NucleoSpin® 8 Food
Contents
1. Genomic DNA from food User manual NucleoSpin 8 Food July 2015 Rev 05 MACHEREY NAGEL www mn net com Genomic DNA from food Table of contents 1 Components 4 1 1 Kit contents 4 1 2 Reagents to be supplied by user 5 2 Product description 6 2 1 The basic principle 6 2 2 Kit specifications 7 2 3 Required hardware 8 2 4 Automated processing on robotic platforms 9 2 5 Sample storage and homogenization 9 2 6 Elution procedures 10 Storage conditions and preparation of working solutions 11 4 Safety instructions 12 5 Protocols 14 5 1 Important information and advice 14 5 2 NucleoSpin 8 Food centrifuge processing 16 5 3 NucleoSpin 8 Food vacuum processing 20 6 Appendix 26 6 1 Troubleshooting 26 6 2 Ordering information 27 6 3 Product use restriction warranty 29 MACHEREY NAGEL 07 2015 Rev 05 3 Genomic DNA from food 1 Components 1 1 Kit contents NucleoSpin 8 Food 12 x 8 preps 60 x 8 preps REF 740975 740975 5 Lysis Buffer CF 100 mL 500 mL Buffer C4 75 mL 250 mL Wash Buffer CQW 125 mL 3x 125 mL Wash Buffer C5 Concentrate 50 mL 2x100 mL Elution Buffer CE 30 mL 125 mL Proteinase K lyophilized 30 mg 2x75 mg Proteinase Buffer PB 1 8 mL 15 mL NucleoSpin Food Binding 12 60 Strips blue rings Round well Blocks 1 5 MN Wash Plate 2 10 Rack of Tube Strips 2 10 Gas permeable Foil 6 18 User manual 1 1 1 For preparation of working solutions and storage conditions see2. 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 8 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 8 Food 12 x 8 preps 60 x 8 preps REF 740975 740975 5 Wash Buffer C5 50 mL 100 mL Concentrate Add 200 mL ethanol Add 400 mL ethanol Proteinase K 30 mg 2x75 mg Add 1 35 mL Add 3 35 mL Proteinase Buffer PB Proteinase Buffer PB to each vial MACHEREY NAGEL 07 2015 Rev 05 11 Genomic DNA from food 4 Safety instructions The following components of the NucleoSpin 8 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 until 125 mL or 125 g Mindergef hrliche Eigenschaften m ssen bis 125 mL oder 125 g nicht mit H und P S tzen gekennzeichnet werden GHS Hazard Precaution Component Hazard contents symbol phrases phrases Inhalt Gefahrstoff GHS Symbo
3. 20 MACHEREY NAGEL 07 2015 Rev 05 NucleoSpin 8 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 07 2015 Rev 05 21 NucleoSpin 8 Food vacuum processing Setup of vacuum manifold Binding Washing steps Elution step Step 4 Place the NucleoSpin Binding Strips inserted the Column Holder A on top of the manifold lid Unused rows have to be filled with NucleoSpin Dummy Strips 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 Step 4 Place the NucleoSpin Binding Strips inserted the Column Holder A on top of the manifold lid Unused rows have to be filled with NucleoSpin Dummy Strips Step 3 Place the manifold lid 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 07 2015 Rev 05 NucleoSpin 8 Food vacuum processing Detailed
4. Nebel Dampf Aerosol vermeiden P264 Wash thoroughly after handling Nach Handhabung gr ndlich waschen P272 Contaminated work clothing should not be allowed out of the workplace Kontaminierte Arbeitskleidung nicht auBerhalb des Arbeitsplatzes tragen P280 Wear protective gloves protective clothing eye protection face protection Schutzhandschuhe Schutzkleidung Augenschutz Gesichtsschutz tragen P301 312 IF SWALLOWED Call a POISON CENTER doctor if you feel unwell BEI VERSCHLUCKEN Bei Unwohlsein GIFTINFORMATIONSZENTRUM Arzt anrufen P302 352 IF ON SKIN Wash with plenty of water BEI BERUHRUNG MIT DER HAUT Mit viel Wasser waschen P3044340 IF INHALED Remove person to fresh air and keep comfortable for breathing BEI EINATMEN Die Person an die frische Luft bringen und f r ungehinderte Atmung sorgen P30543514338 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 P330 Rinse mouth Mund aussp len P3334313 If skin irritation or rash occurs Get medical advice attention Bei Hautreizung oder ausschlag Arztlichen Rat einholen rztliche Hilfe hinzuziehen P337 313 If eye irritation persists Get medical advice attention Bei anhaltender Augenreizung Arztlichen Rat einholen
5. is possible to empty the MN Square well Blocks after every centrifugation step reducing the amount of MN Square well Blocks needed Vacuum processing For processing 8 well strips under vacuum the Starter Set A see ordering infomation containing Column Holders A and NucleoSpin Dummy Strips is required For automation on laboratory platforms with standard 96 well plate manifolds the use of Starter Set A is also required The NucleoSpin 8 Food kit can be used manually with the NucleoVac 96 Vacuum Manifold see ordering information 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 8 MACHEREY NAGEL 07 2015 Rev 05 Genomic DNA from food 2 4 Automated processing on robotic platforms NucleoSpin 8 Food can be used fully automated on many common laboratory workstations For the availability of scripts and general considerations about a
6. 2015 Rev 05 Genomic DNA from food Table 2 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 MACHEREY NAGEL 07 2015 Rev 05 15 NucleoSpin 8 Food centrifuge processing 5 2 NucleoSpin 8 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 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 uL Proteinase K Mix 65 C 30 min 2 Clear lysate 5 600 x g 20 min 3 Adjust DNA binding conditions 300 pL clear lysate 300 pL C4 300 uL ethanol 96 100 Mix 4 Transfer lysates to NucleoSpin Food Binding Strips 5 Bind DNA to silica membrane of the 5 600 x g NucleoSpin Food Binding Strips 10 min 6 Wash silica membrane 500 uL CQW 5 600 x g 2 min 900 uL C5 5 600 x g 5 min 16 MAC
7. CHEREY NAGEL products are suited for QUALIFIED PERSONNEL ONLY MACHEREY NAGEL products shall in any event only be used wearing adequate PROTECTIVE CLOTHING For detailed information please refer to the respective Material Safety Data Sheet of the product MACHEREY NAGEL products shall exclusively be used in an ADEQUATE TEST ENVIRONMENT MACHEREY NAGEL does not assume any responsibility for damages due to improper application of our products in other fields of application Application on the human body is STRICTLY FORBIDDEN The respective user is liable for any and all damages resulting from such application DNA RNA PROTEIN purification products of MACHEREY NAGEL are suitable for IN VITRO USES ONLY ONLY MACHEREY NAGEL products specially labeled as IVD are also suitable for IN VITRO diagnostic use Please pay attention to the package of the product IN VITRO diagnostic products are expressly marked as IVD on the packaging IF THERE IS NO IVD SIGN THE PRODUCT SHALL NOT BE SUITABLE FOR IN VITRO DIAGNOSTIC USE ALL OTHER PRODUCTS NOT LABELED AS IVD ARE NOT SUITED FOR ANY CLINICAL USE INCLUDING BUT NOT LIMITED TO DIAGNOSTIC THERAPEUTIC AND OR PROGNOSTIC USE No claim or representations is intended for its use to identify any specific organism or for clinical use included but not limited to diagnostic prognostic therapeutic or blood banking It is rather in the responsibility of the user or in any case of resale of the products i
8. ER WARRANTIES OF ANY KIND OR NATURE WHATSOEVER DIRECTLY OR INDIRECTLY EXPRESS OR IMPLIED INCLUDING WITHOUT LIMITATION AS TO THE SUITABILITY REPRODUCTIVITY DURABILITY FITNESS FOR A PARTICULAR PURPOSE OR USE MERCHANTABILITY CONDITION OR ANY OTHER MATTER WITH RESPECT TO MACHEREY NAGEL PRODUCTS In no event shall MACHEREY NAGEL be liable for claims for any other damages whether direct indirect incidental compensatory foreseeable consequential or special including but not limited to loss of use revenue or profit whether based upon warranty contract tort including negligence or strict liability arising in connection with the sale or the failure of MACHEREY NAGEL products to perform in accordance with the stated specifications This warranty is exclusive and MACHEREY NAGEL makes no other warranty expressed or implied The warranty provided herein and the data specifications and descriptions of this MACHEREY NAGEL product appearing in MACHEREY NAGEL published catalogues and product literature are MACHEREY NAGEL s sole representations concerning the product and warranty No other statements or representations written or oral by MACHEREY NAGEL s employees agent or representatives except written statements signed by a duly authorized officer of MACHEREY NAGEL are authorized they should not be relied upon by the customer and are not a part of the contract of sale or of this warranty Product claims are subject to change Theref
9. HEREY NAGEL 07 2015 Rev 05 NucleoSpin 8 Food centrifuge processing 7 Dry silica membrane 5 600 x g 15 min or 37 C 20 min 8 Elute DNA 100 pL CE 70 C 5 600 x g 2 min Optional Repeat elution step once Detailed protocol For hardware requirements refer to section 2 3 For processing of NucleoSpin 8 Food with a centrifuge the NucleoVac 96 Vacuum Manifold and the Starter Kit C are required see ordering information The use of NucleoSpin Food Binding Strips in a Column Holder C allows the isolation of up to n x 8 samples n 1 to 6 Insert as many of the NucleoSpin Food Binding Strips as required into the same positions of each one of the two reusable column holders and place column holders onto the MN Square well Blocks Label the column holders or 8 well strips for later identification Always use 2 Column Holders C containing identical numbers of NucleoSpin Food Binding Strips for centrifugation This avoids the need to balance the centrifuge and allows multiples of 16 samples to be processed in parallel We recommend inserting the NucleoSpin Food Binding Strips around the center of the column holder Before starting the preparation Check if 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 Homo
10. URL MACHEREY NAGEL Inc Tel 41 62 388 55 00 Tel 33 388 68 22 68 Tel 1 484 821 0984 E mail sales ch mn net com E mail sales fr mn net com E mail sales us mn net com A043540 0750 1
11. 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 pL 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 8 Food Binding Strips Otherwise prepare 2 aliquots as described in the protocol and load them step by step onto the NucleoSpin 8 Food Binding Strips 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 14 MACHEREY NAGEL 07
12. amplify only short DNA fragments 80 150 bp 6 MACHEREY NAGEL 07 2015 Rev 05 Genomic DNA from food 2 2 Kit specifications NucleoSpin 8 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 8 Food kits can be used for the identification of GMO DNA or animal components in food and feed NucleoSpin 8 Food allows processing of up to 200 mg material Depending on the individual sample typical yields for NucleoSpin 8 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 8 Food allows parallel purification of multiples of 8 samples NucleoSpin 8 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 Anyunusedwells ofthe NucleoSpin 8 Food Binding Strips should be covered with Self adhering PE Foil see ordering information section 6 2 in order to guarantee a proper vacuum and to protect the unused wells from being contaminated Table 1 Kit specifications at a glance Parameter NucleoSpin 8 F
13. ar supernatant to a Round well Block Add 300 pL Buffer C4 and 300 pL ethanol 96 100 95 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 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 07 2015 Rev 05 23 NucleoSpin 8 Food vacuum processing 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 Insert desired number of NucleoSpin Food Binding Strips in the Column Holder A Use NucleoSpin Dummy Strips to seal unused positions in the column holder Place Column Holder A with inserted NucleoSpin Food Binding Strips on top of the manifold 4 Transfer lysates Transfer samples from the Round well Block into the wells of the NucleoSpin Food Binding Strips 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 Strips 0 2 to 0 4 bar Flow through rate should be about 1 2 drops per seco
14. are well Block Round well Block 1 set consists of 1 Round well Block and 12 Cap Strips MN Wash Plate Cap Strips NucleoVac 96 Vacuum Manifold NucleoVac Vacuum Regulator Starter SetA for processing NucleoSpin 8 well strips on NucleoVac 96 Vacuum Manifold Product 740975 740975 5 740976 2 740976 4 740976 24 740946 740931 740313 125 740505 50 740505 740506 740505 740477 740477 24 740481 740481 24 740476 740476 24 740475 740475 24 740479 740479 24 740478 740478 24 740681 740681 740682 Pack of 12 x 8 preps 60 x 8 preps 2 x 96 preps 4 x 96 preps 24 x 96 preps 1L 25 mL 125 mL 50 mg 100 mg 100 mg 100 mg 4 sets 24 sets 24 24 4 sets 24 sets 24 48 288 MACHEREY NAGEL 07 2015 Rev 05 27 Genomic DNA from food Product Product Pack of Starter Set C 740684 1 for processing NucleoSpin 8 well strips under centrifugation MN Frame 740680 1 Self adhering PE Foil 740676 50 Visit www mn net com for more detailed product information 28 MACHEREY NAGEL 07 2015 Rev 05 Genomic DNA from food 6 3 Product use restriction warranty NucleoSpin 8 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 MA
15. arztliche Hilfe hinzuziehen P342 311 If experiencing respiratory symptoms Call a POISON CENTER doctor Bei Symptomen der Atemwege GIFTINFORMATIONSZENTRUM Arzt anrufen P363 Wash contaminated clothing before reuse Kontaminierte Kleidung vor erneutem Tragen waschen P370 378 In case of fire Use to extinguish Bei Brand zum L schen verwenden P403 235 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 MACHEREY NAGEL 07 2015 Rev 05 13 Genomic DNA from food 5 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 protocol or perform
16. cleic 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 8 Food guarantees good recovery rates for small 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
17. dapting NucleoSpin 8 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 for 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 Strips Drying of the NucleoSpin Food Binding Strips 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 8 Food kit on various liquid handling instruments can also be found at www mn
18. ding Strips Do not moisten the rims of the individual wells while dispensing samples After transfer seal the openings of the NucleoSpin Food Binding Strips with Gas permeable Foil The Gas permeable Foil can be cut into appropriate pieces due to the number of NucleoSpin Food Binding Strips which are to be covered When not using air permeable foil pierce foil to achieve air permeability Bind DNA to silica membrane Place the MN Square well Block with Holder C onto the centrifuge carriers and insert them into 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 Strips is 1 mL Higher volumes resulting from steps 1 3 have to be loaded successively until the complete lysis mixture has been applied 18 MACHEREY NAGEL 07 2015 Rev 05 NucleoSpin 8 Food centrifuge processing Wash silica membrane Remove the Gas permeable Foil and add 500 uL Buffer CQW to each well of the NucleoSpin Food Binding Strips Seal the strips with a new Gas permeable Foil and centrifuge again at 5 600 6 000 x g for 2 min Discard waste collected in the MN Square well Block after this wash step Remove the Gas permeable Foil and add 900 uL Buffer C5 to each well of the NucleoSpin Fo
19. e 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 modifications possible Use elution buffer pre heated at 70 C for one of the following procedures High yield By performing two elution steps with 2 x 100 pL 90 100 96 of bound nucleic acids can be eluted Finally combine eluates and measure yield High concentration Perform one elution step with only 6096 of the volume indicated in the individual protocol Concentration of DNA will be about 30 96 higher than with the standard elution procedure Maximum yield of bound nucleic acids is about 80 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 96 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 Howe
20. genize and lyse samples Homogenize up to 200 mg sample using a commercial homogenizer Transfer homogenized samples into Rack of Tube Strips and add 550 pL Buffer CF pre heated 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 Clear lysate Centrifuge the samples for 20 min at full speed 5 600 6 000 x g Remove Cap Strips MACHEREY NAGEL 07 2015 Rev 05 17 NucleoSpin 8 Food centrifuge processing Adjust DNA binding conditions Transfer 300 pL clear supernatant to a Round well Block Add 300 pL Buffer C4 and 300 uL ethanol 96 100 96 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 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 Transfer lysates Insert the desired number of NucleoSpin Food Binding Strips in the Column Holder C and place it on a MN Square well Block supplied with Starter Set C for collection of flow through Transfer samples from the Round well Block into the wells of the NucleoSpin Food Bin
21. ions 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 Strips on top Dispense 100 uL Buffer CE pre heated to 70 C to each well of the NucleoSpin Food Binding Strips 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 s 8 0 Reduction of atmospheric pressure MACHEREY NAGEL 07 2015 Rev 05 25 Genomic DNA from food 6 Appendix 6 1 Troubleshooting Problem Low DNA yield Degraded DNA 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 materia
22. l H S tze P S tze C4 Guanidine hydrochloride 302 319 264 280 36 50 3014312 Guanidinhydrochlorid 36 50 96 30543514338 WARNING CAS 50 01 1 a 330 337 313 caw Guanidine hydrochloride 226 302 210 233 24 36 and Ethanol 301 312 330 20 35 370 378 Guanidinhydrochlorid 24 36 WARNING 4034235 und Ethanol 20 35 96 ACHTUNG CAS 50 01 1 64 17 5 Proteinase K Proteinase K 90 100 0 e 317 334 261 272 280 3024352 CAS 39450 01 6 3044340 WARNING 3334313 ACHTUNG 3424311 363 Hazard phrases H226 Flammable liquid and vapour Fl ssigkeit und Dampf entz nabar 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 H334 May cause allergy or asthma symptoms or breathing difficulties if inhaled Kann bei Einatmen Allergie asthmaartige Symptome oder Atembeschwerden verursachen Precaution phrases P210 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 12 MACHEREY NAGEL 07 2015 Rev 05 Genomic DNA from food P233 Keep container tightly closed Beh lter dicht verschlossen halten P261 Avoid breathing dust fume gas mist vapours spray Einatmen von Staub Rauch Gas
23. l 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 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 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 Sample dependent problem Highly processed samples may be responsible for impossibility to extract high molecular weight DNA Low DNA quality Sample contains DNA degrading contaminants e g phenolic compounds metabolites Repeat washing step with Buffer CQW 26 MACHEREY NAGEL 07 2015 Rev 05 Genomic DNA from food 6 2 Ordering information Product NucleoSpin 8 Food NucleoSpin 96 Food Buffer CF Buffer C5 Concentrate for 125 mL Buffer C5 Buffer CQW RNase A Proteinase K RNase A lyophilized Rack of Tube Strips 1 set consists of 1 rack 12 strips with 8 tubes each and 12 Cap Strips Square well Block MN Squ
24. n the responsibility of the reseller to inspect and assure the use of the DNA RNA protein purification products of MACHEREY NAGEL for a well defined and specific application MACHEREY NAGEL shall only be responsible for the product specifications and the performance range of MN products according to the specifications of in house quality control product documentation and marketing material This MACHEREY NAGEL product is shipped with documentation stating specifications and other technical information MACHEREY NAGEL warrants to meet the stated specifications MACHEREY NAGEL s sole obligation and the customer s sole remedy is limited to replacement of products free of charge in the event products fail to perform as warranted Supplementary reference is made to the general business terms and conditions of MACHEREY NAGEL which are printed on the price list Please contact us if you wish to get an extra copy There is no warranty for and MACHEREY NAGEL is not liable for damages or defects arising in shipping and handling transport insurance for customers excluded or out of accident or improper or abnormal use of this product defects in products or MACHEREY NAGEL 07 2015 Rev 05 29 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 OTH
25. nd Adjust vacuum strength accordingly 6 Wash silica membrane Add 500 uL Buffer CQW to each well of the NucleoSpin Food Binding Strips Apply vacuum 0 2 bar 5 min until all buffer has passed the wells of the NucleoSpin Food Binding Strips Release the vacuum Add 900 pL Buffer C5 to each well of the NucleoSpin Food Binding Strips Apply vacuum 0 2 bar 5 min until all buffer has passed the wells of the NucleoSpin Food Binding Strips Release the vacuum Add 900 pL Buffer C5 to each well of the NucleoSpin Food Binding Strips Apply vacuum 0 2 bar 5 min until all buffer has passed the wells of the NucleoSpin Food Binding Strips Release the vacuum Reduction of atmospheric pressure 24 MACHEREY NAGEL 07 2015 Rev 05 NucleoSpin 8 Food vacuum processing Remove MN Wash Plate After the final washing step close the valve release the vacuum and remove the NucleoSpin Food Binding Strips 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 Dry silica membrane Insert Column Holder A with the NucleoSpin Food Binding Strips again 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 react
26. net com at Bioanalysis Literature 2 5 Sample storage and homogenization 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 sample with lysis buffer mixtures can be cleared easily and effectively by either centrifugation or with a NucleoSpin Filter REF 740606 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 MACHEREY NAGEL 07 2015 Rev 05 9 Genomic DNA from food 2 6 Elution procedures It is possible to adjust the elution method and th
27. od Binding Strips 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 to 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 Strips 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 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 Strips 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 ethanol in Buffer B5 may inhibit enzymatic reactions and should be removed completely before eluting DNA Elute DNA Place the column holder with NucleoSpin Food Binding Strips on an opened Rack of Tube Strips Dispense 100 uL pre heated buffer CE 70 C to each well of the NucleoSpin Food Binding Strips 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 Column Holder C from the Tube Stri
28. ood Tecchnology Silica membrane technology Format 8 well strips Processing Manual and automated vacuum or centrifugation Sample material 200 mg food or feed Typical yield 0 1 10 ug Aoso Aogo 1 6 1 9 Elution volume 100 200 uL Preparation time 60 min 6 strips excl lysis Binding capacity 30 ug MACHEREY NAGEL 07 2015 Rev 05 7 Genomic DNA from food 2 3 Required hardware NucleoSpin 8 Food can be processed under vacuum or with centrifugation Certain hardware for processing is required Centrifugation For processing the 8 well strips under centrifugation the Starter Set C see ordering information section 6 2 containing Column Holders C NucleoSpin Dummy Strips MN Square well Blocks and Rack of Tube Strips is required For centrifugation with Column Holder C with inserted NucleoSpin Food Binding Strips stacked on a MN Square well Block or Rack of Tube Strips a microtiter plate centrifuge is required This centrifuge must be able to accommodate the above mentioned sandwich 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
29. ore 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 mn net com Trademarks 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 30 MACHEREY NAGEL 07 2015 Rev 05 MACHEREY NAGEL Germany and international Tel 49 24 21 969 0 E mail info mn net com MACHEREY NAGEL GmbH amp Co KG Neumann Neander Str 6 8 52355 D ren Germany Switzerland France USA MACHEREY NAGEL AG MACHEREY NAGEL E
30. protocol For hardware requirements refer to section 2 3 For processing of NucleoSpin 8 Food under vacuum the NucleoVac 96 Vacuum Manifold and the Starter Kit A are required see ordering information Starter Kit A contains the Column Holders A and NucleoSpin Dummy Strips to seal unused rows The use of NucleoSpin Food Binding Strips in a Column Holder A allows the isolation of up to n x 8 samples n 1 to 6 Insert as many NucleoSpin Food Binding Strips as required into the reusable column holder Seal unused wells of NucleoSpin Food Binding Strips with Self adhering PE Foil and close unused wells with NucleoSpin Dummy Strips Place the Column Holder A on the NucleoVac 96 manifold Before starting the preparation Check if Buffer C5 and Proteinase K were prepared according to section 3 Pre heat Buffer CF to 65 C and Buffer CE to 70 C 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 pL cle
31. ps Yields will be 10 20 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 07 2015 Rev 05 19 NucleoSpin 8 Food vacuum processing 5 3 NucleoSpin 8 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 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 uL pre heated 65 C CF 10 uL Proteinase K Mix 65 C 30 min 2 Clear lysate 5 600 x g 20 min 3 Adjust DNA binding conditions 300 pL clear lysate 300 pL C4 300 uL ethanol 96 100 96 Mix Prepare the NucleoVac 96 Vacuum Manifold 4 Transfer lysates to NucleoSpin Food Binding Strips 5 Bind DNA to silica membrane of the 0 2 bar 5 min NucleoSpin Food Binding Strips Reduction of atmospheric pressure
32. section 3 Elution Buffer CE 5 mM Tris HCl pH 8 5 3 Including 12 Cap Strips for each block 4 For use with vacuum only 5 Sets of 1 rack 12 strips with 8 tubes each including Cap Strips 4 MACHEREY NAGEL 07 2015 Rev 05 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 MACHEREY NAGEL 07 2015 Rev 05 5 Genomic DNA from food 2 Product description 2 1 The basic principle NucleoSpin 8 Food is designed for the isolation of genomic DNA from food samples preferably of plant or animal origin The NucleoSpin 8 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 and 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 nu
33. ver EDTA interferes depending on the final concentration with certain downstream applications For optimal performance of isolated DNA in 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 10 MACHEREY NAGEL 07 2015 Rev 05 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
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