NucleoSpin® 96 Tissue - MACHEREY
Contents
1. Step 3 Place the manifold lid on top of the manifold base Step 2 Place the MN Wash Plate in the manifold Step 2 Place the Rack of Tube Strips in the manifold Step 1 Insert spacers MTP MULTI 96 PLATE and waste container in the manifold base Step 1 Insert spacers MICROTUBE RACK in the manifold base Final setup Final setup MACHEREY NAGEL 05 2014 Rev 07 23 NucleoSpin 96 Tissue vacuum processing Detailed protocol For hardware requirements refer to section 2 3 For detailed information regarding the vacuum manifold setup see page 26 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 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 Lyse samples 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 well of the Round well Block containing the samples Close the wells with2. Genomic DNA from tissue User manual NucleoSpin 96 Tissue NucleoSpin 96 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 96 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 14 5 Protocols 16 5 1 NucleoSpin 96 Tissue centrifuge processing 16 5 2 NucleoSpin 96 Tissue vacuum processing 21 6 Appendix 28 6 1 Troubleshooting 28 6 2 Ordering information 30 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 96 Tissue 2 x 96 preps 4x96preps 24x96 preps REF 740741 2 740741 4 740741 24 Lysis Buffer T1 50 mL 100 mL 6x 100 mL Binding Buffer BQ1 50 mL 100 mL 6x100 mL Wash Buffer B5 Concentrate 100 mL 2x100mL 12 x 100 mL Wash Buffer BW 125 mL 2x 125 mL 12 x 125 mL Elution Buffer BE 60 mL 125 mL 6x 125 mL Proteinase K lyophilized 2x75mg 4 x 75 mg 24 x 75 mg Proteinase Buffer PB 8 mL 15 mL 6x15 mL oo 2 a Round well Blocks 2 4 24 MN Square well Blocks 2 4 24 MN Wash Plates 2 4 24
3. Rev 07 Genomic DNA from tissue Protocol step Adjust binding conditions Bind DNA to the membrane Elute DNA Suitable consumables not supplied with the core kits 48 x Cap Strips per 4 x 96 preps 4 x MN Square well Block per 4 x 96 preps 4 x MN Wash Plate per 4 x 96 preps 2 x MN Square well Block 4 x Rack of Tubes Strips with Cap Strips per 4 x 96 preps or 4 x Round well Block with Cap Strips per 4 x 96 preps centrifugation reusable Remarks When using Round well Block or Tube Strips for lysis new Cap Strips are required for sealing of wells after adding Buffer BQ1 and ethanol Recommended for automated processing only MN Wash Plate minimizes the risk of cross contamination vacuum processing only For waste collection during MACHEREY NAGEL 05 2014 Rev 07 Genomic DNA from tissue 2 5 Automated processing on robotic platforms NucleoSpin 96 Tissue can be used fully automated on many common laboratory workstations For the availability of scripts and general considerations about adapting NucleoSpin 96 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 elu
4. 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 amp Co KG All used names and denotations can be brands trademarks or registered labels of their respective owner a
5. Rack of Tube Strips 2 4 24 Cap Strips 24 48 288 Self adhering PE Foil 5 10 60 User manual 1 1 6 1 The kit for 24 x 96 preparations REF 740741 24 consists of 6 x REF 740741 4 For preparation of working solutions and storage conditions see section 3 3 Elution Buffer BE 5 mM Tris HCl 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 05 2014 Rev 07 Genomic DNA from tissue 1 4 Kit contents continued NucleoSpin 96 Tissue Core Kit 4 x 96 preps REF 740454 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 15mL NucleoSpin Tissue Binding 4 Plates green rings User manual 1 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 96 Tissue Core Kit reduced kit composition REF 740454 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 ti
6. 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 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
7. 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 6 000 x g Clogged wells Transfer lysates to a new Round well Block without disturbing the debris pellet Incomplete passage of lysate in step 4 If no more than 300 500 uL 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 MACHEREY NAGEL 05 2014 Rev 07 29 Genomic DNA from tissue 6 2 Ordering information Product REF Pack of 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 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 Buffer T1 740940 25 25 mL Buffer BQ1 740923 1 1L Buffer B5 Concentrate 740921 100 100 mL for 500 mL Buffer B5 Buffer BW 740922 500 500 mL 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 Round well Block 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
8. 0 C for some minutes and mix well until all of the precipitate is redissolved The performance of the kits is not affected by the salt precipitates Before starting any NucleoSpin 96 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 96 Tissue 2 x 96 preps 4 x 96 preps 24 x 96 preps REF 740741 2 740741 4 740741 24 Wash Buffer B5 100 mL 2x100 mL 12x 100 mL Concentrate Add 400 mL ethanol Add 400 mL ethanol Add 400 mL ethanol to each bottle to each bottle Proteinase K 2x75mg 4 x 75 mg 24x 75 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 to each vial 12 MACHEREY NAGEL 05 2014 Rev 07 Genomic DNA from tissue NucleoSpin 96 Tissue Core Kit 4 x 96 preps REF 740454 4 Wash Buffer B5 2 x 100 mL Concentrate Add 400 mL ethanol to each bottle Proteinase K 4 x 75 mg lyophilized Add 2 6 mL Proteinase Buffer to each vial MACHEREY NAGEL 05 2014 Rev 07 13 Genomic DNA from tissue 4 Safety instructions The f
9. 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 30 MACHEREY NAGEL 05 2014 Rev 07 Genomic DNA from tissue 6 3 Product use restriction warranty NucleoSpin 96 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 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
10. Cap Strips 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 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 Incubate the Round well Block 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 Place a weight on top of the Round well Block in order to prevent the Cap Strips from popping off occasionally Centrifuge the Round well Block 15 s 1 500 x g to collect any condensate from the Cap Strips Remove Cap Strips 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 a new Round well Block not supplied with the kit 24 MACHEREY NAGEL 05 2014 Rev 07 NucleoSpin 96 Tissue vacuum processing 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 individual wells with new Cap Strips supplied Mix by vigorous shaking for 10 15 s Spin bri
11. ble Use elution buffer preheated 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 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 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 down
12. during centrifugation After transfer seal the openings of the plate with Self adhering PE Foil For transfer of the lysate from the Round well Block to the NucleoSpin Tissue Binding Plate we recommend use of an electronic eight channel pipetting device with extra long tips capable of holding more than 650 uL Bind DNA to silica membrane Place the MN Square well Blocks with NucleoSpin Tissue Binding Plates 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 MACHEREY NAGEL 05 2014 Rev 07 19 NucleoSpin 96 Tissue centrifuge processing 6 Wash silica membrane Remove the Self adhering PE Foil and add 500 uL Buffer BW to each well of the NucleoSpin Tissue Binding Plate Seal the plate with a new Self adhering PE Foil and centrifuge again at 5 600 6 000 x g for 2 min Remove the Self adhering PE Foil and add 700 pL Buffer B5 to each well of the NucleoSpin Tissue Binding Plate Seal the plate 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 7 Dry silica membrane Remove the Self adhering PE Foil and place the NucleoSpin Tissue Binding Plate on an
13. efly 10 s 1 500 x g to collect any sample from the Cap Strips Ethanol and Buffer BQ1 can be premixed before addition to the samples if the mixture is to be used up 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 Tissue Binding Plate on top of the manifold Transfer lysates Remove the first Cap Strip and transfer the lysates resulting from step 2 carefully from the Round well Block into the wells of the NucleoSpin Tissue Binding Plate Continue with the next eight samples Do not moisten the rims of the individual wells while dispensing the samples moistened rims may cause cross contamination For transfer of the lysate from the Round well Block to the NucleoSpin Tissue Binding Plate we recommend use of an electronic eight channel pipetting device with extra long tips capable of holding more than 650 uL Bind DNA to silica membrane Apply vacuum until all lysates have passed through the wells of the NucleoSpin Tissue Binding Plate 0 2 bar 5 min Release the vacuum Reduction of atmospheric pressure MACHEREY NAGEL 05 2014 Rev 07 25 NucleoSpin 96 Tissue vacuum processing 6 Wash si
14. ency under vacuum Reduction of atmospheric pressure 26 MACHEREY NAGEL 05 2014 Rev 07 NucleoSpin 96 Tissue vacuum processing Elute DNA Insert spacers Microtube rack into the NucleoVac Vacuum Manifold s short sides Place a Rack of Tube Strips onto the spacer Close the vacuum manifold and place the NucleoSpin Tissue Binding Plate on top Dispense 100 uL preheated Buffer BE onto the membrane Incubate for 3 min at room temperature Apply vacuum for elution 0 4 bar 2 min Release the vacuum and repeat the elution step once For alternative elution procedures see section 2 3 Finally close the Tube Strips with Cap Strips for storage Centrifuge 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 Reage
15. enomic DNA can be prepared typical yields 15 25 ug NucleoSpin 96 Tissue can be processed by vacuum or in a centrifuge The kit allow easy automation on common liquid handling instruments The NucleoSpin 96 Tissue kits allow for the purification of multiples of 96 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 96 Tissue Core Kit provides the buffers Proteinase K and NucleoSpin Tissue Binding Plate 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 96 Tissue Format 96 well plates 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 Aoso Aogo 1 8 1 9 Elution volume 100 200 uL Preparation time 60 min plate excl lysis Binding capacity 40 ug 2 3 Required hardware NucleoSpin 96 Tissue can be processed under vacuum or with centrifugation Certain hardware for processing is required Centrifugation For centrifugation a microtiterplate ce
16. h 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 well of the Round well Block containing the samples Close the wells with Cap Strips 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 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 Incubate the Round well Block 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 Place a weight on top of the Round well Block 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 Round well Block 15 s 1 500 x g to collect any condensate from the Cap Strips Remove Cap St
17. hing difficulties if inhaled Kann bei Einatmen Allergie asthmaartige Symptome oder Atembeschwerden verursa chen 14 MACHEREY NAGEL 05 2014 Rev 07 Genomic DNA from tissue Hazard phrases H 335 May cause respiratory irritation Kann die Atemwege reizen Precaution phrases P 210 P 233 P 261 P 280 P 301 312 P 302 352 P 304 340 P 305 351 338 P 312 P 330 P 332 313 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 rin
18. issue 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 96 Tissue Core Kit The NucleoSpin 96 Tissue Core Kit provides buffers Proteinase K and NucleoSpin Tissue Binding Plates 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 96 Tissue Core Kit follow the standard protocols see section 5 1 and 5 2 Recommended accessories for use of the NucleoSpin 96 Tissue Core Kit are available from MACHEREY NAGEL see ordering information Protocol step Suitable consumables not Remarks supplied with the core kits Lyse samples 4x Round well Block with Cap Strips per 4x 96 preps or For sample lysis 4 x Rack of Tube Strips with Cap Strips per 4 x 96 preps 8 MACHEREY NAGEL 05 2014
19. lica membrane Add 600 pL Buffer BW to each well of the NucleoSpin Tissue Binding Plate Apply vacuum 0 2 bar 5 min until all buffer has passed through the wells of the NucleoSpin Tissue Binding Plate Release the vacuum Add 900 uL Buffer B5 to each well of the NucleoSpin TissueBinding Plate Apply vacuum 0 2 bar 5 min until all buffer has passed through the wells of the NucleoSpin Tissue Binding Plate Release the vacuum Add 900 uL Buffer B5 to each well of the NucleoSpin Tissue Binding Plate Apply vacuum 0 2 bar 5 min until all buffer has passed through the wells of the NucleoSpin Tissue Binding Plate Release the vacuum Remove MN Wash Plate After the final washing step close the valve release the vacuum and remove the NucleoSpin Tissue Binding Plate 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 the NucleoSpin Tissue Binding Plate 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 effici
20. lso 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
21. nded 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 cultured 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 Plate 5 Bind DNA to silica membrane of the 5 600 x g NucleoSpin Tissue Binding Plate 10 min 16 MACHEREY NAGEL 05 2014 Rev 07 NucleoSpin 96 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 MACHEREY NAGEL 05 2014 Rev 07 17 NucleoSpin 96 Tissue centrifuge processing Detailed protocol For hardware requirements refer to section 2 3 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 1 Prepare samples For eac
22. ntrifuge is required This centrifuge must be able to accomodate the NucleoSpin Tissue Binding Plate stacked on a Round or Square well Block and reach accelerations of 5 600 6 000 x g is 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 The NucleoSpin 96 Tissue kit can be used with the NucleoVac 96 Vacuum Manifold see ordering information When using NucleoSpin 96 Tissue 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 Tissue 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 MACHEREY NAGEL 05 2014 Rev 07 7 Genomic DNA from t
23. nts not applied properly Prepare Buffer B5 and Proteinase K solution according to instructions See section 3 Add Buffer BQ1 and ethanol to the lysates before loading them to the wells of the NucleoSpin Tissue Binding Plate 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 lt 7 Use slightly alkaline elution buffer like Buffer BE pH 8 5 RNA contamination 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 28 MACHEREY NAGEL 05 2014 Rev 07 Genomic DNA from tissue Problem Possible cause and suggestions Carry over of ethanol After washing with Buffer B5 centrifuge gt 4 min at 5 600 Poor 6 000 x g in order to remove ethanolic Buffer B5 completely performance and evaporate residual sla by incubating the NucleoSpin of genomic Tissue Binding Plate at 70 C for 10 min DNA in Increase vacuum drying time to 15 min enzymatic reactions Contamination of DNA with inhibitory substances Do not elute DNA with TE buffer EDTA may inhibit enzymatic reactions Repurify DNA and elute in Buffer BE Too much starting material e Repeat the procedure using two mouse
24. 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 perform as warranted Supplementary reference is made to the general business terms and conditions of MACHEREY NAGEL which are printed
25. ollowing components of the NucleoSpin 96 Tissue and NucleoSpin 96 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 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 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 D 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 breat
26. 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 8 Elute DNA Dispense 100 uL preheated Buffer BE 70 C to each well of the NucleoSpin Tissue Binding Plate 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 the NucleoSpin Tissue Binding Plate from the Rack of Tube Strips by lifting the plate at one side carefully as the Tube Strips may stick to the outlets of the NucleoSpin Tissue Binding Plate 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 NucleoSpin 96 Tissue vacuum processing 5 2 NucleoSpin 96 Tissue vacuum processing For hardware requirements refer to section 2 3 For detailed information regarding the vacuum manifold setup see page 26 For detailed information on each step see page 27 For use of the NucleoSpin 96 Tissue Core Kit REF 740454 4 refer to section 2 4 regarding recommended accessories Before starting the
27. 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 Plate 5 Bind DNA to silica membrane of the 0 2 bar NucleoSpin Tissue Binding Plate 5 min Reduction of atmospheric pressure MACHEREY NAGEL 05 2014 Rev 07 21 NucleoSpin 96 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 Reduction of atmospheric pressure 22 MACHEREY NAGEL 05 2014 Rev 07 NucleoSpin 96 Tissue vacuum processing Setup of vacuum manifold Binding Washing steps Elution step Step 4 Step 4 EE Place the NucleoSpin EE Place the NucleoSpin Binding Plate on top of S Paz Binding Plate on top of the manifold lid the manifold lid Step 3 Place the manifold lid on top of the manifold base
28. rips 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 a new Round well Block not supplied with the kit 18 MACHEREY NAGEL 05 2014 Rev 07 NucleoSpin 96 Tissue centrifuge processing 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 individual wells with new Cap Strips supplied Mix by vigorous shaking for 10 15 s Spin briefly 10 s 1 500 x g to collect any sample from the Cap Strips 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 Place a NucleoSpin Tissue 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 lysates Remove the first Cap Strip and transfer the lysates resulting from step 2 carefully from the Round well Block into the wells of the NucleoSpin Tissue Binding Plate Continue with the next eight samples Do not moisten the rims of the individual wells while dispensing the samples moistened rims may cause cross contamination
29. sing 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 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 MACHEREY NAGEL 05 2014 Rev 07 15 NucleoSpin 96 Tissue centrifuge processing 5 1 Protocols NucleoSpin 96 Tissue centrifuge processing For hardware requirements refer to section 2 3 For detailed information on each step see page 21 For use of the NucleoSpin 96 Tissue Core Kit REF 740454 4 refer to section 2 4 regarding recomme
30. ssue 2 Product description 2 1 The basic principle The NucleoSpin 96 Tissue kit is designed for the efficient isolation of high molecular weight genomic DNA from tissue samples or cells With the NucleoSpin 96 Tissue procedure sample 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 Plate 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 96 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 g
31. stream 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 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 96 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 4
32. ted 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 Tissue Binding Plate Drying of the NucleoSpin Tissue 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 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 possi
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