NucleoSpin® 96 Food - MACHEREY
Contents
1. Genomic DNA from food User manual NucleoSpin 96 Food April 2014 Rev 04 MACHEREY NAGEL www mn net com Genomic DNA from food Table of contents 1 Components 1 1 Kit contents 1 2 Reagents to be supplied by user 5 2 Product description 5 2 1 The basic principle 5 2 2 Kit specifications 6 2 3 Required hardware 7 2 4 Automated processing on robotic platforms 7 2 5 Sample storage and homogenization 8 2 6 Elution procedures 8 3 Storage conditions and preparation of working solutions 10 4 Safety instructions 11 5 Protocols 13 5 1 Important information and advice 13 5 2 NucleoSpin 96 Food centrifuge processing 15 5 3 NucleoSpin 96 Food vacuum processing 20 6 Appendix 26 6 1 Troubleshooting 26 6 2 Ordering information 27 6 3 Product use restriction warranty 28 MACHEREY NAGEL 04 2014 Rev 04 3 Genomic DNA from food 1 Components 1 1 Kit contents NucleoSpin 96 Food 2 x 96 preps 4x96preps 24x96 preps REF 740976 2 740976 4 740976 24 Lysis Buffer CF 2x100 mL 300 mL 6x 300 mL Binding Buffer C4 75 mL 150 mL 6x150mL Wash Buffer CQW 125 mL 250 mL 6x250mL Wash Buffer C5 Concentrate 100 mL 2 x100 mL 12x 100 mL Elution Buffer CE 60 mL 125 mL 6x125mL Proteinase K lyophilized 2x30 mg 4 x 30 mg 24 x 30 mg Proteinase Buffer PB 8mL 15 mL 6x15mL NucleoSpin Food Binding 2 4 24 Plates blue rings Round well Blocks with Cap Strips 4 24 MN Square well Blocks 2 4 24 52. 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 04 2014 Rev 04 29
3. MN Wash Plates 2 4 24 including six Paper Sheets Racks of Tube Strips with Cap 4 8 48 Strips Gas permeable Foil 6 12 72 User manual 1 1 6 1 The kit for 24 x 96 preparations REF 740976 24 consists of 6 x REF 740976 4 2 For preparation of working solutions and storage conditions see section 3 3 Elution Buffer CE 5 mM Tris HCI pH 8 5 Including 12 Cap Strips for each block 5 For use with vacuum only Sets of 1 rack 12 strips with 8 tubes each including Cap Strips 4 MACHEREY NAGEL 04 2014 Rev 04 Genomic DNA from food 1 2 Reagents to be supplied by user 96 100 ethanol for preparation of working solutions see section 3 For more detailed information regarding special hardware required for centrifuge or vacuum processing see section 2 3 2 Product description 2 1 The basic principle NucleoSpin 96 Food is designed for the isolation of genomic DNA from food samples preferably of plant or animal origin The NucleoSpin 96 Food kits combine the NucleoSpin technology from MACHEREY NAGEL GmbH and GMO experience from GEN IAL GmbH to provide an optimal lysis and purification system for nearly all types of food samples Resulting eluates are ready to use in all types of subsequent detection methods especially in real time and basic PCR technologies GEN IAL is a company which offers contract research and molecular testing services in food and feed stuff Special areas of interest are the development a
4. 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 pre heated 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 Sample was contaminated with DNase Check working area and pipettes Degraded DNA Sample dependent problem Highly processed samples may be responsible for impossibility to extract high molecular weight DNA Low DNA Sample contains DNA degrading contaminants e g phenolic OW compounds metabolites quality Repeat washing step with Buffer CQW 26 MACHEREY NAGEL 04 2014 Rev 04 Genomic DNA from food 6 2 Ordering information Product REF Pack of NucleoSpin 96 Food 740976 2 2 x 96 preps 740976 4 4 x 96 preps 740976 24 24 x 96 preps NucleoSpin 8 Food 740975 12 x 8 preps 740975 5 60 x 8 preps Buffer CF 740946 1L Buffer B5 Concentrate 740921 100 100 mL for 500 mL Buffer B5 Buffer BW 740922 500 500 mL RNase A 740505 50 50 mg 740505 100 mg Proteinase K 740506 100 mg RNase A lyophilized 740505 100 mg Rack of Tube Strips 740477 4 sets 1 set consists of 1 rack 740477 24 24 sets 12 strips with 8 tubes each and 12 Cap Strips Square well Block 740481 4 740481 24 24 MN Square well Block 740476 4 740476 24 24 Round well Blo
5. ACHEREY NAGEL products specially labeled as IVD are also suitable for N VITRO diagnostic use Please pay attention to the package of the product N VITRO diagnostic products are expressly marked as IVD on the packaging IF THERE IS NO IVD SIGN THE PRODUCT SHALL NOT BE SUITABLE FOR N 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 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 perf
6. AGEL 04 2014 Rev 04 13 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 Plant and animal ingredients e g in creme or powder Bacteria Starter cultures etc 14 MACHEREY NAGEL 04 2014 Rev 04 NucleoSpin 96 Food centrifuge processing 5 2 NucleoSpin 96 Food centrifuge processing For hardware requirements refer to section 2 3 For detailed information on each step see page 18 Before starting the preparation Check if Buffer C4 Buffer C5 and Proteinase K were prepared according to section 3 Pre heat Buffer CF to 65 C and Buffer CE to 70 C Protocol at a glance 1 Homogenize samples 0 2 g sample 500 pL pre heated 65 C CF 10 pL Proteinase K Mix 65 C 30 min 2 Clear lysate 5 600 x g 20 min 3 Adjust DNA binding conditions 300 pL clear Iysate 300 uL C4 200 uL ethanol 96 100 96 Mix 4 Transfer lysates to NucleoSpin Food Binding Plate 5 Bind DNA to silica membrane of the 5 600 x g NucleoSpin Food Binding Plate 10 min MACHEREY NAGEL 04 2014 Rev 04 15 NucleoSpin 96 Food centrifuge processi
7. ck 740475 4 sets 1 set consists of 1 Round well 740475 24 24 sets Block and 12 Cap Strips MN Wash Plate 740479 4 740479 24 24 Cap Strips 740478 48 740478 24 288 NucleoVac 96 Vacuum Manifold 740681 1 NucleoVac Vacuum Regulator 740641 1 Self adhering PE Foil 740676 50 Visit www mn net com for more detailed product information MACHEREY NAGEL 04 2014 Rev 04 27 Genomic DNA from food 6 3 Product use restriction warranty NucleoSpin 96 Food 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 respective user is liable for any and all damages resulting from such application DNA RNA PROTEIN purification products of MACHEREY NAGEL are suitable for N VITRO USES ONLY ONLY M
8. ded successively until the complete lysis mixture has been applied Wash silica membrane Remove the Gas permeable Foil and add 500 uL Buffer CQW to each well of the NucleoSpin Food Binding Plate Seal the plate with a new Gas permeable Foil and centrifuge again at 5 600 6 000 x g for 2 min Remove the Gas permeable Foil and add 900 uL Buffer C5 to each well of the NucleoSpin Food Binding Plate Centrifuge for 5 15 min at full speed 5 600 6 000 x g in order to remove Buffer C5 For critical ethanol sensitive applications it is recommended 1o prolong the centrifugation time up to 15 min or incubate at higher temperature Remove the adhesive foil and place the column holder with the NucleoSpin Food Binding Plates into an incubator for 20 min at 37 C to evaporate residual ethanol Removal of ethanol by evaporation at 37 C is more effective than additional prolonged centrifugation 15 min 6 000 x g 18 MACHEREY NAGEL 04 2014 Rev 04 NucleoSpin 96 Food centrifuge processing Dry silica membrane For critical ethanol sensitive applications it is recommended to prolong the centrifugation time up to 15 min or incubate at higher temperature Remove the adhesive foil and place the NucleoSpin Food Binding Plates into an incubator for 20 min at 37 C to evaporate residual ethanol Removal of ethanol by evaporation at 37 C is more effective than prolonged centrifugation 15 min 6 000 x g Note The ethano
9. downstream applications we recommend eluting with the supplied elution buffer and storing it especially long term at 20 C Several freeze thaw cycles will not interfere with most downstream applications Performance of long range PCR e g gt 10 kb or the detection limit of trace amount of DNA species may be reduced after multiple freeze thaw cycles or prolonged storage of eluted DNA at 4 C or room temperature This is due to shearing of DNA or adsorption to surfaces Due to the dead volume of the silica membrane please note that the difference between the dispensed elution buffer volume and the recovered elution buffer volume containing genomic DNA is approximately 20 uL recovered elution volume dispensed elution volume 20 uL MACHEREY NAGEL 04 2014 Rev 04 9 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 Nucl
10. ed with Self adhering PE Foil see ordering information in order to guarantee a proper vacuum and to protect the unused wells from being contaminated Kit specifications at a glance Parameter NucleoSpin 96 Food Tecchnology Silica membrane technology Format 96 well plates Processing Manual and automated vacuum or centrifugation Sample material 200 mg food or feed Typical yield 0 1 10 ug A260 A280 1 6 1 9 Elution volume 100 200 uL Preparation time 120 min plate excl lysis Binding capacity 30 ug MACHEREY NAGEL 04 2014 Rev 04 Genomic DNA from food 2 3 Required hardware NucleoSpin 96 Food can be processed under vacuum or with centrifugation Certain hardware for processing is required Centrifugation For centrifugation a microtiterplate centrifuge is required This centrifuge must be able to accomodate the NucleoSpin Food Binding Plate stacked on a Round or Square well Block and reach accelerations of 5 600 6 000 x g bucket height 85 mm Regarding waste collection suitable consumables e g MN Square well Blocks are necessary and they are not included in the kit For the most convenient handling without the need of emptying and reusing the MN Square well Blocks we recommend using six MN Square well Blocks if two 96 well plates are processed at once see ordering information Alternatively it is possible to empty the MN Square well Blocks after every centrifugation step reducing t
11. ed to 70 C to each well of the NucleoSpin Food Binding Plate Pipette buffer directly onto the membrane Incubate at room temperature for 3 min Apply vacuum for elution 0 4 bar until all the samples have passed For optimal yield it is recommended to repeat this step once incubation not necessary Finally close the Tube Strips with Cap Strips for storage Yields will be 10 20 6 higher when eluting in 200 uL Buffer CE depending on the total amount of DNA The concentration of DNA however will be much lower than with 100 uL Elution can also be done in TE buffer at least pH 8 0 as well Elution efficiency will decrease when using elution buffers with pH x 8 0 Reduction of atmospheric pressure MACHEREY NAGEL 04 2014 Rev 04 25 Genomic DNA from food Genomic DNA from food 6 Appendix 6 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 5 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 Sample contains too much RNA Low DNAyield Add 10 20 uL RNase A solution to the lysis buffer before heat incubation If this is not successful add the enzyme to the cleared lysate and incubate for 30 min
12. eiBen 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 Wear protective gloves eye protection Schutzhandschuhe Augenschutz 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 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 doctor if you feel unwell Bei Unwohlsein GIFTINFORMATIONSZENTRUM Arzt anrufen Rinse mouth Mund aussp len If skin irritation occurs Get medical advice attention Bei Hautreizung Arztlichen Rat einholen rztliche Hilfe hinzuziehen IF skin irritation or a rash occurs Get medical advice attention Bei Hautreizung oder ausschlag Arztlichen Rat einholen rztliche Hilfe hinzuz
13. enize samples 0 2 g sample 500 pL pre heated 65 C CF 10 pL Proteinase K Mix 65 C 30 min 2 Clear lysate 5 600 x g 20 min 3 Adjust DNA binding conditions 300 pL clear Iysate 300 uL C4 200 uL ethanol 96 100 96 Mix Prepare the NucleoVac 96 Vacuum Manifold 4 Transfer lysates to NucleoSpin Food Binding Plate 5 Bind DNA to silica membrane of the 0 2 bar 5 min NucleoSpin Food Binding Plate Reduction of atmospheric pressure 20 MACHEREY NAGEL 04 2014 Rev 04 NucleoSpin 96 Food vacuum processing 6 Wash silica membrane 500 uL COW 0 2 bar 5 min 900 uL C5 0 2 bar 5 min 900 uL C5 0 2 bar 5 min 7 Dry silica membrane 0 6 bar 10 min or 37 C 20 min 8 Elute DNA 100 pL CE 70 C 0 6 bar 2 min Optional Repeat elution step once Reduction of atmospheric pressure MACHEREY NAGEL 04 2014 Rev 04 21 NucleoSpin 96 Food vacuum processing Setup of vacuum manifold Binding Washing steps Step 4 Place the NucleoSpin Binding Plate on top of the manifold lid Step 3 Place the manifold lid on top of the manifold base Step 2 Place the MN Wash Plate in the manifold Step 1 Insert spacers MTP MULTI 96 PLATE and waste container in the manifold base Final setup Elution step Step 4 Place the NucleoSpin Binding Plate on top of the manifold lid Step 3 Place the manifold l
14. eoSpin 96 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 for 6 months at 20 C NucleoSpin 96 Food 2 x 96 preps 4x 96 preps 24 x 96 preps REF 740976 2 740976 4 740976 24 Wash Buffer C5 80 mL 2x 100 mL 12 x 100 mL Concentrate Add 320 mL ethanol Add 100 mL ethanol Add 400 mL ethanol to each bottle to each bottle Proteinase K 2x30 mg 4x 30 mg 24 x 30 mg Add 1 35 mL Add 1 35 mL Add 1 35 mL Proteinase Buffer PB Proteinase Buffer PB Proteinase Buffer PB to each vial to each vial to each vial 10 MACHEREY NAGEL 04 2014 Rev 04 Genomic DNA from food 4 Safety instructions The following components of the NucleoSpin 96 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 Hazard contents GHS symbol Hazard Precaution phrases phrases Inhalt Gefahrs
15. es if the mixture is to be used during the next 3 months Never centrifuge at higher g forces or for longer periods as DNA will precipitate Prepare the NucleoVac 96 Vacuum Manifold Place waste tray into vacuum manifold base Insert spacers labeled MTP MULTI 96 PLATE notched side up and place the MN Wash Plate on them Close the manifold with the manifold lid and place a NucleoSpin Food Binding Plate on top of the manifold MACHEREY NAGEL 04 2014 Rev 04 23 NucleoSpin 96 Food vacuum processing 4 Transfer lysates Place the NucleoSpin Food Binding Plate on aMN Square well Block If using more than one plate label the plates for later identification The use of a second plate placed on a MN Square well Block avoids the need to balance the centrifuge Transfer samples from the Round well Block into the wells of the NucleoSpin Food Binding Plate Do not moisten the rims of the individual wells while dispensing samples 5 Bind DNA to silica membrane Apply vacuum until all lysates have passed through the wells of the NucleoSpin Food Binding Plate 0 2 to 0 4 bar Flow through rate should be about 1 2 drops per second Adjust vacuum strength accordingly 6 Wash silica membrane Add 500 pL Buffer CQW to each well of the NucleoSpin Food Binding Plate Apply vacuum 0 2 bar 5 min until all buffer has passed the wells of the NucleoSpin Food Binding Plate Release the vacuum Add 900 pL Buffer C5
16. he amount of MN Square well Blocks needed Vacuum processing The NucleoSpin 96 Food kit can be used with the NucleoVac 96 Vacuum Manifold see ordering information When using NucleoSpin 96 Food with less than 96 samples Self adhering PE Foil see ordering information should be used in order to close and protect non used wells of the NucleoSpin Food Binding Plate and thus guarantee proper vacuum Establish a reliable vacuum source for the NucleoVac 96 Vacuum Manifold The manifold may be used with a vacuum pump house vacuum or water aspirator We recommend a vacuum of 0 2 to 0 4 bar reduction of atmospheric pressure The use of the NucleoVac Vacuum Regulator see ordering information is recommended Alternatively adjust the vacuum so that during the purification the sample flows through the column with a rate of 1 2 drops per second Depending on the amount of sample being used the vacuum times may need to be increased for complete filtration Additionally a suitable centrifuge for sample preparation steps may be required 2 4 Automated processing on robotic platforms NucleoSpin 96 Food can be used fully automated on many common laboratory workstations For the availability of scripts and general considerations about adapting NucleoSpin 96 Food on a certain workstation please contact MN Full processing under vacuum enables complete automation without the need for centrifugation steps for drying of the binding membrane or f
17. id on top of the manifold base Step 2 Place the Rack of Tube Strips in the manifold Step 1 Insert spacers MICROTUBE RACK in the manifold base Final setup 22 MACHEREY NAGEL 04 2014 Rev 04 NucleoSpin 96 Food vacuum processing Detailed protocol For hardware requirements refer to section 2 3 Before starting the preparation Check if Buffer C4 Buffer C5 and Proteinase K were prepared according to section 3 Pre heat Buffer CF to 65 C and Buffer CE to 70 C 1 Homogenize and lyse samples Homogenize up to 200 mg sample using a commercial homogenizer Transfer homogenized samples into Rack of Tube Strips and add 550 uL Buffer CF pre fheated to 65 C Add 10 uL of Proteinase K solution Close the Tube Strips using Cap Strips and mix by vigorous shaking for 15 30 s Spin briefly for 30 s at 1 500 x g to collect any sample at the bottom of the Tube Strips Incubate at 65 C for 30 min 2 Clear Iysate Centrifuge the samples for 20 min at full speed 5 600 6 000 x g Remove Cap Strips 3 Adjust DNA binding conditions Transfer 300 uL clear supernatant to a Round well Block Add 300 uL Buffer C4 and 200 pL ethanol 96 100 C Close the individual wells with Cap Strips Mix by vigorous vortexing for 15 30 s or by pipetting up and down Spin briefly for 30 s at 1 500 x g to collect any sample from Cap Strips Ethanol and Buffer C4 can be premixed before addition to the sampl
18. iehen 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 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 12 MACHEREY NAGEL 04 2014 Rev 04 Genomic DNA from food 5 1 Protocols Important information and advice Due to the low DNA content in processed food this protocol should be started with up to 0 2 g 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 pre culture 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 see ordering information 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 p
19. l in Buffer B5 may inhibit enzymatic reactions and should be removed completely before eluting DNA Elute DNA Place the NucleoSpin Food Binding Plate on an opened Rack of Tube Strips Dispense 100 pL pre heated buffer CE 70 C to each well of the NucleoSpin Food Binding Plate Dispense the buffer directly onto the membrane Incubate at room temperature for 2 3 min Centrifuge at 5 600 6 000 x g for 2 min Remove the plate from the Tube Strips Yields will be 10 20 6 higher when eluting in 200 uL Buffer CE depending on the total amount of DNA The concentration of DNA however will be much lower than with 100 uL Elution can also be done in TE buffer at least pH 8 0 as well Elution efficiency will decrease when using elution buffers with pH x 8 0 Clean the MN Square well Blocks with detergent and hot water and incubate for 1 5 min in 0 4 M HCI Rinse with water again and autoclave before next use MACHEREY NAGEL 04 2014 Rev 04 19 NucleoSpin 96 Food vacuum processing 5 3 NucleoSpin 96 Food vacuum processing For hardware requirements refer to section 2 3 For detailed information regarding the vacuum manifold setup see page 23 For detailed information on each step see page 24 Before starting the preparation Check if Buffer C4 Buffer C5 and Proteinase K were prepared according to section 3 Pre heat Buffer CF to 65 C and Buffer CE to 70 C Protocol at a glance 1 Homog
20. mall genomic DNA fragments 1 kb from processed complex food matrices e g ketchup or spices which generally have very low DNA contents as well as poor quality degraded DNA We thus recommend the selection of primers which amplify only short DNA fragments 80 150 bp MACHEREY NAGEL 04 2014 Rev 04 5 Genomic DNA from food 2 2 Kit specifications NucleoSpin 96 Food is designed for the 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 NucleoSpin 96 Food kits can be used for the identification of GMO DNA or animal components in food and feed NucleoSpin 96 Food allows processing of up to 200 mg material Depending on the individual sample typical yields for NucleoSpin 96 Food are in the range of 0 1 10 ug DNA The eluted DNA is ready for use in subsequent reactions such as real time PCR GMO detection etc NucleoSpin 96 Food allows parallel purification of multiples of 96 samples NucleoSpin 96 Food can be processed by centrifugation or under vacuum Processing under vacuum allows easy automation on common liquid handling instruments For more information about the automation process and the availability of ready to run scripts for certain platforms please refer to section 2 4 and contact your local distributor or MN directly Any unused wells ofthe NucleoSpin 96 Food Binding Plate should be cover
21. nce 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 Rev 03 Please contact MACHEREY NAGEL GmbH amp Co KG Tel 49 0 24 21 969 270 e mail tech bio 9 mn net com Trademarks NucleoSpin is a registered trademark of MACHEREY NAGEL GmbH
22. nd standardization of detection methods for GMOs as well as animal and microbial species identification and differentiation NucleoSpin silica membrane technology from MACHEREY NAGEL allows fast and effective purification of nucleic acids from various matrices The silica membranes are optimized for high DNA recoveries and low unspecific binding of impurities Nucleic acid extraction After the food samples have been homogenized the DNA can be extracted with lysis buffers containing chaotropic salts denaturing agents and detergents The standard isolation ensures lysis using Lysis Buffer CF a proprietary buffer developed by GEN IAL for food matrices patented Lysis mixtures have to be cleared by centrifugation or filtration in order to remove contaminants and residual cellular debris Afterwards the clear supernatant is mixed with binding buffer and ethanol to adjust binding conditions for optimal binding of DNA 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 and is ready to use in subsequent reactions Food samples are very heterogeneous and contain many different compounds such as fat cocoa or polysaccharides which can lead to suboptimal extraction or subsequent processing of DNA NucleoSpin 96 Food guarantees good recovery rates for s
23. ng Wash silica membrane 500 uL COW 5 600 x g 2 min 900 uL C5 5 600 x g 5 min Dry silica membrane 5 600 x g 15 min or 37 C 20 min Elute DNA 100 pL CE 70 C 5 600 x g 2 min Optional Repeat elution step once 16 MACHEREY NAGEL 04 2014 Rev 04 NucleoSpin 96 Food centrifuge processing Detailed protocol For hardware requirements refer to section 2 3 Before starting the preparation Check if Buffer C4 Buffer C5 and Proteinase K were prepared according to section 3 Pre heat Buffer CF to 65 C and Buffer CE to 70 C For each preparation collect up to 200 mg of sample into an appropriate lysis vessel e g Rack of Tube Strips Homogenize and lyse samples Homogenize up to 200 mg sample using a commercial homogenizer Transfer homogenized samples into Rack of Tube Strips and add 550 uL Buffer CF pre heated to 65 C Add 10 pL of Proteinase K solution Close the Tube Strips using Cap Strips and mix by vigorous shaking for 15 30 s Spin briefly for 30 s at 1 500 x g to collect any sample at the bottom of the Tube Strips Incubate at 65 C for 30 min Clear lysate Centrifuge the samples for 20 min at full speed 5 600 6 000 x g Remove Cap Strips Adjust DNA binding conditions Transfer 300 uL clear supernatant to a Round well Block Add 300 uL Buffer C4 and 200 pL ethanol 96 100 C Close the individual wells with Cap Strips Mix by vigoro
24. or elution However a final elution step by centrifugation is recommended in order to achieve higher concentrated eluted DNA The risk of cross contamination is reduced by optimized vacuum settings during the elution step and by the improved shape of the outlets of the NucleoSpin Food Binding Plate MACHEREY NAGEL 04 2014 Rev 04 7 Genomic DNA from food Drying of the NucleoSpin Food Binding Plates under vacuum is sufficient because the bottom of the plate is protected by the MN Wash Plate during the washing steps As a result it is recommended to integrate the MN Wash Plate into the automated procedure to protect against these wash buffer residues The MN Frame see ordering information can be used to position the disposable MN Wash Plate inside the vacuum chamber This also reduces the risk of cross contamination as common metal adaptors tend to get contaminated by gDNA Thorough cleaning of the vacuum chamber is recommended after each run to prevent DNA containing aerosols from forming Visit MN online at www mn net com or contact your local MACHEREY NAGEL distributor for technical support regarding hardware software setup instructions and selection of the protocol Several application notes of the NucleoSpin 96 Food kit on various liquid handling instruments can also be found at www mn net com at Bioanalysis Literature 2 5 Sample storage and homogenization The lysis procedure is most effective when well homogenized po
25. orm 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 28 MACHEREY NAGEL 04 2014 Rev 04 Genomic DNA from food 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 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 accorda
26. rotocol or perform a RNase A digestion in the eluate before further use Ketchup sauce and similar fluid samples 0 2 g 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 additional 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 Lysis Buffer CF Extraction can be improved by pre incubation 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 up scaled lysis buffer volumes We recommend using a single 300 uL aliquot step 3 of the clear supernatant for further processing with the NucleoSpin 96 Food Binding Plate Otherwise prepare 2 aliquots as described in the protocol and load them step by step onto the NucleoSpin 96 Food Binding Plate For processing large samples of for example 1 g lysis buffer and Proteinase K have to be upscaled per 0 1 g of sample add 275 uL Buffer CF and 5 uL Proteinase K After incubation and clearance of the lysate proceed with step 3 in the protocol and further use of 300 uL of the lysate In order to increase the sensitivity a repeated loading repeated performance of step 3 5 is possible MACHEREY N
27. s possible Use elution buffer pre heated at 70 C for one of the following procedures High yield Perform two elution steps with the volume indicated in the individual protocol About 90 100 of bound nucleic acids can be eluted 8 MACHEREY NAGEL 04 2014 Rev 04 Genomic DNA from food High concentration Perform one elution step with only 60 of the volume indicated in the individual protocol Concentration of DNA will be about 3096 higher than with the standard elution procedure Maximum yield of bound nucleic acids is about 8096 High yield and high concentration Apply half the volume of elution buffer as indicated in the individual protocol incubate for 3 min and centrifuge Apply a second aliquot of elution buffer incubate and centrifuge again Thus about 85 100 of bound nucleic acids are eluted in the standard elution volume at a high concentration Convenient elution For convenience elution buffer of ambient temperature may be used This will result in a slightly lower yield approximately 20 compared to elution with heated elution buffer Elution may also be performed with Tris EDTA buffer TE of pH equal or higher than 8 This will increase DNA stability during long term or multi use storage at 4 C or ambient temperature by inhibiting omnipresent DNases However EDTA interferes depending on the final concentration with certain downstream applications For optimal performance of isolated DNA in
28. to each well of the NucleoSpin Food Binding Plate Apply vacuum 0 2 bar 5 min until all buffer has passed the wells of the NucleoSpin Food Binding Plate Release the vacuum Add 900 pL Buffer C5 to each well of the NucleoSpin Food Binding Plate Apply vacuum 0 2 bar 5 min until all buffer has passed the wells of the NucleoSpin Food Binding Plate Release the vacuum Remove MN Wash Plate After the final washing step close the valve release the vacuum and remove the NucleoSpin Food Binding Plate from the vacuum manifold Put it on a clean paper towel to remove residual EtOH containing wash buffer Remove manifold lid MN Wash Plate and waste container from the vacuum manifold Reduction of atmospheric pressure 24 MACHEREY NAGEL 04 2014 Rev 04 NucleoSpin 96 Food vacuum processing Dry silica membrane Insert the NucleoSpin Food Binding Plate into the lid and close the manifold Apply maximum vacuum at least 0 6 bar for 15 min to dry the membrane completely This step is necessary to eliminate traces of ethanol Note The ethanol in Buffer C5 inhibits enzymatic reactions and has to be removed completely before eluting DNA Finally release the vacuum Elute DNA Insert spacers MICROTUBE RACK into the NucleoVac Vacuum Manifold base Place the Rack of Tube Strips onto the spacer Close manifold and place the NucleoSpin Food Binding Plate on top Dispense 100 pL Buffer CE pre heat
29. toff GHS Symbol H S tze P S tze C4 Guanidine hydrochloride Warning 302 319 264 280 36 50 301 312 Guanidinhydrochlorid 36 50 Achtung 305 351 338 330 337 313 CQW Guanidine hydrochloride Warning 226 302 210 233 24 36 ethanol 35 55 301 312 330 Guanidinhydrochlorid 24 36 Achtung 403 235 Ethanol 24 36 Proteinase K Proteinase K Iyophilized Danger 315 319 261 280 Proteinase K Iyophilisiert D Gefahr 334 335 302 352 304 340 305 351 338 312 332 313 337 313 3424311 4034233 Hazard phrases H 226 H 302 H 315 H 319 H 334 H 335 Flammable liquid and vapour Fl ssigkeit und Dampf entz ndbar Harmful if swallowed Gesundheitssch dlich bei Verschlucken Causes skin irritation Verursacht Hautreizungen Causes serious eye irritation Verursacht schwere Augenreizung May cause allergy or asthma symptoms or breathing difficulties if inhaled Kann bei Einatmen Allergie asthmaartige Symptome oder Atembeschwerden verursachen May cause respiratory irritation Kann die Atemwege reizen MACHEREY NAGEL 04 2014 Rev 04 11 Genomic DNA from food Precaution phrases P 210 P 233 P 261 P 280 P 3014312 P 3024352 P 3044340 P 305 351 338 P 312 P 330 P 3324313 P 3334313 P 3374313 P 3424311 P 4034233 P 4034235 Keep away from heat hot surfaces sparks open flames and other ignition Sources No smoking Von Hitze h
30. us vortexing for 15 30 s or by pipetting up and down Spin briefly for 30 s at 1 500 x g to collect any sample from Cap Strips Ethanol and Buffer C4 can be premixed before addition to the samples if the mixture is to be used during the next 3 months Never centrifuge at higher g forces or for longer periods as DNA will precipitate MACHEREY NAGEL 04 2014 Rev 04 17 NucleoSpin 96 Food centrifuge processing Transfer lysates Place the NucleoSpin Food Binding Plate on a MN Square well Block If using more than one plate label the plates for later identification The use of a second plate placed on a MN Square well Block avoids the need to balance the centrifuge Transfer samples from the Round well Block into the wells of the NucleoSpin Food Binding Plate Do not moisten the rims of the individual wells while dispensing samples After transfer seal the openings of the NucleoSpin Food Binding Plate with Gas permeable Foil Bind DNA to silica membrane Place the NucleoSpin Food Binding Plate on a MN Square well Block and place both in the rotor buckets Centrifuge at 5 600 6 000 x g for 5 min Typically the lysates will have passed through the silica membrane within a few minutes The centrifugation process can be extended to 20 min if the lysates have not passed completely The volume of each well of the NucleoSpin Food Binding Plate is 1 mL Higher volumes resulting from steps 1 3 have to be loa
31. wdered 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 sample with lysis buffer mixtures can be cleared easily and effectively by either centrifugation or with a NucleoSpin Plasmid Filter Plate REF 740483 Methods to homogenize sample Commercial homogenizers for example Crush Express for 96 well homogenization Saaten Union Resistenzlabor GmbH Tissue Striker KisanBiotech or Geno Grinder 2000 are suitable Homogenizing samples by VA steel beads diameter 7 mm Put 4 5 beads and food material together into a 15 mL plastic tube Falcon chill the tube in liquid nitrogen and vortex for about 30 seconds e g with a Multi Pulse Vortexer Repeat this chilling and vortexing procedure until the entire material is ground to a powder Chill the tube once more and remove the beads by rolling them out gently or with 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 material attached to the beads 2 6 Elution procedures It is possible to adjust the elution method and the volume of the elution buffer to the subsequent application of interest In addition to the standard method described in the protocols recovery rate about 70 90 96 there are several modification
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