NucleoSpin® 8 Tissue - MACHEREY
Contents
1. Reduction of atmospheric pressure 22 MACHEREY NAGEL 05 2014 Rev 07 NucleoSpin 8 Tissue 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 to ace the NucleoSpin inding Strips inserted e Column Holder A on p of the manifold lid Unused rows have to be fil PI tol led with NucleoSpin Dummy Strips Step 3 ace the manifold lid on p of the manifold base Step 2 P ace the Rack of Tube Strips in the manifold Step 1 In sert spacers MICROTUBE RACK in Fi the manifold base nal setup MACHEREY NAGEL 05 2014 Rev 07 23 NucleoSpin 8 Tissue vacuum processing Detailed protocol For hardware requirements refer to section 2 3 For processing of NucleoSpin 8 Tissue 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 Nuc2. Genomic DNA from tissue User manual NucleoSpin 8 Tissue NucleoSpin 8 Tissue Core Kit May 2014 Rev 07 MACHEREY NAGEL MN www mn net com Genomic DNA from tissue Table of contents 1 Components 1 1 Kit contents 1 2 Reagents to be supplied by user 5 2 Product description 6 2 1 The basic principle 6 2 2 Kit specifications 6 2 3 Required hardware 7 2 4 Accessories supplied for use of the NucleoSpin 8 Tissue Core Kit 8 2 5 Automated processing on robotic platforms 10 2 6 Elution procedures 10 3 Storage conditions and preparation of working solutions 12 4 Safety instructions 13 5 Protocols 15 5 1 NucleoSpin 8 Tissue centrifuge processing 15 5 2 NucleoSpin 8 Tissue vacuum processing 21 6 Appendix 28 6 1 Troubleshooting 28 6 2 Ordering information 29 6 3 Product use restriction warranty 31 MACHEREY NAGEL 05 2014 Rev 07 3 Genomic DNA from tissue 1 Components 1 1 Kit contents NucleoSpin 8 Tissue 12 x 8 preps 60 x 8 preps Cat No 740740 740740 5 Lysis Buffer T1 50 mL 125 mL Binding Buffer BQ1 25 mL 125 mL Wash Buffer B5 Concentrate 50 mL 2x 100 mL Wash Buffer BW 75 mL 3x 125 mL Elution Buffer BE 60 mL 2x 125 mL Proteinase K Iyophilized 75 mg 5x75 mg Proteinase Buffer PB 8 mL 35 mL NucleoSpin Tissue Binding Strips green rings 2 80 MN Square well Blocks 2 10 MN Wash Plates 1 5 Rack of Tube Strips 1 5 Self adhering PE Foil 5 25 User Manual 4 1 1 For prep
3. 8 preps or 8 x Rack of Tube Strips with Cap Strips E per 48 x 8 preps or 8 x MN Square well Block per 48 x 8 preps Remarks If residual hair and or bones in the lysate must be removed by centrifugation and transfer of the supernatant an additional Round well Block per 96 preps is necessary Bind DNA to the membrane Elute DNA 8x MN Wash Plate per 48 x 8 preps 2 x MN Square well Block 4 x Rack of Tubes Strips with Cap Strips per 4 x 96 preps or 8 x Round well Block with Cap Strips per 48 x 8 preps or 8 x Round well Block Low per 48 x 8 preps MN Wash Plate minimizes the risk of cross contamination vacuum processing only For waste collection during centrifugation reusable For processing under centrifugation MACHEREY NAGEL 05 2014 Rev 07 Genomic DNA from tissue 2 5 Automated processing on robotic platforms NucleoSpin 8 Tissue can be used fully automated on many common laboratory workstations For the availability of scripts and general considerations about adapting NucleoSpin 8 Tissue 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 co
4. NucleoSpin 8 Tissue vacuum processing 5 2 NucleoSpin 8 Tissue vacuum processing For hardware requirements refer to section 2 3 For detailed information regarding the vacuum manifold setup see page 24 For detailed information on each step see page 25 For use of the NucleoSpin 96 Tissue Core Kit REF 740454 4 refer to section 2 4 regarding recommended accessories Before starting the preparation Check if Buffer B5 and Proteinase K were prepared according to section 3 Set incubator or oven to 56 C Preheat Elution Buffer BE to 70 C Protocol at a glance 1 Prepare samples 2 x 0 5 cm mouse tail or up to 20 mg tissue 10 cultured cells or bacteria 2 Lyse samples 180 pL T1 25 uL Proteinase K Mix 56 C gt 6 h 3 Adjust DNA binding conditions 200 uL BQ1 200 uL ethanol 96 100 96 Mix Prepare the NucleoVac 96 Vacuum Manifold 4 Transfer lysates to NucleoSpin Tissue Binding Strips 5 Bind DNA to silica membrane of the 0 2 bar NucleoSpin Tissue Binding Strips 5 min Reduction of atmospheric pressure MACHEREY NAGEL 05 2014 Rev 07 21 NucleoSpin 8 Tissue vacuum processing 6 Wash silica membrane 600 pL BW 900 uL B5 900 uL B5 0 2 bar 5 min each step Remove MN Wash Plate 7 Dry silica membrane 0 6 bar 10 min 8 Elute DNA 100 pL BE 70 C 0 4 bar 2 min Optional Repeat elution step once
5. Proteinase K working solution more than 15 min before addition to the samples Proteinase K tends to self digestion when incubated in Buffer T1 without substrate 24 MACHEREY NAGEL 05 2014 Rev 07 NucleoSpin 8 Tissue vacuum processing Incubate the tubes plate containing the samples at 56 C for at least 6 h for mammalian cells reduce incubation to 10 min bacterial cells may require pre lysis with e g lysozyme or overnight until the samples are completely lysed For optimal lysis mix occasionally during incubation Make sure that the lysis tubes plate are securely closed When using Rack of Tube Strips place a weight on top in order to prevent the Cap Strips from popping off occasionally Centrifuge the tubes plate 15 s 1 500 x g to collect any condensate from the lid of the tube plate Residual hair and or bones in the lysate can be removed by centrifugation 2 min 5 600 6 000 x g and transfer of the supernatant to new microtubes or a new Rack of Tube Strips not supplied with the kit Adjust DNA binding conditions Add 200 uL Buffer BQ1 and 200 pL 96 100 96 ethanol to each sample Again take care not to moisten the rims of the individual wells while dispensing the buffer Close the tubes plate Mix by vigorous shaking for 10 15 s Spin briefly 10 s 1 500 x g to collect any sample from the lid Ethanol and Buffer BQ1 can be premixed before addition to the samples if the mixture is to be used up d
6. well until all of the precipitate is redissolved The performance of the kits is not affected by the salt precipitates Before starting any NucleoSpin 8 Tissue protocol prepare the following Wash Buffer B5 Add the indicated volume of ethanol 96 100 96 to Buffer B5 Concentrate before use Mark the label of the bottle to indicate that ethanol was added Store Wash Buffer B5 at room temperature 18 25 C for up to one year Before first use of the kit add the indicated volume of Proteinase Buffer PB to lyophilized Proteinase K Proteinase K solution is stable at 20 C for up to 6 months NucleoSpin NucleoSpin NucleoSpin 8 Tissue 8 Tissue 8 Tissue Core Kit 12 x 8 preps 60 x 8 preps 4 x 96 preps REF 740740 740740 5 740453 5 Wash Buffer B5 50 mL 2x100 mL 2x 100 mL Concentrate Add 200 mL ethanol Add 400 mL ethanol Add 400 mL ethanol to each bottle to each bottle Proteinase K 75 mg 4 x 75 mg 4x75 mg lyophilized Add 2 6 mL Add 2 6 mL Add 2 6 mL Proteinase Buffer PB Proteinase Buffer PB Proteinase Buffer PB to each vial to each vial 12 MACHEREY NAGEL 05 2014 Rev 07 Genomic DNA from tissue 4 Safety instructions The following components of the NucleoSpin 8 Tissue and NucleoSpin 8 Tissue Core kits contain hazardous contents Wear gloves and goggles and follow the safety instructions given in this section GHS classification Only harmful features need not be labeled with H and P phrases until 125 mL o
7. 33 P 261 P 280 P 3014312 P 3024352 P 304 340 P 305 351 338 P 312 P 330 P 3324313 P 337 313 P 3424311 P 403 233 P 4034235 Keep away from heat hot surfaces sparks open flames and other ignition Sources No smoking Von Hitze heiBen 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 irri
8. Spin 8 Tissue centrifuge processing Incubate the tubes plate containing the samples at 56 C for at least 6 h for mammalian cells reduce incubation to 10 min bacterial cells may require pre lysis with e g lysozyme or overnight until the samples are completely lysed For optimal lysis mix occasionally during incubation Make sure that the lysis tubes plates are securely closed When using Rack of Tube Strips place a weight on top in order to prevent the Cap Strips from popping off occasionally After lysis set the incubator to 70 C for the membrane drying step Centrifuge the tubes plate 15 s 1 500 x g to collect any condensate from the lid of tubes plate Residual hair and or bones in the lysate can be removed by centrifugation 2 min 5 600 6 000 x g and transfer of the supernatant to new microtubes or to a new Rack of Tube Strips not supplied with the kit Adjust DNA binding conditions Add 200 uL Buffer BQ1 and 200 pL 96 100 ethanol to each sample Again take care not to moisten the rims of the individual wells while dispensing the buffer Close the tubes plate Mix by vigorous shaking for 10 15 s Spin briefly 10 s 1 500 x g to collect any sample from the lid Ethanol and Buffer BQ1 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 Insert desired number of Nu
9. aration of working solutions and storage conditions see section 3 2 Elution Buffer BE 5 mM Tris HCl pH 8 5 3 For use with vacuum only 4 Set of 1 rack 12 strips with 8 tubes each Cap Strips included 4 MACHEREY NAGEL 05 2014 Rev 07 Genomic DNA from tissue 1 4 Kit contents continued NucleoSpin 8 Tissue Core Kit 48 x 8 preps REF 740453 4 Lysis Buffer T1 100 mL Binding Buffer BQ1 100 mL Wash Buffer B5 Concentrate 2 x 100 mL Wash Buffer BW 2x 125 mL Elution Buffer BE 125 mL Proteinase K lyophilized 4x 75 mg Proteinase Buffer PB 15 mL NucleoSpin Tissue Binding 48 Strips green rings User manual 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 please see section 2 3 For recommended accessories for use of the flexible NucleoSpin 8 Tissue Core Kit reduced kit composition REF 740453 4 please see section 2 4 1 For preparation of working solutions and storage conditions see section 3 2 Elution Buffer BE 5 mM Tris HCl pH 8 5 MACHEREY NAGEL 05 2014 Rev 07 5 Genomic DNA from tissue 2 Product description 2 1 The basic principle The NucleoSpin 8 Tissue kit is designed for the efficient isolation of high molecular weight genomic DNA from tissue samples or cells With the NucleoSpin 8 Tissue procedure sample
10. ated 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 High concentration Perform one elution step with only 6096 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 80 96 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 10 MACHEREY NAGEL 05 2014 Rev 07 Genomic DNA from tissue Convenient elution For convenience elution buffer of ambient temperature may be used This will result in a slightly lower yield approximately 2096 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 downstream applications we recomm
11. ble 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 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
12. cleoSpin Tissue Binding Strips in the Column Holder C and place it on an MN Square well Block for collection of flow through If using more than one plate label the plates for later identification Transfer lysates Transfer the lysates resulting from step 2 carefully into the wells of the NucleoSpin Tissue Binding Strips When using the Rack of Tube Strips for lysis remove the first Cap Strip and transfer lysates before removing the next Cap Strip Do not moisten the rims of the individual wells while dispensing the samples moistened rims may cause cross contamination during centrifugation After transfer seal the openings of the inserted NucleoSpin Tissue Binding Strips with Self adhering PE Foil 18 MACHEREY NAGEL 05 2014 Rev 07 NucleoSpin 8 Tissue centrifuge processing Bind DNA to silica membrane Place the MN Square well Block with Column Holder C onto the centrifuge carriers and insert them into the rotor buckets Centrifuge at 5 600 6 000 x g for 10 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 Wash silica membrane Remove the Self adhering PE Foil and add 500 uL Buffer BW to each well of the NucleoSpin Tissue Binding Strips Seal strips with a new Self adhering PE Foil and centrifuge again at 5 600 6 000 x g for 2 min Remove the Self adheri
13. cumentation 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 05 2014 Rev 07 31 Genomic DNA from tissue 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 foreseea
14. d 12 Cap Strips Round well Block Low 740487 4 sets 1 set consists of 1 Round well 740487 24 24 sets Block Low and Self adhering PE Foil MN Wash Plate 740479 4 740479 24 24 Cap Strips 740478 48 740478 24 288 Starter Set A 740682 1 for processing NucleoSpin 8 well strips on NucleoVac 96 Vacuum Manifold Starter Set C 740684 1 for processing NucleoSpin 8 well strips under centrifugation MN Frame 740680 1 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 30 MACHEREY NAGEL 05 2014 Rev 07 Genomic DNA from tissue 6 3 Product use restriction warranty NucleoSpin 8 Tissue Core Kit components are intended developed designed and sold FOR RESEARCH PURPOSES ONLY except however any other function of the product being expressly described in original MACHEREY NAGEL product leaflets MACHEREY NAGEL products are intended for GENERAL LABORATORY USE ONLY MACHEREY NAGEL products are suited for QUALIFIED PERSONNEL ONLY MACHEREY NAGEL products shall in any event only be used wearing adequate PROTECTIVE CLOTHING For detailed information please refer to the respective Material Safety Data Sheet of the product MACHEREY NAGEL products shall exclusively be used in an ADEQUATE TEST ENVIRONMENT MACHEREY NAGEL does not assume any responsibility for damages due to improper application of our products
15. e vacuum processing 8 Elute DNA Insert spacers MICROTUBE RACK into the NucleoVac 96 Vacuum Manifold s short sides Place a Rack of Tube Strips onto the spacer Close the vacuum manifold and place the Column Holder A with the NucleoSpin Tissue Binding Strips on top Dispense 100 uL preheated 70 C Buffer BE directly to the bottom of each well Incubate for 3 min at room temperature Apply vacuum for elution 0 4 bar 2 min Release vacuum and repeat elution step once For alternative elution procedures see section 2 3 Finally close Tube Strips with Cap Strips for storage Centrifuge the Rack of Tube Strips shortly to collect all sample at the bottom of the Tube Strips Reduction of atmospheric pressure MACHEREY NAGEL 05 2014 Rev 07 27 Genomic DNA from tissue 6 Appendix 6 1 Troubleshooting Problem Possible cause and suggestions No or poor DNA yield Incomplete lysis Sample has not completely been submerged during heat incubation Cut samples into small pieces Mix well Be sure that the samples are fully submerged in Buffer T1 Proteinase K mixture Incubate until the samples are completely lysed Buffer T1 and Proteinase K have been premixed more than 15 min before addition to the substrate Proteinase K tends to self digestion under optimal reaction conditions in Buffer T1 without substrate Reagents not applied properly Prepare Buffer B5 and Proteinase K solution acco
16. end 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 05 2014 Rev 07 11 Genomic DNA from tissue 3 Storage conditions and preparation of working solutions Attention Buffer BQ1 and BW contain chaotropic salts Wear gloves and goggles CAUTION Buffers BQ1 and BW 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 Storage conditions All components of the NucleoSpin 8 Tissue kits should be stored at room temperature 18 25 C for a maximum of 1 year Storage at lower temperatures may cause precipitation of salts If a salt precipitation is observed incubate the bottle at 30 40 C for some minutes and mix
17. ication of multiples of 8 samples The kits are supplied with accessory plates for highest convenience The kits are designed for manual or automated use in a centrifuge or for use with a vacuum manifold The NucleoSpin 8 Tissue Core Kit provides the buffers Proteinase K and NucleoSpin Tissue Binding Strips only Accessory components e g lysis plates elution plates are not provided with the core kit but can be individually selected from a variety of suitable accessories see section 2 4 for further information This allows highest flexibility for the user 6 MACHEREY NAGEL 05 2014 Rev 07 Genomic DNA from tissue Kit specifications at a glance Parameter NucleoSpin 8 Tissue Format 8 well strips Processing Manual and automated vacuum or centrifugation Sample material Up to 20 mg tissue up to 10 cultured cells bacteria Typical yield 15 25 ug Aoeo Aogo 1 8 1 9 Elution volume 100 200 uL Preparation time 20 min 6 strips excl lysis Binding capacity 40 ug 2 3 Required hardware NucleoSpin 8 Tissue 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 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 Tiss
18. 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 MACHEREY 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 do
19. ips Release the vacuum Add 900 uL Buffer B5 to each well of the NucleoSpin Tissue Binding Strips Apply vacuum 0 2 bar 5 min until all buffer has passed through the wells of the NucleoSpin Tissue Binding Strips Release the vacuum Add 900 uL Buffer B5 to each well of the NucleoSpin Tissue Binding Strips Apply vacuum 0 2 bar 5 min until all buffer has passed through the wells of the NucleoSpin Tissue Binding Strips Release the vacuum Remove MN Wash Plate After the final washing step close the valve release the vacuum and remove the Column Holder A with inserted NucleoSpin Tissue 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 7 Dry silica membrane Insert Column Holder A with the NucleoSpin Tissue Binding Strips again into the lid and close the manifold Apply maximum vacuum at least 0 6 bar for 10 min to dry the membrane completely This step is necessary to eliminate traces of ethanol Note The ethanol in Buffer B5 inhibits enzymatic reactions and has to be removed completely before eluting DNA Finally release the vacuum Buffer volumes are increased compared to processing under centrifugation to improve washing efficiency under vacuum Reduction of atmospheric pressure 26 MACHEREY NAGEL 05 2014 Rev 07 NucleoSpin 8 Tissu
20. leoSpin Tissue 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 Tissue Binding Strips as required into the reusable column holder Seal unused wells of NucleoSpin Tissue Binding Strips with Self adhering PE Foil and close unused wells with Dummy Strips Place the Column Holder on the NucleoVac 96 manifold For detailed information on each step see page 25 For use of the NucleoSpin 8 Tissue Core Kit REF 740453 4 refer to section 2 4 regarding recommended accessories Before starting the preparation Check if Buffer B5 and Proteinase K were prepared according to section 3 Set incubator or oven to 56 C Preheat Elution Buffer BE to 70 C 1 Prepare samples For each preparation cut up to two 0 5 cm pieces 20 mg of mouse tail into appropriate lysis tubes or plates If preparing DNA from rat tails one 0 5 cm piece is sufficient Tissue samples should not exceed 20 mg cultured cells and bacteria should not exceed 10 cells 2 Lysesamples Prepare a Proteinase K working solution For each sample mix 25 uL Proteinase K with 180 uL Buffer T1 and vortex Transfer 200 uL of the resulting solution to each lysis tube containing the samples Close the individual tubes and mix by vigorous shaking for 10 15 s Spin briefly 15 s 1 500 x g to collect any sample at the bottom of the wells The samples must be submerged in the solution Never prepare the
21. lysis is achieved by incubation of the samples in a solution containing SDS and Proteinase K Appropriate conditions for binding of DNA to the silica membrane in the NucleoSpin Tissue Binding Strips are created by addition of large amounts of chaotropic salt and ethanol to the Iysate The binding process is reversible and specific to nucleic acids While DNA is kept on the silica membrane contaminations are removed by washing with two different wash buffers Pure genomic DNA is finally eluted under low ionic strength conditions in a slightly alkaline elution buffer 2 2 Kit specifications NucleoSpin 8 Tissue is designed for the rapid preparation of highly pure genomic DNA from tissue for example mouse and rat tails organ tissue or animal or bacterial cells The purified DNA can be used directly as template for PCR blotting or any kind of enzymatic reactions This kit provides reagents and consumables for purification of up to 40 ug average 20 ug of pure genomic DNA from up to 20 mg tissue samples with an Aggo Acgo ratio between 1 8 and 1 9 and a typical concentration of 100 200 ng uL From up to two 0 5 cm long mouse tail tip section age of mice 4 6 weeks up to 35 ug of pure genomic DNA can be prepared typical yields 15 25 ug NucleoSpin 8 Tissue can be processed by vacuum or in a centrifuge The kit allow easy automation on common liquid handling instruments The NucleoSpin 8 Tissue kits allow for the purif
22. ng PE Foil and add 700 pL Buffer B5 to each well of the NucleoSpin Tissue Binding Strips Seal strips with a new Self adhering PE Foil and centrifuge again at 5 600 6 000 x g for 4 min During this step as much ethanolic Buffer B5 as possible is removed by centrifugation Dry silica membrane Remove the Self adhering PE Foil and place the Column Holder C holding the NucleoSpin Tissue Binding Strips on an opened Rack of Tube Strips Place it in an incubator for 10 min at 70 C to evaporate residual ethanol Removal of ethanol by evaporation at 70 C is more effective than prolonged centrifugation Note The ethanol in Buffer B5 may inhibit enzymatic reactions and should be removed completely before eluting DNA MACHEREY NAGEL 05 2014 Rev 07 19 NucleoSpin 8 Tissue centrifuge processing Elute DNA Dispense 100 uL preheated Buffer BE 70 C to each well of the NucleoSpin Tissue Binding Strips Dispense the buffer directly onto the membrane Incubate at room temperature for 1 min Centrifuge at 5 600 6 000 x gfor 2 min Repeat elution step once Remove Column Holder C with inserted NucleoSpin Tissue Binding Strips from the Rack of Tube Strips For alternative elution procedures see section 2 3 If elution in small volume tubes is desired place a 96 PCR plate not supplied on top of a Round well Block or a Rack of Tube Strips and elute into the PCR plate 20 MACHEREY NAGEL 05 2014 Rev 07
23. ng two mouse tail sections of maximally 4 6 mm length If processing rat tails one 0 5 cm long tail tip section is sufficient Hair or bones left in the lysate after step 2 Centrifuge the Round well Block for 3 min at 5 600 Clogged wells 6 000 x g Transfer lysates to a new Round well Block without disturbing the debris pellet Incomplete passage of lysate in step 4 f no more than 300 500 LL of lysate is remaining in the columns continue with step 5 Through the addition of Buffer BW the sample is diluted and thus the sample will pass the column more easily 6 2 Ordering information Product REF Pack of NucleoSpin 8 Tissue 740740 12 x 8 preps 740740 5 60 x 8 preps NucleoSpin 8 Tissue Core Kit 740453 4 48 x 8 preps NucleoSpin 96 Tissue 740741 2 2 x 96 preps 740741 4 4 x 96 preps 740741 24 24 x 96 preps NucleoSpin 96 Tissue Core Kit 740454 4 4 x 96 preps Buffer T1 740940 25 25 mL Buffer BQ1 740923 1 1L Buffer B5 Concentrate 740921 100 100 mL for 500 mL Buffer B5 MACHEREY NAGEL 05 2014 Rev 07 29 Genomic DNA from tissue Product REF Pack of Buffer BW 740922 500 500 mL Proteinase K 740506 100 mg RNase A lyophilized 740505 100 mg MN Square well Block 740476 4 740476 24 24 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 Round well Block 740475 4 sets 1 set consists of 1 Round well 740475 24 24 sets Block an
24. 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 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 32 MACHEREY NAGEL 05 2014 Rev 07
25. ntamination is reduced by optimized vacuum settings during the elution step and by the improved shape of the outlets of the NucleoSpin Tissue Binding Strips Drying of the NucleoSpin Tissue Binding Strips under vacuum is sufficient because the bottom of the strips 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 gDNA 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 Tissue kit on various liquid handling instruments can also be found at www mn net com at Bioanalysis Literature 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 modifications possible Use elution buffer prehe
26. r 125 g Mindergef hrliche Eigenschaften m ssen bis 125 mL oder 125 g nicht mit H und P S tzen gekennzeichnet werden Component Hazard contents GHS symbol Hazard Precaution phrases phrases Inhalt Gefahrstoff GHS Symbol H S tze P S tze Guanidine hydrochloride Warning 280 301 4312 50 66 lt gt 319 302 352 Guanidinhydrochlorid 50 66 Achtung 305 351 338 330 332 313 337 313 Guanidine hydrochloride 36 50 isopropanol 210 233 280 319 301 312 lt gt Warning 20 50 305 351 338 Guanidinhydrochlorid 36 50 Achtung 330 337 313 Isopropanol 20 50 403 235 315 319 261 280 Proteinase K Proteinase K lyophilized Danger Proteinase K lyophilisiert lt gt Gefahr 334 335 302 352 304 340 305 351 338 312 332 313 337 313 342 311 403 233 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 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 verursa chen H 335 May cause respiratory irritation Kann die Atemwege reizen MACHEREY NAGEL 05 2014 Rev 07 13 Genomic DNA from tissue Precaution phrases P 210 P 2
27. rding to instructions see section 3 Add Buffer BQ1 and ethanol to the lysates before loading them to the wells of the NucleoSpin Tissue Binding Strips Suboptimal elution of DNA from the column Preheat Buffer BE to 70 C before elution Apply Buffer BE directly onto the center of the silica membrane Elution efficiencies decrease dramatically if elution is done with buffers with pH 7 Use slightly alkaline elution buffer like Buffer BE pH 8 5 RNA contamination Poor performance of genomic DNA in enzymatic reactions RNA in sample If DNA free of RNA is desired cool down to room temperature after lysis incubation and add 20 uL of an RNase A solution 20 mg mL see ordering information Incubate for 15 min with moderate shaking Carry over of ethanol After washing with Buffer B5 centrifuge Amin at 5 600 6 000 x g in order to remove ethanolic Buffer B5 completely and evaporate residual ethanol by incubating the NucleoSpin Tissue Binding Strips at 70 C for 10 min Increase vacuum drying time to 15 min 28 MACHEREY NAGEL 05 2014 Rev 07 Genomic DNA from tissue Problem Possible cause and suggestions Poor performance of genomic DNA Contamination of DNA with inhibitory substances Do not elute DNA with TE buffer EDTA may inhibit in enzymatic enzymatic reactions Repurify DNA and elute in Buffer BE reactions continued Too much starting material Repeat the procedure usi
28. re 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 For general consumables and equipment needed please see section 1 2 2 4 Accessories supplied for use of the NucleoSpin 8 Tissue Core Kit The NucleoSpin 8 Tissue Core Kit provides buffers Proteinase K and NucleoSpin Tissue Binding Strips Accessory plates e g lysis plates elution plates are not provided with the core kit The reduced kit composition along with a variety of separately available accessories allow optimal adjustment of the kit to individual user needs The user can select additional consumables according to his her requirements for highest flexibility For use of NucleoSpin 8 Tissue Core Kit follow the standard protocols see section 5 1 and 5 2 Recommended accessories for use of the NucleoSpin 8 Tissue Core Kit are available from MACHEREY NAGEL see ordering information 8 MACHEREY NAGEL 05 2014 Rev 07 Genomic DNA from tissue Protocol step Lyse samples Suitable consumables not supplied with the core kits 8x Round well EN Block with Cap d al Strips AN per 48 x
29. red cells or bacteria 2 Lysesamples 180 uL T1 25 uL Proteinase K Mix 56 C z6h 3 Adjust DNA binding conditions 200 uL BQ1 200 uL ethanol 96 100 96 Mix 4 Transfer lysates to NucleoSpin Tissue Binding Strips 5 Bind DNA to silica membrane of the 5 600 x g NucleoSpin Tissue Binding Strips 10 min MACHEREY NAGEL 05 2014 Rev 07 15 NucleoSpin 8 Tissue centrifuge processing Wash silica membrane 500 uL BW 5 600 x g 2 min 700 uL B5 5 600 x g 4 min Dry silica membrane 70 C 10 min Elute DNA 100 uL BE 70 C 5 600 x g 2 min Optional Repeat elution step once 16 MACHEREY NAGEL 05 2014 Rev 07 NucleoSpin 8 Tissue centrifuge processing Detailed protocol For processing under centrifugation the Starter Kit C and a suitable centrifuge are required see section 2 3 For handling of the 8 well strips and the column holders refer to the protocol of the Starter Kit C The use of NucleoSpin Tissue 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 Tissue 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 Tissue Binding Strips for cent
30. rifugation This avoids the need to balance the centrifuge and allows multiples of 16 samples to be processed in parallel We recommend inserting the NucleoSpin Tissue Binding Strips around the center of the column holder For use of the NucleoSpin 8 Tissue Core Kit REF 740453 5 refer to section 2 4 regarding recommended accessories Before starting the preparation Check if Buffer B5 and Proteinase K were prepared according to section 3 Set incubator or oven to 56 C Preheat Elution Buffer BE to 70 C 1 Prepare samples For each preparation cut up to two 0 5 cm pieces 20 mg of mouse tail into appropriate lysis tubes or plates If preparing DNA from rat tails one 0 5 cm piece is sufficient Tissue samples should not exceed 20 mg cultured cells and bacteria should not exceed 10 cells 2 Lysesamples Prepare a Proteinase K working solution For each sample mix 25 uL Proteinase K with 180 uL Buffer T1 and vortex Transfer 200 uL of the resulting solution to each lysis tube containing the samples Close the individual tubes and mix by vogorous shaking for 10 15 s Spin briefly 15 s 1 500 x g to collect any sample at the bottom of the wells The samples must be submerged in the solution Never prepare the Proteinase K working solution more than 15 min before addition to the samples Proteinase K tends to self digestion when incubated in Buffer T1 without substrate MACHEREY NAGEL 05 2014 Rev 07 17 Nucleo
31. tation occurs Get medical advice attention Bei Hautreizung Arztlichen Rat einholen rztliche Hilfe hinzuziehen Get medical advice attention Bei anhaltender Augenreizung 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 geschlossen 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 14 MACHEREY NAGEL 05 2014 Rev 07 NucleoSpin 8 Tissue centrifuge processing 5 Protocols 5 1 NucleoSpin 8 Tissue centrifuge processing For hardware requirements refer to section 2 3 For detailed information on each step see page 20 For use of the NucleoSpin 8 Tissue Core Kit REF 740453 5 refer to section 2 4 regarding recommended accessories Before starting the preparation Check if Buffer B5 and Proteinase K were prepared according to section 3 Set incubator or oven to 56 C Preheat Elution Buffer BE to 70 C Protocol at a glance 1 Prepare samples 2 x 0 5 cm mouse tail or up to 20 mg tissue 108 cultu
32. ue Binding Strips stacked on a MN Square well Block or Rack of Tube Strips a microtiter plate centrifuge is required which is able to accommodate the above mentioned sandwich and reach accelerations of 5 600 6 000 x gis required 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 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 wit standard 96 well plate manifolds the use of Starter Set Ais also required MACHEREY NAGEL 05 2014 Rev 07 7 Genomic DNA from tissue The NucleoSpin 8 Tissue 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 pressu
33. uring 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 Insert desired number of NucleoSpin Tissue 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 Tissue Binding Strips on top of the manifold Transfer lysates Transfer the lysates resulting from step 2 carefully into the wells of the NucleoSpin Tissue Binding Strips When using the Rack of Tube Strips remove the first Cap Strip and transfer lysates before removing the next Cap Strip Do not moisten the rims of the individual wells while dispensing the samples moistened rims may cause cross contamination MACHEREY NAGEL 05 2014 Rev 07 25 NucleoSpin 8 Tissue vacuum processing 5 Bind DNA to silica membrane Apply vacuum until all lysates have passed through the wells of the NucleoSpin Tissue Binding Strips 0 2 bar 5 min Release the vacuum 6 Wash silica membrane Add 600 uL Buffer BW to each well of the NucleoSpin Tissue Binding Strips Apply vacuum 0 2 bar 5 min until all buffer has passed through the wells of the NucleoSpin Tissue Binding Str
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