 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 4 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 5 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 6 for more information Make sure that the NucleoSpin Bead Tube is fixed horizontally on the vortexer
20. ision whether the yield as determined from Azs readings can be trusted or not the ratio of the absorption at 260 nm and 230 nm can be used The ratio Azgo Az39 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 Axsgo Azaq ratio Purity ratio A250 A280 Another indicator of DNA purity is the ratio Asgo Azg9 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 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 A B Se Figure 3 Gel analysis of A pure and B contaminated genomic DNA from soil 10 uLofeach sample were run on a 1 TAE
21. ke sure to dry the silica membrane and the NucleoSpin Soil Column completely before elution to avoid carry over of Suboptimal ethanolic Wash Buffer SW2 performance Check if Buffer SW2 has been equilibrated to room temperature of DNA in 18 25 C before use Washing at lower temperatures downstream decreases the efficiency of salt removal experiments Contamination with PCR inhibitors The DNA purity can be increased by lowering the amount of starting material see section 2 4 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 5 for more information Make sure to carefully follow the washing instructions e Dilute DNA 1 10 to reduce concentration of inhibitors 22 MACHEREY NAGEL 11 2011 Rev 03 Genomic DNA from soil 6 2 Ordering information Product REF Pack of NucleoSpin Soil 740780 10 50 250 10 50 250 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 Collection Tubes 2 mL 740600 1000 Visit www mn net com for more detailed product information 6 3 Product use restriction warranty NucleoSpin Soil kit components are intended developed designed and sold FOR RESEARCH PURPOSES ONLY except however any other function of the product being expressly described
22. mple material the Enhancer SX might also reduce 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 Aaso Aaa0 Of the sample significantly Table 2 might 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 8 MACHEREY NAGEL 11 2011 Rev 03 Genomic DNA from soil 1 m cn aai Figure 1 Total DNA purified from wheat field soil with four different Iysis buffer combinations 20 of 100 uL eluate were analyzed on a 1 TAE agarose gel Lane 1 Marker X HindIII 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 gt Yield 2 3 ug 2 3 ug 1 4 ug 3 1 ug Apso Aag 1 69 1 60 1 76 1 72 Apso Asso 1 85 0 96 1 78 0 99 MACHEREY NAGEL 11 2011 Rev 03 Genomic DNA from soil 2 6 Mechanical sample lysis A thorough mechanical lysis step is essential to break up the soil crumbs to free the cells within
23. ors 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 Column 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 Column 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 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 tyoes of 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 Column completely remove humic substances and
24. 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 6 MACHEREY NAGEL 11 2011 Rev 03 Genomic DNA from soil Table 1 Kit specifications at a glance Parameter NucleoSpin Soil Format Mini spin columns Sample material lt 500 mg soil or sediment Typical yield 2 10 ug Elution volume 30 100 uL Preparation time 90 min 10 preps Binding capacity 50 ug 2 3 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 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 wit
25. 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 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 230nm In comparison the spectrum of a sample that is contaminated with humic acids demonstrates only a small shoulder at 260 nm it lacks the minimum 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 14 12 1 0 0 8 0 6 0 4 0 2 A 0 0 Absorption 210 220 230 240 250 260 270 280 290 300 Wave length nm Figure 2 UV VIS quantification of A pure DNA and B contaminated DNA A 7 7 ug in 100 uL 1 84 Aggo Asgo 1 71 A260 A230 B 9 3 ug in 100 UL 1 35 Aggo Aago 0 27 A260 A230 MACHEREY NAGEL 11 2011 Rev 03 11 Genomic DNA from soil Purity ratio A260 A230 To facilitate the dec
26. the soil and to break up cells and spores Ceramic beads have proven to be most effective in combination with a bead mill a FastPrep 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 7 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 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
27. tion tube Add 550 pL Buffer SW1 to the NucleoSpin Soil Column Centrifuge for 30 s at 11 000 x g Discard flow through and place the column back into the collection tube 250 pL SB Vortex 5s Load 550 pL sample 11 000 x g 1 min Load remaining sample 11 000 x g 1 min 500 pL SB 11 000 x g 30s 550 pL SW1 11 000 x g 30s MACHEREY NAGEL 11 2011 Rev 03 19 NucleoSpin Soil 10 Add 700 pL Buffer SW2 to the NucleoSpin Soil Column Close the lid and vortex for 2 s Centrifuge for 30 s at 11 000 x g Discard flow through and place the column back into the collection tube Add 700 uL Buffer SW2 to the NucleoSpin Soil Column Close the lid and vortex for 2 s Centrifuge for 30 s at 11 000 x g Discard flow through and place the column back into the collection tube Note The same collection tube is used throughout the entire washing procedure to reduce plastic waste If new collection tubes are to be used for each step see section 6 2 for ordering information Dry silica membrane Centrifuge for 2 min at 11 000 x g If for any reason the liquid in the collection tube has touched the NucleoSpin Soil Column after the drying step discard flow through and centrifuge again Elute DNA Place the NucleoSpin Soil Column into a new microcen trifuge tube not provided Add 30 uL for high concentration 50 uL for medium concentration and yield or 100 uL for hig
28. ucts 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 24 MACHEREY NAGEL 11 2011 Rev 03 Genomic DNA from soil 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 o
Download Pdf Manuals
Related Search