NucleoSpin® Food - MACHEREY
Contents
1. Genomic DNA from food User manual NucleoSpin Food July 2014 Rev 11 MACHEREY NAGEL www mn net com Genomic DNA from food Protocol at a glance Rev 11 NucleoSpin Food 1 Homogenize sample Homogenize 200 mg material 2 Lyse cells 550 uL CF 65 C 10 uL Proteinase K 65 C 30 min cS gt 10 000 x g 10 min Take clear supernatant 1 vol and continue with step 3 3 Adjust DNA binding conditions 1 vol C4 1 vol ethanol 4 Bind DNA Load sample stepwise maximum loading capacity 750 uL es 11 000 x g 1 min 5 Wash and dry silica 400 uL CQW t membrane 1 wash 11 000 x g 1 min 700 uL C5 d ae Wash 11 000 x g 1 min 200 uL C5 3 wash 11 000 x g 2 min 6 Elute DNA 100 uL CE 70 C RT 5 min 11 000 x g ed 1 min 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 food Table of contents 1 Components 1 1 Kit contents 1 2 Reagents consumables and equipment to be supplied by user 1 3 About this user manual 2 Product description 2 1 The basic principle 2 2 Kit specifications 2 3 Storage and homogenization of samples 2 4 Methods to homogenize samples 2 5 Elution procedures 3 Storage conditions and preparation of working solutions 4 Safety instructions 5 General remarks 5 1 Important infor2. 342 311 403 233 405 Hazard phrases H 226 Flammable liquid and vapour Fl ssigkeit und Dampf entz ndbar H 302 Harmful if swallowed Gesundheitssch dlich bei Verschlucken H 315 Causes skin irritation Verursacht Hautreizungen H 317 May cause an allergic skin reaction Kann allergische Hautreaktionen verursachen H 319 Causes serious eye irritation Verursacht schwere Augenreizung H 334 May cause allergy or asthma symptoms or breathing difficulties if inhaled Kann bei Einatmen Allergie asthmaartige Symptome oder Atembeschwerden verursachen H 335 May cause respiratory irritation Kann die Atemwege reizen 10 MACHEREY NAGEL 07 2014 Rev 11 Genomic DNA from food Precaution phrases P 210 P 233 P 261 P271 P 280 P 301 312 P 302 352 P 304 340 P 305 351 338 P 312 P 330 P 333 313 P 337 313 P 342 311 P 403 233 P 403 235 P 405 Keep away from heat hot surfaces sparks open flames and other ignition sources No smoking Von Hitze hei en Oberfl chen Funken offenen Flammen sowie anderen Z ndquellenarten fernhalten Nicht rauchen Keep container tightly closed Beh lter dicht verschlossen halten Avoid breathing dust Einatmen von Staub vermeiden Use only outdoors or in a well ventilated area Nur im Freien oder in gut bel fteten R umen verwenden Wear protective gloves eye protection Schutzhandschuhe Augenschutz tragen IF SWALLOWED Call a PO
3. an einem gut bel fteten Ort aufbewahren Store locked up Unter Verschluss 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 A The symbol shown on labels refers to further safety information in this section Das auf Etiketten dargestellte Symbol weist auf weitere Sicherheitsinformationen dieses Kapitels hin MACHEREY NAGEL 07 2014 Rev 11 11 Genomic DNA from food 5 1 General remarks Important information and advice Due to the low DNA content in processed food this protocol should be started with up to 200 mg of material Lysis buffer was tested see list on the next page for extraction of DNA from various types of samples including food of plant and animal origin and bacteria To detect bacterial DNA in food samples we recommend an overnight preculture of sample and appropriate culture medium Centrifuge an aliquot of the culture and start the preparation with the bacterial pellet RNase A not included in the kit addition may be recommended for RNA rich samples Add 10 uL 20 mg mL stock solution per 550 uL lysis buffer in step 2 of the protocol or perform an RNase A digestion in the eluate before further use A vacuum manifold can optionally be used for acceleration of washing steps Loading and elution steps should be done by centrifugation as described in the protocol Ke
4. 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 MACHEREY NAGEL 07 2014 Rev 11 17 Genomic DNA from food 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 con
5. for 6 months at 20 C NucleoSpin Food 10 preps 50 preps 250 preps REF 740945 10 740945 50 740945 250 nn 6 mL 12 mL 50 mL Add 24 mL ethanol Add 48 mL ethanol Add 200 mL ethanol Concentrate 1 2 mg 6 mg 30 mg Proteinase K Add 120 uL Add 600 uL Add 2 7 mL Proteinase Buffer Proteinase Buffer Proteinase Buffer MACHEREY NAGEL 07 2014 Rev 11 9 Genomic DNA from food 4 Safety instructions The following components of the NucleoSpin Food 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 Component Inhalt Precaution phrases P Satze Hazard phrases H S tze Hazard contents GHS symbol Gefahrstoff GHS Symbol C4 Guanidine hydrochloride Warning 302 319 280 301 312 36 50 D 305 351 338 Guanidinhydrochlorid 36 50 Achtung 330 337 313 CQW Guanidine hydrochloride Warning 226 302 210 233 24 36 ethanol 35 55 D D 301 312 330 Guanidinhydrochlorid 24 36 Achtung 403 235 Ethanol 24 36 Proteinase K Proteinase K lyophilized Danger 315 317 261 264 271 Proteinase K lyophilisiert D Gefahr 319 334 280 302 352 335 304 340 305 351 338 312 332 313 337 313
6. not provided capable of holding at least 3 sample volumes Add 1 vol Buffer C4 and 1 vol ethanol e g take 300 uL sample and add 300 uL Buffer C4 and 300 uL ethanol Vortex the mixture for 30 s 4 Homogenize samples 550 pL CF 65 C y 10 pL Proteinase K 65 C 30 min gt 10 000 x g 10 min 1 vol C4 1 vol ethanol 14 MACHEREY NAGEL 07 2014 Rev 11 NucleoSpin Food 4 Bind DNA For each preparation take one NucleoSpin Food Column placed in a Collection Tube Pipette 700 uL mixture onto the column Centrifuge for 1 min at 11 000 x g Discard flow through Repeat the procedure to load the remaining sample 5 Wash and dry silica membrane Pipette 400 pL Buffer CQW onto the NucleoSpin Food Column Centrifuge for 1 min at 11 000 x g Discard flow through Pipette 700 uL Buffer C5 onto the NucleoSpin Food Column Centrifuge for 1 min at 11 000 x g Discard flow through Pipette another 200 pL Buffer C5 onto the NucleoSpin Food Column Centrifuge for 2 min at 11 000 x g in order to remove Buffer C5 completely Residual ethanol from Wash Buffer C5 may inhibit enzymatic reactions 6 Elute DNA Place the NucleoSpin Food Column in a new 1 5 mL microcentrifuge tube not provided Pipette 100 pL Elution Buffer CE preheated to 70 C onto the membrane Incubate for 5 min at room temperature 18 25 C Centrifuge for 1 min at 11 000 x g to elute the DNA For alternati
7. ISON CENTER doctor if you feel unwell BEI VERSCHLUCKEN Bei Unwohlsein GIFTINFORMATIONSZENTRUM Arzt anrufen IF ON SKIN Wash with plenty of water BEI KONTAKT MIT DER HAUT Mit viel Wasser waschen IF INHALED Remove victim to fresh air and keep at rest in a position com fortable for breathing BEI EINATMEN An die frische Luft bringen und in einer Position ruhigstellen die das Atmen erleichtert 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 sp len Call a POISON CENTER or doctor physician if you feel unwell Bei Unwohlsein GIFTINFORMATIONSZENTRUM oder Arzt anrufen Rinse mouth Mund aussp len IF skin irritation or a rash occurs Get medical advice attention Bei Hautreizung oder ausschlag Arztlichen Rat einholen rztliche Hilfe hinzuziehen Get medical advice attention Bei anhaltender Hautreizung Arztlichen Rat einholen rztliche Hilfe hinzuziehen If experiencing respiratory symptoms Call a POISON CENTER doctor Bei Symptomen der Atemwege GIFTINFORMATIONSZENTRUM Arzt anrufen Store in a well ventilated place Keep container tightly closed Beh lter dicht verschlossen an einem gut bel fteten Ort aufbewahren Store in a well ventilated place Keep cool K hl
8. ant and animal ingredients e g in creme or powder Bacteria Starter cultures etc MACHEREY NAGEL 07 2014 Rev 11 13 NucleoSpin Food 6 Protocol for genomic DNA purification from food Before starting the preparation Check if Wash Buffer C5 was prepared according to section 3 Preheat Lysis Buffer CF to 65 C and Elution Buffer CE to 70 C Ethanol 96 100 Homogenize sample Homogenize about 200 mg material with a commercial homogenizer Lyse cells Transfer the resulting powder to a Collection Tube 2 mL and add 550 uL Buffer CF preheated to 65 C Mix carefully 15 s add 10 pL Proteinase K and mix again 2 3 s If the lysis buffer volume is not large enough to dissolve the sample completely add more buffer and Proteinase K proportionally until sample has been totally resuspended Incubate at 65 C for 30 min Afterwards centrifuge the mixture for 10 min gt 10 000 x g to pellet contaminants and cell debris Optional If RNA free DNA is crucial for downstream applications an RNase digest may be performed After incubation at 65 C for 30 min add 10 uL RNase A 20 mg mL stock solution not provided see ordering information per 550 uL lysis buffer mix well and incubate at RT 18 25 C for 30 min Proceed with the protocol with the centrifugation step Adjust DNA binding conditions Transfer clear supernatant from step 2 into a microcen trifuge tube
9. ental 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 18 MACHEREY NAGEL 07 2014 Rev 11 Genomic DNA from food 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
10. for sample disruption and homogenization see section 2 4 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 Food 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 07 2014 Rev 11 5 Genomic DNA from food 2 Product description NucleoSpin isolation technology from MACHEREY NAGEL GmbH and GMO experience from GEN IAL GmbH were combined to provide an optimal Iysis and purification system for nearly alltypes of food samples Resulting eluates are ready to use for all types of subsequent detection methods especially for real time and basic PCR technologies GEN IAL is an established startup company which offers contract research and molecular testing services in food and feed stuff Special areas of interest are the development and standardization of detection methods for GMOs as well as animal and microbial species identification and differentiation NucleoSpin silica memb
11. g in highly concentrated eluates Elution Buffer CE 5 mM Tris HCl pH 8 5 can be replaced by TE buffer or water as well If water is used the pH should be checked and adjusted to pH 8 8 5 since deionized water usually exhibits a pH below 7 Furthermore absorption of CO leads to a decrease in pH of unbuffered solutions MACHEREY NAGEL 07 2014 Rev 11 Genomic DNA from food 3 Storage conditions and preparation of working solutions Attention Buffers C4 and CQW contain guanidine hydrochloride and detergents Wear gloves and goggles CAUTION Buffers C4 and CQW contain guanidine hydrochloride which can form highly reactive compounds when combined with bleach sodium hypochlorite DO NOT add bleach or acidic solutions directly to the sample preparation waste All kit components can be stored at room temperature 18 25 C and are stable up to one year If there is any precipitate present in the buffers warm the buffer to 25 37 C to dissolve the precipitate before use Before starting any NucleoSpin Food protocol prepare the following Wash Buffer C5 Add the indicated volume of ethanol 96 100 to Buffer C5 Concentrate Mark the label of the bottle to indicate that ethanol was added Store Wash Buffer C5 at room temperature 18 25 C for at least one year Before first use of the kit add the indicated volume of Proteinase Buffer PB to dissolve lyophilized Proteinase K Proteinase K solution is stable
12. gation speed was too high Centrifuge at the speed indicated in the protocol Higher velocities and prolonged vortexing can lead to shearing of the DNA DNA lit Sample contains DNA degrading contaminants e g phenolic com is quality pounds metabolites is low Repeat washing step with Buffer CQW 16 MACHEREY NAGEL 07 2014 Rev 11 Genomic DNA from food 7 2 Ordering information Product REF Pack of NucleoSpin Food 740945 10 50 250 10 50 250 NucleoSpin Filters 740606 50 Buffer CF 740946 1L Buffer C4 740366 250 250 mL Proteinase K 740506 100 mg RNase A 740505 50 50 mg 740505 100 mg Collection Tubes 2 mL 740600 1000 Visit www mn net com for more detailed product information 7 3 Product use restriction warranty NucleoSpin Food 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
13. 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 disclaimer NucleoSpin is a trademark of MACHEREY NAGEL GmbH amp Co KG All used names and denotations can be brands trademarks or registered labels of their respective owner also if they are not special denotation To mention products and brands is only a kind of information i e it does not offend against trademarks and brands and can not be seen as a kind of recommendation or assessment Regarding these products or services we can not grant any guarantees regarding selection efficiency or operation MACHEREY NAGEL 07 2014 Rev 11 19
14. mation and advice 6 Protocol for genomic DNA purification from food 7 Appendix 7 1 Troubleshooting 7 2 Ordering information 7 3 Product use restriction warranty ana A A N NOQA 10 12 12 14 16 16 17 17 MACHEREY NAGEL 07 2014 Rev 11 Genomic DNA from food 1 Components 1 1 Kit contents NucleoSpin Food 10 preps 50 preps 250 preps REF 740945 10 740945 50 740945 250 Lysis Buffer CF 12 mL 100 mL 300 mL Buffer C4 10 mL 30 mL 150 mL Wash Buffer CQW 6 mL 30 mL 125 mL Wash Buffer C5 6 mL 12 mL 50 mL Concentrate Elution Buffer CE 13 mL 13 mL 60 mL NucleoSpin Food Columns plus 10 50 250 Collection Tubes Proteinase K Iyophilized 1200 emg 30mg Proteinase Buffer PB 1 8 mL 1 8 mL 8 mL Collection Tubes 2 mL 10 50 250 User manual 1 1 1 For preparation of working solutions and storage conditions see section 3 Composition of Elution Buffer CE 5 mM Tris HCl pH 8 5 4 MACHEREY NAGEL 07 2014 Rev 11 Genomic DNA from food 1 2 Reagents consumables and equipment to be supplied by user Reagents 96 100 ethanol Consumables 1 5 mL microcentrifuge tubes for sample lysis and DNA elution e Disposable pipette tips Equipment Manual pipettors Centrifuge for microcentrifuge tubes Vortex mixer Heating block for incubation at 65 C Incubator or water bath for preheating Lysis Buffer CF to 65 C and Elution Buffer CE to 70 C Equipment
15. ple with lysis buffer mixtures can be cleared easily and effectively by either centrifugation or with NucleoSpin Filters see ordering information MACHEREY NAGEL 07 2014 Rev 11 7 Genomic DNA from food 2 4 2 5 Methods to homogenize samples Pestle and mortar in the presence of liquid nitrogen Commercial homogenizers for example bead mills VA steel beads diameter 7 mm sample available on request Put 4 5 beads and food material together in a 15 mL plastic tube Falcon chill the tube in liquid nitrogen Vortex for about 30 s e g with a Multi Pulse Vortexer contact Sch tt Labortechnik GmbH Postfach 3454 D 37024 G ttingen Germany Repeat this chilling and vortexing procedure until the entire sample is ground to a powder Chill the tube once more and remove the beads by rolling them out gently or by using a magnet Keep the material frozen throughout the whole homogenization procedure Do not add nitrogen to the tube This leads to sticking and loss of sample as it attaches to the beads Elution procedures It is possible to adapt the elution method and volume of elution buffer for the subsequent application of interest Complete yields By performing two elution steps with 2 x 100 uL 90 100 of bound nucleic acids can be eluted Finally combine eluates and measure yield Highly concentrated eluates With minimal elution volumes 25 50 uL about 60 80 of bound nucleic acids can be eluted resultin
16. rane technology from MACHEREY NAGEL allows fast and effective purification of nucleic acids from various matrices The silica membranes are optimized for high DNA recovery and low binding efficiency for impurities 2 1 The basic principle After the food samples have been homogenized the DNA can be extracted with Iysis buffers containing chaotropic salts denaturing agents and detergents The standard isolation ensures lysis using Lysis Buffer CF which was especially developed by GEN IAL for food matrices patent pending Lysis mixtures should be cleared by centrifugation or filtration in order to remove contaminants and residual cellular debris The clear supernatant is then mixed with binding buffer and ethanol to create conditions for optimal binding to the NucleoSpin Silica Membrane which was selected for this purpose due to its unique DNA binding properties After washing with two different buffers for efficient removal of potential PCR inhibitors DNA can be eluted in low salt buffer or water see section 2 5 for details and is ready to use for subsequent reactions Food samples are very heterogeneous and contain many different compounds like fat cocoa or polysaccharides which can lead to suboptimal extraction or subsequent processing of DNA NucleoSpin Food guarantees good recovery for small genomic DNA fragments lt 1 kbp out of processed complex food matrices e g ketchup or spices which generally have very low DNA con
17. tchup sauce and similar fluid samples 200 mg equivalents can be mixed with lysis buffer 500 1000 uL each and incubated with Proteinase K as described in the protocol see ordering information for separately available Lysis Buffer CF For powdered hygroscopic samples more lysis buffer than indicated in the protocol can be used until the lysis solution is at least semi fluid and can be pipetted see ordering information for separately available Lysis Buffer CF Extraction can be improved by preincubation of sample with lysis buffer for 1 2 h According to local law regulations different amounts of sample have to be analyzed for GMO detection for example up to 1 2 g of sample can be used with upscaled lysis buffer volumes We recommend to use a single 300 uL aliquot section 6 step 3 of the clear supernatant for further processing with NucleoSpin Food Columns Otherwise prepare 2 aliquots as described in the protocol and load them step by step onto the NucleoSpin Food Column 12 MACHEREY NAGEL 07 2014 Rev 11 Genomic DNA from food Positively tested samples PCR Food plant origin Raw products maize soja rape etc powder or oil Chocolate products cocoa nougat products Breakfast cereals muesli nut chocolate spread Jam and fruit concentrates Cookies cakes and biscuits Pollen Lecithine Spices Bread Food animal origin Raw and processed products meat sausage pie Cosmetics Pl
18. tents as well as poor quality degraded DNA Because of this we recommend the selection of primers which amplify only short DNA fragments 80 150 bp 6 MACHEREY NAGEL 07 2014 Rev 11 Genomic DNA from food 2 2 Kit specifications NucleoSpin Food is designed for isolation of genomic DNA from food samples preferably of plant or animal origin However bacteria can also be processed see section 5 1 for details The NucleoSpin Food kit can be used for the identification of GMO DNA or animal components in food and feed NucleoSpin Food standard procedure allows processing of up to 200 mg material Depending on the individual sample typical yields for NucleoSpin Food are in the range of 0 1 10 ug DNA The eluted DNA is ready to use for subsequent reactions like real time PCR GMO detection etc Table 1 Kit specifications at a glance Parameter NucleoSpin Food Format Mini spin column Sample material 5 200 mg Fragment size 300 bp approx 50 kbp Typical yield 0 1 10 ug A seo Avan 1 6 1 9 Elution volume 100 uL Preparation time 30 min 6 preps Binding capacity 30 ug 2 3 Storage and homogenization of samples The lysis procedure is most effective when well homogenized powdered samples are used To achieve this we recommend grinding with a pestle and mortar in the presence of liquid nitrogen or using steel beads Commercial homogenizers can also be used After homogenization and treatment of the sam
19. trol product documentation and marketing material This MACHEREY NAGEL product is shipped with documentation stating specifications and other technical information MACHEREY NAGEL warrants to meet the stated specifications MACHEREY NAGEL s sole obligation and the customer s sole remedy is limited to replacement of products free of charge in the event products fail to perform as warranted Supplementary reference is made to the general business terms and conditions of MACHEREY NAGEL which are printed on the price list Please contact us if you wish to get an extra copy There is no warranty for and MACHEREY NAGEL is not liable for damages or defects arising in shipping and handling transport insurance for customers excluded or out of accident or improper or abnormal use of this product defects in products or components not manufactured by MACHEREY NAGEL or damages resulting from such non MACHEREY NAGEL components or products MACHEREY NAGEL makes no other warranty of any kind whatsoever and SPECIFICALLY DISCLAIMS AND EXCLUDES ALL OTHER WARRANTIES OF ANY KIND OR NATURE WHATSOEVER DIRECTLY OR INDIRECTLY EXPRESS OR IMPLIED INCLUDING WITHOUT LIMITATION AS TO THE SUITABILITY REPRODUCTIVITY DURABILITY FITNESS FOR A PARTICULAR PURPOSE 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 incid
20. ve elution procedures see section 2 6 Load sample 11 000 x g 1 min 400 uL caw 11 000 x g 1 min 700 uL C5 11 000 x g 1 min 200 uL C5 11 000 x g 2 min Load sample RT 5 min 11 000 x g 1 min MACHEREY NAGEL 07 2014 Rev 11 15 Genomic DNA from food 7 Appendix 7 1 Troubleshooting Problem Possible cause and suggestions Homogenization of food material was not sufficient For most species we recommend grinding with steel beads see section 2 4 or with commercial bead mills mixers or homogenizers Extraction of DNA from food material during lysis was not sufficient To obtain higher yields of DNA the incubation time in lysis buffer can be prolonged up to overnight DNA yield is Sample contains too much RNA low Add 10 20 uL RNase A solution to the lysis buffer after heat incubation If this is not successful add the enzyme to the cleared lysate and incubate for 30 min at 37 C Suboptimal elution The DNA can be either eluted in higher volumes up to 300 uL or by repeating the elution step up to three times Remember that the elution buffer must be preheated to 70 C prior to elution Also check the pH of the used elution buffer which should be in the range of pH 8 0 8 5 To ensure correct pH use supplied Elution Buffer CE 5 mM Tris HCI pH 8 5 Sample was contaminated with DNase Check working area and pipettes DNA is i degraded Centrifu
Download Pdf Manuals
Related Search