NucleoSpin® Microbial DNA - MACHEREY
Contents
1. Genomic DNA from microoganisms User manual NucleoSpin Microbial DNA August 2015 Rev 01 MACHEREY NAGEL MN www mn net com Genomic DNA from microorganisms Protocol at a glance Rev 01 NucleoSpin Microbial DNA lt 40 mg microbial pellet wet weight 1 Prepare sample 100 uL BE Transfer sample in NucleoSpin Bead Tube Type B 40 uL Buffer MG 2 Sample lysis eS 10 uL Liquid Proteinase K Agitate on a swing mill or similar device 4 12 min 11 000 x g 30 s w 600 uL Buffer MG 3 Adjust binding conditions Vortex 3 s 11 000 x g 30s S Load 500 600 uL sample on 4 Bind DNA NucleoSpin Microbial DNA Column z 11 000 x g 30 s m 5 Wash silica es EG 500 u Bw 11 000 x g 30 s S membrane amp BJ 500 ut B5 11 000 x g 30s 6 Dry silica membrane 11 000 x g 30 s AA 100 uL BE 7 Elute DNA e gt RT 1 min 11 000 x g 30 s 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 microorganisms 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 7 2 3 Handling preparation and storage of starting materials 7 2 4 Lysis and disruption of sample m2. 333 313 363 CAS 39450 01 6 WARNING ACHTUNG Hazard phrases H226 Flammable liquid and vapour Fl ssigkeit und Dampf entz ndbar H302 Harmful if swallowed Gesundheitssch dlich bei Verschlucken H317 May cause an allergic skin reaction Kann allergische Hautreaktionen verursachen H319 Causes serious eye irritation Verursacht schwere Augenreizung H336 May cause drowsiness or dizziness Kann Schl frigkeit und Benommenheit verursachen H412 Harmful to aquatic life with long lasting effects Sch dlich f r Wasserorganismen mit langfristiger Wirkung EUHO31 Contact with acids liberates toxic gas Entwickelt bei Ber hrung mit S ure giftige Gase MACHEREY NAGEL 09 2015 Rev 01 11 Genomic DNA from microorganisms Precaution phrases P210 P233 P260 P261 P264 P272 P273 P280 P301 312 P302 352 P305 351 338 P330 P333 313 P337 313 P363 P370 378 P403 235 Keep away from heat hot surfaces sparks open flames and other ignition sources No smoking Von Hitze heissen Oberfl chen Funken offenen Flammen sowie anderen Z ndquellenarten fernhalten Nicht rauchen Keep container tightly closed Beh lter dicht verschlossen halten Do not breathe dust fume gas mist vapours spray Staub Rauch Gas Nebel Dampf Aerosol nicht einatmen Avoid breathing dust fume gas mist vapours spray Einatmen von Staub Rauch Gas Nebel Dampf Aerosol vermeiden Wash t
3. Store in a well ventilated place Keep cool An einem gut bel fteten Ort aufbewahren K hl halten 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 ee symbol shown on labels refers to further safety information in this section Das auf Etiketten dargestellte Symbol weist auf weitere Sicherheitsinformationen dieses Kapitels hin 12 MACHEREY NAGEL 09 2015 Rev 01 NucleoSpin Microbial DNA 5 Standard protocol for gram positive and gram negative bacteria Before starting the preparation Check if Buffer B5 was prepared according to section 3 Check section 2 4 for lysis and disruption of sample material 1 Prepare sample Harvest cells from a culture by centrifugation in a microcentrifuge tube not provided Discard supernatant Up to approximately 40 mg of wet weight microbial y 100 pL Buffer cell culture pellet can be used as sample material BE Add 100 uL Elution Buffer BE and resuspend cells Alternatively high quality grade water not provided can be used 2 Lyse sample Transfer the cell suspension into the NucleoSpin Bead Tube Type B provided Add 40 uL Buffer MG Then add 10 pL Liquid Proteinase K and close the tube 40 uL Buffer MG 10 uL Liquid Proteinase K Note It is not necessary to vortex here Agitate the NucleoSpin Bead Tube o
4. 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 18 MACHEREY NAGEL 09 2015 Rev 01 Genomic DNA from microorganisms components not manufactured by MACHEREY NAGEL or damages resulting from such non MACHEREY NAGEL components or products MACHEREY NAGEL makes no other warranty of any kind whatsoever and SPECIFICALLY DISCLAIMS AND EXCLUDES ALL OTHER WARRANTIES OF ANY KIND OR NATURE WHATSOEVER DIRECTLY OR INDIRECTLY EXPRESS OR IMPLIED INCLUDING WITHOUT LIMITATION AS TO THE SUITABILITY REPRODUCTIVITY DURABILITY FITNESS FOR A PARTICULAR PURPOSE OR USE MERCHANTABILITY CONDITION OR ANY OTHER MATTER WITH RESPECT TO MACHEREY NAGEL PRODUCTS In no event shall MACHEREY NAGEL be liable for claims for any other damages whether direct indirect incidental compensatory foreseeable consequential or special including but not limited to loss of use revenue or
5. Bead Tube Disruption time Gram negative bacteria NucleoSpin Bead Tubes 4 min E g Escherichia coli Vibrio Type B fischeri Alternative Type A Type C Gram positive bacteria NucleoSpin Bead Tubes 12 min E g Bacillus subtilis Type B Alternative Type A Corynebacterium glutamicum Yeast NucleoSpin Bead Tubes 12 min E g Saccharomyces cerevisiae TypeC Filamentous fungi NucleoSpin Bead Tubes 12 min E g Aspergillus spec Rhizopus Type C spec Note Performance and stability testing has been conducted on the NucleoSpin Bead Tubes A B and C on a Retsch Schwingm hle MM300 at highest frequency 30 Hertz for up to 15 minutes for optimal sample disruption avoidance of DNA fragmentation and tube durability Other disruption devices see section 2 4 1 will require different settings regarding frequency and duration for optimal performance with the selected sample material Please note that the position of the tube within the machine Retsch Schwingm hle is important for optimal performance Please consult instruction manual of the machine WARNING Many modern disruption devices can cause very high energy input in bead tubes Depending on bead tube type and content beads liquid volume sample type especially high frequency of shaking and or long shaking duration can cause breaking up of the bead tubes It is the responsibility of the user to perform initial stability test for the used bead tubes under the conditions u
6. Rev 03 Please contact MACHEREY NAGEL GmbH amp Co KG Tel 49 24 21 969 270 tech bio mn net com MACHEREY NAGEL 09 2015 Rev 01 19 Genomic DNA from microorganisms Trademarks Retsch is a registered trademark of Retsch GmbH FastPrep is a registered trademark of MP Biomedicals LLC Precellys is a registered trademark of Bertin Technologies MagNA Lyser is a trademark of Roche Diagnostics GmbH Bullet Blender is a registered trademark of Next Advance Mini Beadbeater is a trademark of Biospec Products Vortex Genie is a registered trademark of Scientific Industries NucleoSpin is a registered trademark of MACHEREY NAGEL GmbH amp Co KG All used names and denotations can be brands trademarks or registered labels of their respective owner also if they are not special denotation To mention products and brands is only a kind of information i e it does not offend against trademarks and brands and can not be seen as a kind of recommendation or assessment Regarding these products or services we can not grant any guarantees regarding selection efficiency or operation 20 MACHEREY NAGEL 09 2015 Rev 01 MACHEREY NAGEL MACHEREY NAGEL GmbH amp Co KG Neumann Neander Str 6 8 52355 D ren Germany Germany Switzerland France USA and international MACHEREY NAGEL AG MACHEREY NAGEL EURL MACHEREY NAGEL Inc Tel 49 24 21 969 0 Tel 41 62 388 55 00 Tel 33 388 68 22 68 Tel 1 484 821 0984 E
7. yeasts Sample amount Up to approx 40 mg wet weight Typical yield Varies by sample and disruption device 5 25 ug DNA from approx 30 mg wet weight microbial pellet can be obtained A260 A280 1 6 2 0 Elution volume 100 200 uL Preparation time 35 min 6 preps Binding capacity 60 ug 2 3 Handling preparation and storage of starting materials Cells should be harvested from fresh microbial cultures by sedimentation via centrifugation Supernatant should be removed by aspiration Microbial cell pellets can be used fresh or stored at 20 C to 80 C before starting DNA isolation 2 4 Lysis and disruption of sample material In order to obtain optimal yields of DNA from sample material a complete disruption of the sample material is necessary Sample disruption efficiency depends on the following parameters and can be achieved by following suggestions outlined in the subsequent sections MACHEREY NAGEL 09 2015 Rev 01 7 Genomic DNA from microorganisms 2 4 1 Sample type and disruption device Sample and disruption device are to be supplied by user The following devices are compatible with NucleoSpin Bead Tubes Schwingm hle MM200 MM300 MM400 Retsch FastPrep System MP Biomedicals Precellys Bertin Technologies MagNa Lyser Roche TissueLyser Il and Tissue Lyser LT QIAGEN Bullet Blender Next Advance Mini Beadbeater Biospec Products Speed Mill Analytik Jena Vort
8. aterial 7 2 5 Elution procedures 9 3 Storage conditions and preparation of working solutions 10 4 Safety instructions 11 5 Standard protocol for gram positive and gram negative bacteria 13 6 Support protocols 15 6 1 Support protocol for yeast e g Saccharomyces cerevisiae 15 7 Appendix 16 7 1 Troubleshooting 16 7 2 Ordering information 17 7 3 Product use restriction warranty 18 MACHEREY NAGEL 09 2015 Rev 01 3 Genomic DNA from microorganisms 1 Components 1 1 Kit contents NucleoSpin Microbial DNA 10 preps 50 preps REF 740235 10 740235 50 Lysis Buffer MG 10 mL 38 mL Wash Buffer BW 6 mL 30 mL Wash Buffer B5 Concentrate 6 mL 6 mL Elution Buffer BE 13 mL 30 mL Liquid Proteinase K 120 uL 600 uL NucleoSpin Bead Tubes Type B 10 50 NucleoSpin Microbial DNA Columns 10 50 light green rings Collection Tubes 2 mL 20 100 User manual 1 1 For preparation of working solutions and storage see section 3 Composition of Elution Buffer BE 5 mM Tris HCI pH 8 5 4 MACHEREY NAGEL 09 2015 Rev 01 Genomic DNA from microorganisms 1 2 Reagents consumables and equipment to be supplied by user Reagents 96 100 ethanol Consumables 1 5 mLor 2 mL microcentrifuge tubes for microbial sample sedimentation Disposable tips Equipment Manual pipettors Centrifuge for microcentrifuge tubes Vortex mixer Sample disruption device swing mill or similar device e g Schwingm
9. e that ethanol was added Wash Buffer B5 can be stored at room temperature 18 25 C for at least one year Liquid Proteinase K is ready to use After first time use store Liquid Proteinase K at 4 C or 20 C NucleoSpin Microbial DNA 10 preps 50 preps REF 740235 10 740235 50 Wash Buffer B5 6 mL 6 mL Concentrate Add 24 mL ethanol Add 24 mL ethanol 10 MACHEREY NAGEL 09 2015 Rev 01 Genomic DNA from microorganisms 4 Safety instructions The following components of the NucleoSpin Microbial DNA 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 GHS Hazard Precaution Component Hazard contents symbol phrases phrases Inhalt Gefahrstoff GHS Symbol H S tze P S tze BW Guanidine hydrochloride 226 302 210 233 264 36 50 2 propanol amp D 319 336 280 301 312 20 50 305 351 338 Guanidinhydrochlorid 36 50 WARNING 330 337 313 2 Propanol 20 50 ACHTUNG 370 378 CAS 50 01 1 ar MG Guanidinium thiocyanate 302 412 260 273 30 60 EUH031 301 312 330 Guanidinthiocyanat 30 60 WARNING CAS 593 84 0 ACHTUNG Proteinase K Proteinase K liquid 1 3 317 261 272 280 Proteinase K fl ssig 1 3 302 352
10. ex Adapter for Vortex Genie 2 X MoBio 2 4 2 Lysis buffer composition sample amount volume of lysate and temperature Lysis buffer maximal sample amount and volume of liquid in the bead tube sample amount water Iysis buffer Proteinase K are specified in the corresponding NucleoSpin kit Room temperature 18 25 C is recommended as the working temperature 2 4 3 Type of bead tube time of disruption and frequency of disruption Bead type disruption time and frequency speed must be optimized for a given sample for maximal results of DNA yield and quality Type of bead tube NucleoSpin Bead Tubes Type A 0 6 0 8 mm ceramic beads Recommended for soil and sediment included in NucleoSpin Soil see ordering information section 7 2 NucleoSpin Bead Tubes Type B 40 400 um glass beads Recommended for gram positive and negative bacteria included in NucleoSpin Microbial DNA see ordering information section 7 2 NucleoSpin Bead Tubes Type C 1 3 mm corundum Recommended for yeast see ordering information section 7 2 Time and frequency of disruption The following recommendations have been established for a Retsch Schwingm hle MM300 operating at highest frequency 30 Hertz For using other disruption devices and other sample materials time and frequency have to be optimized 8 MACHEREY NAGEL 09 2015 Rev 01 Genomic DNA from microorganisms Sample material NucleoSpin
11. he microbes of interest should be in pellet format Preliminary data also indicate the usability of the kit for DNA isolation from fungal mycelia e g Aspergillus nidulans from bacterial spore suspensions e g Geobacillus stearothermophilus and from plant pollen e g honey bee pollen baskets For optimal DNA yield bead tubes different from the ones included in the kit might be required for such applications see section 2 4 Microbial samples such as gram positive bacteria yeast and spores can be difficult to lyse due to their strong complex cell wall structures The NucleoSpin Microbial DNA kit replaces enzymatic lysis by utilizing mechanical disruption of cell wall structures with the NucleoSpin Bead Tubes The NucleoSpin Bead Tubes can be used in combination with many compatible disruptive devices see section 2 4 1 High DNA yields can be obtained with the NucleoSpin Bead Tubes from a large variety of sample types enabling the procedure to be convenient fast and easy Alternative bead types can be ordered separately for select sample types see section 2 4 3 for recommendations 6 MACHEREY NAGEL 09 2015 Rev 01 Genomic DNA from microorganisms 2 2 Kit specifications Table 1 Kit specifications at a glance Parameter NucleoSpin Microbial DNA Technology Silica membrane technology Format Mini spin column Sample material Microbial cell culture pellets of gram positive and gram negative bacteria
12. hle MM200 MM300 MM400 Retsch FastPrep System MP Biomedicals Precellys Bertin Technologies MagNA Lyser Roche TissueLyser QIAGEN Bullet Blender Next Advance Mini Beadbeater Biospec Products Speed Mill Analytik Jena Vortex Adapter for Vortex Genie 2 X MoBio Personal protection equipment lab coat gloves goggles 1 3 About this user manual It is strongly recommended reading the detailed protocol sections of this user manual if the NucleoSpin Microbial DNA kit is used for the first time 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 09 2015 Rev 01 5 Genomic DNA from microorganisms 2 Product description 2 1 The basic principle The NucleoSpin Microbial DNA kit is designed for efficient isolation of genomic DNA from microbial samples DNA can be isolated from a wide variety of microorganisms such as gram negative and gram positive bacteria as well as yeasts e g Escherichia coli Bacillus subtilis Corynebacterium glutamicum Saccharomyces cerevisiae Preparation of the collected samples containing t
13. horoughly after handling Nach Handhabung gr ndlich waschen Contaminated work clothing should not be allowed out of the workplace Kontaminierte Arbeitskleidung nicht au erhalb des Arbeitsplatzes tragen Avoid release to the environment Freisetzung in die Umwelt vermeiden Wear protective gloves protective clothing eye protection face protection Schutzhandschuhe Schutzkleidung Augenschutz Gesichtsschutz tragen IF SWALLOWED Call a POISON CENTER doctor if you feel unwell BEI VERSCHLUCKEN Bei Unwohlsein GIFTINFORMATIONSZENTRUM Arzt anrufen IF ON SKIN Wash with plenty of water BEI BERUHRUNG MIT DER HAUT Mit viel Wasser waschen IF IN EYES Rinse cautiously with water for several minuts Remove contact lenses if present and easy to do Continue rinsing BEI KONTAKT MIT DEN AUGEN Einige Minuten lang behutsam mit Wasser aussp len Eventuell vorhandene Kontaktlinsen nach M glichkeit entfernen Weiter aussp len Rinse mouth Mund aussp len If skin irritation or rash occurs Get medical advice attention Bei Hautreizung oder ausschlag Arztlichen Rat einholen rztliche Hilfe hinzuziehen If eye irritation persists Get medical advice attention Bei anhaltender Augenreizung Arztlichen Rat einholen arztliche Hilfe hinzuziehen Wash contaminated clothing before reuse Kontaminierte Kleidung vor erneutem Tragen waschen In case of fire Use to extinguish Bei Brand zum L schen verwenden
14. ive 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 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
15. l incubate the lysate after the disruption step for 5 min at 70 C in order to inactivate the Proteinase K After cooling to room temperature add 20 uL RNase A 20 mg mL and incubate 5 min Continue with the application of the lysate onto the column Reagents not applied properly Prepare Buffer B5 according to instructions see section 3 Too much sample material used Clogged columns Make sure to centrifuge the lysate after cell disruption in order to sediment beads and cell debris Only transfer cleared supernatant onto the column 16 MACHEREY NAGEL 09 2015 Rev 01 Genomic DNA from microorganisms Carry over of ethanol or salt Make sure to centrifuge 1 min at 11 000 x g in order to remove all of ethanolic Buffer B5 before eluting the DNA If for any reason the level of Buffer B5 has reached the column Suboptimal outlet after drying repeat the centrifugation performance of genomic DNA Do not chill Buffer B5 before use Cold buffer will not remove in enzymatic salt effectively Equilibrate Buffer B5 to room temperature reactions before use Contamination of DNA with inhibitory substances Do not elute DNA with TE buffer EDTA may inhibit enzymatic reactions Repurify DNA and elute in Buffer BE 7 2 Ordering information Product REF Pack of NucleoSpin Microbial DNA 740235 10 50 10 50 preps NucleoSpin Soil 740780 10 50 250 10 50 250 preps NucleoSpin Bead Tube 740786 50 50
16. lt instruction manual of the machine If bead carryover is observed in the eluate transfer the eluate into anew 1 5 mL nuclease free tube carefully avoid disturbing the pellet MACHEREY NAGEL 09 2015 Rev 01 15 Genomic DNA from microorganisms 7 Appendix 7 1 Troubleshooting Problem Possible cause and suggestions Incomplete lysis Adjust lysis conditions bead tube type agitation device duration or frequency Reagents not applied properly Prepare Buffer B5 according to instructions section 3 Suboptimal elution of DNA from the column Noor poor DNA For certain sample types preheat Buffer BE to 70 C before yield elution Apply Buffer BE directly onto the center of the silica membrane Elution efficiencies decrease dramatically if elution is done with buffers with a pH lt 7 0 Use slightly alkaline elution buffers like Buffer BE pH 8 5 Especially when expecting high yields from large amounts of material we recommend elution with 200 uL Buffer BE and incubation of the closed columns in an incubator at 70 C for 5 min before centrifugation High Azgq Azgo ratio Ratios gt 1 9 can be caused by RNA contamination Usually such RNA contamination do not interfere with downstream application Depending on sample type amount and disruption procedure preparations might contain small amounts of RNA If it is necessary to reduce RNA contamination to the Poor DNA quality lowest possible leve
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18. n a swing mill or similar device Note Optimal agitation duration speed frequency depends on the machine used On a Retsch Schwingm hle MM200 MM300 MM400 e g Agitate 4 min at maximal frequency 30 Hertz is adequate for E coli 12 min for B subtilis see section 2 4 On the swing mill position of the tube in the mill can considerably influence the result Please consult the instruction manual of the device used Centrifuge the NucleoSpin Bead Tube 30 s at 11 000 x g to clean the lid gt 11 000xg Note In this step foam is displaced from the screw 30 s cap so that the cap can be removed in a clean way MACHEREY NAGEL 09 2015 Rev 01 13 NucleoSpin Microbial DNA 3 Adjust DNA binding conditions Add 600 uL Buffer MG and mix e g vortex for 3 s Note Glass beads should be resuspended some residual pellet cell debris may remain on the bottom 600 pL MG of the tube Mix Centrifuge for 30 s at 11 000 x g 11 000 x g Note This centrifugation step is performed in order Cc 30s to clean the lid and sediment glass beads and cell debris 4 Bind DNA Transfer the supernatant 500 600 uL onto the NucleoSpin Microbial DNA Column placed in a 2 mL Collection Tube provided Load samples Centrifuge for 30 s at 11 000 x g Discard collection asi tube with flow through Put column into a fresh Collection Tube 2 mL provided 5 Wash silica membrane 1 wa
19. pieces Type A 0 6 0 8 mm ceramic beads recommended for soil and sediments NucleoSpin Bead Tube 740812 50 50 pieces Type B 40 400 um glass beads recommended for bacteria NucleoSpin Bead Tube 740813 50 50 pieces Type C 1 3 mm corundum recommended for yeasts Buffer BE 740306 100 125 mL Buffer B5 Concentrate 740921 25 mL for 125 mL Buffer B5 Buffer BW 740922 100 mL Liquid Proteinase K 740396 5 mL RNase A 740505 50 50 mg 740505 Collection Tubes 2 mL 740600 1000 MACHEREY NAGEL 09 2015 Rev 01 17 Genomic DNA from microorganisms 7 3 Product use restriction warranty NucleoSpin Microbial DNA 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 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 respect
20. 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 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
21. sed Perform initial test with water instead of lysis buffer and moderate machine setting low frequency short time in order to avoid spillage of chaotropic lysis buffer in case of tube breakage 2 5 Elution procedures In addition to the standard method several modifications are possible to increase yield concentration and convenience Convenient elution standard elution For convenience elution can be performed by one time addition of 100 uL elution buffer onto the column High yield Two serial elutions of 100 uL each for total elution volume of 200 uL e High concentration Use initial 100 uL eluate for second elution 100 uL total elution volume 2 elutions MACHEREY NAGEL 09 2015 Rev 01 9 Genomic DNA from microorganisms 3 Storage conditions and preparation of working solutions Attention Lysis MG and Wash Buffer BW contain chaotropic salt Wear gloves and goggles CAUTION Buffers MG and BW contain chaotropic salts which can form highly reactive compounds when combines with bleach sodium hypochlorite DO NOT add bleach or acidic solutions directly to the sample preparation waster All kit components can be stored at room temperature 18 25 C and are stable for at least one year Before starting any NucleoSpin Microbial DNA protocol prepare the following Wash Buffer B5 Add the indicated volume of ethanol 96 100 to Wash Buffer B5 Concentrate Mark the label of the bottle to indicat
22. sh 500 pL BW Add 500 uL Buffer BW Centrifuge for 30s at 11 089 g 11 000 x g Discard flow through and place the 30s column back into the Collection Tube ER eS 500 pL BS Add 500 uL Buffer B5 to the column and centrifuge for 30s at 11 000 x g Discard flow through and 11 000 x g place the column back into the Collection Tube 30s 6 Dry silica membrane Centrifuge the column for 30 s at 11 000 x g 11 000 x g Note Residual wash buffer is removed in this step ate 7 Elute highly pure DNA 100 pL BE Place the NucleoSpin Microbial DNA Column into a 1 5 mL nuclease free tube not provided and add RT 100 uL Buffer BE onto the column Incubate at room 1 min temperature for 1 min Centrifuge 30 s at 11 000 x g 11 000 x g For alternative elution procedures see section 2 5 CD 30 s 14 MACHEREY NAGEL 09 2015 Rev 01 NucleoSpin Microbial DNA 6 Support protocols 6 1 Support protocol for yeast e g Saccharomyces cerevisiae Optimal DNA yields from yeast samples can be obtained by following the standard protocol using NucleoSpin Bead Tube Type C see ordering information on section 7 2 instead of NucleoSpin Bead Tube Type B provided with the NucleoSpin Microbial DNA kit The agitation is recommended at a Retsch Schwingm hle MM300 12 min at 30 Hz For other disruption devices please check section 2 4 Please note that the position of the tube within the machine is important for optimal performance please consu
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