NucleoSpin® 8 PCR Clean-up - MACHEREY
Contents
1. PCR clean up User manual NucleoSpin 8 PCR Clean up NucleoSpin 8 PCR Clean up Core Kit April 2014 Rev 03 MACHEREY NAGEL www mn net com PCR clean up 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 8 2 4 Recommended accessories for use of the NucleoSpin 8 PCR Clean up 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 14 5 1 NucleoSpin 8 PCR Clean up manual vacuum processing 14 5 2 NucleoSpin 8 PCR Clean up elution of DNA using a centrifuge 20 6 Appendix 21 6 1 Troubleshooting 21 6 2 Ordering information 22 6 3 Reference 23 6 4 Product use restriction warranty 23 MACHEREY NAGEL 04 2014 Rev 03 3 PCR clean up 1 Components 1 1 Kit contents NucleoSpin 8 PCR Clean up 12 x 8 preps 60 x 8 preps REF 740668 740668 5 Binding Buffer NT 25 mL 2x75mL Wash Buffer NTS 100 mL 2x 100 mL Concentrate Elution Buffer NE 30 mL 125 mL NucleoSpin PCR Clean up 12 60 Binding Strips yellow rings MN Wash Plates 1 5 Rack of Tube Strips 1 5 Elution Plate U bottom 1 5 User manual 1 1 1 For preparation of working solutions and storage conditions see section 3 2 Composition of Elution Buffer NE 5 mM Tris HCl2. This kit is intended for use under vacuum A support protocol for elution under centrifu gation is included see section 5 2 A support protocol for complete processing under centrifugation is available from our technical service tech bio mn net com Vacuum processing The NucleoSpin 8 PCR Clean up kits can be used manually with the NucleoVac 96 Vacuum Manifold see ordering information For processing the 8 well strips under vacuum the Starter Set A see ordering information containing Column Holders A and NucleoSpin Dummy Strips is required For automation on laboratory platforms with standard 96 well plate manifolds Starter Set A is also required 2 4 Recommended accessories for use of the NucleoSpin 8 PCR Clean up Core Kit The NucleoSpin 8 PCR Clean up Core Kit provides all necessary buffers and NucleoSpin Binding Strips Accessory plates e g mixing plates waste collection plates elution plates or tubes are not provided with the Core Kit This condensed kit composition along with a large variety of separately available accessories allows an 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 PCR Clean up Core Kit follow the protocols see section 5 1 or 5 2 Recommended accessories for use of the NucleoSpin 8 PCR Clean up Core Kit are available from MACHEREY NAGEL For o
3. pH 8 5 3 Including six paper sheets Set of 1 rack 12 strips with 8 tubes each and 12 cap strips 5 Including one Self adhering PE foil 4 MACHEREY NAGEL 04 2014 Rev 03 PCR clean up NucleoSpin 8 PCR Clean up Core Kit 48 x 8 preps REF 740463 4 Binding Buffer NT 2x75 mL Wash Buffer NT3 Concentrate 2 x 100 mL Elution Buffer NE 125 mL NucleoSpin PCR Clean up Binding 48 Strips yellow rings User manual 1 1 2 Reagents to be supplied by user 96 100 ethanol 1 For preparation of working solutions and storage conditions see section 3 Composition of Elution Buffer NE 5 mM Tris HCl pH 8 5 MACHEREY NAGEL 04 2014 Rev 03 5 PCR clean up 2 2 1 Product description The basic principle The NucleoSpin 8 PCR Clean up kit is designed for vacuum based clean up of DNA after amplification in PCR reactions or after enzymatic treatment As described in the NucleoSpin 8 PCR Clean up procedure the addition of chaotropic salt Buffer NT leads to a reversible adsorption of the DNA to the silica membrane in the NucleoSpin 8 PCR Clean up Binding Strips Primers salts nucleotides and proteins polymerases BSA are removed in subsequent washing steps using Buffer NTS Finally highly pure DNA is eluted in Elution Buffer NE 5 mM Tris HCI pH 8 5 or water pH 8 5 and can be used directly for further applications 2 2 Kit specifications NucleoSpin 8 PCR Clean up is designed for
4. removed before eluting the DNA Finally release the vacuum Reduction of atmospheric pressure 18 MACHEREY NAGEL 04 2014 Rev 03 NucleoSpin 8 PCR Clean up manual vacuum processing 8 Insert Rack of Tube Strips Insert spacers Microtube rack notched side up into the grooves located at the short sides of the vacuum manifold Place the Rack of Tube Strips on the spacers inside the manifold base and insert the appropriate number of Tube Strips Insert the column holder with the NucleoSpin PCR Clean up Binding Strips in the manifold lid and place it on the manifold base Alternatively elution can be performed into a 96 well microtiter plate supplied with the kit Insert spacers MTP Multi 96 Plate notched side up into the grooves located at the short sides of the vacuum manifold Place the Elution Plate U bottom on the spacers inside the manifold base Insert the column holder with the NucleoSpin PCR Clean up Binding Strips in the manifold lid and place it on the manifold base 9 Elute DNA Add 75 150 uL Elution Buffer NE 5 mM Tris HCl pH 8 5 or water pH 8 5 to each well of the NucleoSpin PCR Clean up Binding Strips The buffer should be dispensed onto the center of the silica membrane Incubate for 1 3 min at room temperature optionally apply vacuum and collect the eluted DNA After the elution buffer has passed the columns release the vacuum 0 4 to 0 6 bar 1 2 min Re
5. harmful features do not need to be labeled with H and P phrases up to 125 mL or 125 g Mindergef hrliche Eigenschaften m ssen bis 125 mL oder 125 g nicht mit H und P S tzen gekennzeichnet werden Component Hazard contents GHS symbol Hazard Precaution phrases phrases Inhalt Gefahrstoff GHS Symbol H S tze P S tze NT Guanidinium thiocyanate Warning 302 412 260 273 30 60 031 301 312 Guanidiniumthiocyanat 30 60 Achtung 330 Hazard phrases H 031 Contact with acids liberates toxic gas Entwickelt bei Ber hrung mit S ure giftige Gase H 302 Harmful if swallowed Giftig bei Verschlucken H 412 Harmful to aquatic life with long lasting effects Sch dlich f r Wasserorganismen mit langfristiger Wirkung Precaution phrases P 260 Do not breathe dust fume gas mist vapours spray Staub Rauch Gas Nebel Dampf Aerosol nicht einatmen P 273 Avoid release to the environment Freisetzung in die Umwelt vermeiden Mund aussp len P 301 312 IF SWALLOWED Call a POISON CENTER doctor if you feel unwell Bei Verschlucken Bei Unwohlsein GIFTINFORMATIONSZENTRUM Arzt anrufen P 330 Rinse mouth Mund aussp len 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 04 2014 Rev 03 13 NucleoSpin 8 PCR Clean up manual vacuum processing 5 1 Protocols Nucle
6. 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 24 21 969 270 tech bio mn net com MACHEREY NAGEL 04 2014 Rev 03 25
7. the price list Please contact us if you wish to get an extra copy There is no warranty for and MACHEREY NAGEL is not liable for damages or defects arising in shipping and handling transport insurance for customers excluded or out of accident or improper or abnormal use of this product defects in products or components not manufactured by MACHEREY NAGEL or damages resulting from such non MACHEREY NAGEL components or products MACHEREY NAGEL makes no other warranty of any kind whatsoever and SPECIFICALLY DISCLAIMS AND EXCLUDES ALL OTHER WARRANTIES OF ANY KIND OR NATURE WHATSOEVER DIRECTLY OR INDIRECTLY EXPRESS OR IMPLIED INCLUDING WITHOUT LIMITATION AS TO THE SUITABILITY REPRODUCTIVITY DURABILITY FITNESS FOR A PARTICULAR PURPOSE OR USE MERCHANTABILITY CONDITION OR ANY OTHER MATTER WITH RESPECT TO MACHEREY NAGEL PRODUCTS In no event shall MACHEREY NAGEL be liable for claims for any other damages whether direct indirect 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 24 MACHEREY NAGEL 04 2014 Rev 03 PCR clean up the stated specifications This warranty is exclusive and MACHEREY NAGEL makes no other warranty expressed or implied The warranty
8. DNA clean up such as purification of PCR products in the convenient 8 well strip format The 8 well strip format allows the highest flexibility in sample throughput The kit is designed for vacuum use with the NucleoVac 96 Vacuum Manifold and Starter Set A see ordering information section 6 2 This kit provides reagents and consumables for purification of up to 15 ug highly pure PCR products DNA recovery of 75 90 is obtained for DNA fragments of 65 10 000 bp Primers primer dimers nucleotides salts and polymerase are removed effectively The final concentration of the eluted DNA is 50 150 ng uL depending on elution buffer volume used Typically the Aggo Aago ratio is gt 1 8 Eluted PCR products are ready to use for several applications including automated fluorescent sequencing cloning or microarray technology Using the NucleoSpin 8 PCR Clean up kit allows simultaneous processing of up to 48 samples typically within 30 minutes MACHEREY NAGEL 04 2014 Rev 03 PCR clean up Kit specifications at a glance Parameter NucleoSpin 8 PCR Clean up Format 8 well strips Processing Manual and automated vacuum Sample material lt 100 uL PCR reaction mixture Fragment size 65 bp 10 kbp Typical recovery 75 95 A260 A280 1 70 1 80 Elution volume 75 150 uL Preparation time 30 min 6 strips Binding capacity 15 ug MACHEREY NAGEL 04 2014 Rev 03 7 PCR clean up 2 3 Required hardware
9. Prepare the NucleoVac 96 Vacuum Manifold Insert an appropriate number of NucleoSpin PCR Clean up Binding Strips yellow rings into a Column Holder A Close any unused openings ofthe Column Holder A with NucleoSpin Dummy Strips Note Make sure that the NucleoSpin PCR Clean up Binding Strips are inserted tightly into the column holder Uneven or not properly inserted strips may prevent sealing when vacuum is applied to the manifold Insert spacers MTP Multi 96 Plate notched side up into the grooves located on the short sides of the manifold Insert deep waste reservoir into the center of the manifold Place the MN Wash Plate on top of the spacers Insert Column Holder A with inserted NucleoSpin PCR Clean up Binding Strips into the manifold lid and place lid on the manifold base Check and adjust the vacuum pressure difference 0 2 bar Release the vacuum 2 Dispense binding buffer to the NucleoSpin PCR Clean up Binding Strips Add 200 uL Buffer NT to each well of the NucleoSpin PCR Clean up Binding Strips 3 Transfer PCR samples to the NucleoSpin PCR Clean up Binding Strips and mix column wise processing is recommended Mix by pipetting up and down 5 times Optional Pre mix PCR reaction and Buffer NT in a Square well Block etc not supplied MACHEREY NAGEL 04 2014 Rev 03 17 NucleoSpin 8 PCR Clean up manual vacuum processing 4 Bind DNA to silica membrane Apply vacuum by openin
10. age DNA recovery rate 64 bp 60 80 164 bp 70 85 200 bp 70 85 490 bp 85 95 982 bp 85 95 1500 bp 80 2000 bp 75 4000 bp 50 60 MACHEREY NAGEL 04 2014 Rev 03 11 PCR clean up 3 Storage conditions and preparation of working solutions Attention Storage conditions NucleoSpin 8 PCR Clean up 8 PCR Clean up Core Kits should be stored at room temperature 18 25 C and are stable for up to one year Before starting any NucleoSpin 8 PCR Clean up 8 PCR Clean up Core Kit purifica tion prepare the following Wash Buffer NT3 Add the indicated volume of ethanol 96 100 to Buffer NT3 Concentrate Mark the label of the bottle to indicate that ethanol was added Wash Buffer NT3 is stable at room temperature 18 25 C for up to one year NucleoSpin 8 PCR Clean up 12 x 8 preps 60 x 8 preps REF 740668 740668 5 Wash Buffer NT3 100 mL 2 x 100 mL Concentrate Add 400 mL ethanol Add 400 mL ethanol to each bottle NucleoSpin 8 PCR Clean up Core Kit 48 x 8 preps REF 740463 4 Wash Buffer NT3 2x 100 mL Concentrate Add 400 mL ethanol to each bottle 12 MACHEREY NAGEL 04 2014 Rev 03 PCR clean up 4 Safety instructions The following components of the NucleoSpin 8 PCR Clean up and the NucleoSpin 8 PCR Clean up Core Kits contain hazardous contents Wear gloves and goggles and follow the safety instructions given in this section GHS classification Only
11. aper before applying vacuum run pump continuously 8 Insert Rack of Tube Strips 9 Elute DNA 75 150 uL NE Optional Incubate 1 3 min 0 4 to 0 6 bar 1 min Reduction of atmospheric pressure MACHEREY NAGEL 04 2014 Rev 03 15 NucleoSpin 8 PCR Clean up manual vacuum processing Setup of vacuum manifold Binding Washing Elution steps Column Holder A with NucleoSpin Binding Strips inserted Unused rows have to be filled with NucleoSpin Dummy Strips Manifold base with spacers Manifold base with spacers for MTP Multi 96 Plate inserted Microtube Rack inserted Binding Washing step Elution step 16 MACHEREY NAGEL 04 2014 Rev 03 NucleoSpin 8 PCR Clean up manual vacuum processing Detailed protocol For processing of NucleoSpin 8 PCR Clean up under vacuum the NucleoVac 96 Vacuum Manifold is required Before starting the preparation Check if Buffer NT3 was prepared according to section 3 1 Adjust the volume of reaction mixture For PCR reaction volumes below 100 uL Before starting the purification procedure add Tris buffer 10 mM pH 7 0 nuclease free water pH 7 0 7 5 or Elution Buffer NE to adjust the reaction mixture to a final volume of 100 UL Note If less than 100 uL of PCR reaction mixture is used the volume of Binding Buffer NT has to be adjusted It is mandatory that the ratio of Buffer NT PCR reaction mixture is 2 1
12. e outlets Place the Column Holder C with the NucleoSpin PCR Clean up Binding Strips on top of a MN Square well Block not supplied with the kit see ordering information and centrifuge for 10 min at maximum speed gt 4 000 x g optimal 5 800 x g Note Do not use a microtiter plate as a support for the NucleoSpin PCR Clean up Binding Strips Microtiter plates may crack under centrifugation at gt 1 500 x g Place the Column Holder C with the NucleoSpin PCR Clean up Binding Strips on top of a Rack of Tube Strips Dispense Elution Buffer NE 50 150 uL directly onto the silica membrane and incubate for 1 3 min at room temperature Centrifuge for 2 min at maximum speed gt 4 000 x g optimal 5 800 x g to collect the DNA Remove the Rack of Tube Strips containing eluted DNA and close them with Cap Strips for further storage Reduction of atmospheric pressure 20 MACHEREY NAGEL 04 2014 Rev 03 PCR clean up 6 Appendix 6 1 Troubleshooting Problem Possible cause and suggestions Poor DNA yield No ethanol added to Buffer NT3 Concentrate ethanol evapo rated Add indicated volume of ethanol to Buffer NT3 Concentrate and mix Keep bottle tightly closed to prevent evaporation of ethanol Elution conditions are not optimal If possible use a slightly alkaline elution buffer like Buffer NE 5 mM Tris HCl pH 8 5 When using nuclease free water for elution make sure the pH value
13. g the valve and press down the Column Holder A slightly until flow through starts Allow the samples to pass the columns and release vacuum by closing the valve 0 2 to 0 4 bar 1 min 5 Wash silica membrane Add 900 uL Buffer NT3 with ethanol added to each well of the NucleoSpin PCR Clean up Binding Strips Apply vacuum by opening the valve Press down the Column Holder A slightly until flow through starts Allow the buffer to pass the columns Release the vacuum 0 2 to 0 4 bar 1 min Repeat this washing step once 6 Remove MN Wash Plate After the final washing step close the valve release the vacuum and remove the Column Holder A with the inserted NucleoSpin PCR Clean up Binding Strips Remove manifold lid MN Wash Plate and waste container from the vacuum manifold 7 Dry NucleoSpin PCR Clean up Binding Strips Remove any residual washing buffer from the NucleoSpin PCR Clean up Binding Strips If necessary tap the outlets of the NucleoSpin PCR Clean up Binding Strips onto a clean paper sheet supplied with the MN Wash Plate or soft tissue until the droplets stop Insert the Column Holder A with the NucleoSpin PCR Clean up Binding Strips into the lid and close the manifold Apply vacuum of 0 3 to 0 4 bar for at least 10 min to dry the membrane completely This step is necessary to eliminate traces of ethanol Note The ethanol in Buffer NT3 inhibits enzymatic reactions and has to be completely
14. he product IN VITRO diagnostic products are expressiy 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 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 on
15. imer dimers Minimize amount of primers in PCR reaction mixture Make sure that the ratio of binding buffer NT PCR reaction mixture is 2 1 6 2 Ordering information Product REF Pack of NucleoSpin 8 PCR Clean up 740668 12 x 8 preps 740668 5 60 x 8 preps NucleoSpin 8 PCR Clean up Core Kit 740463 4 48 x 8 preps NucleoSpin 96 PCR Clean up 740658 1 1 x 96 preps 740658 2 2 x 96 preps 740658 4 4x 96 preps 740658 24 24 x 96preps NucleoSpin 96 PCR Clean up Core Kit 740464 4 4 x 96 preps MN Wash Plate 740479 4 740479 24 24 Round well Block with Cap Strips 740475 4 sets set consists of 1 Round well Block and 740475 24 24 sets 12 Cap Strips Rack of Tube Strips 740477 4 sets 1 set consists of 1 rack 12 strips with 8 740477 24 24 sets tubes each and 12 Cap Strips Cap Strips 740478 48 740478 24 288 MN Square well Block 740476 4 740476 24 24 Round well Block Low 740487 4 sets set consists of Round well Block Low and 740487 24 24 sets Self adhering Foil 22 MACHEREY NAGEL 04 2014 Rev 03 PCR clean up Product REF Pack of Elution Plate U bottom 740486 24 24 sets with Self adhering Foil Cap Strips 740638 30 Self adhering PE Foil 740676 50 MN Frame 740680 1 NucleoVac 96 Vacuum Manifold 740681 1 NucleoVac Vacuum Regulator 740641 1 Starter Set A 740682 1 for processing NucleoSpin 8 well strips on NucleoVac 96 Vacuum Manifold Starter Set C 740684 1 for processing NucleoSpi
16. is 8 5 Elution efficiencies drop dramatically at pH lt 7 Elution buffer volume is insufficient Optimal elution is achieved for an elution buffer volume of 100 150 uL Do not use less than 75 uL elution buffer Suboptimal performance of PCR product in sequencing reactions problems with downstream applications Carryover of ethanol Be sure to remove all of ethanolic Buffer NT3 after the final washing step Dry the NucleoSpin PCR Clean up Binding Strips for at least 10 min with maximum vacuum Elution of PCR products with TE buffer EDTA may inhibit enzymatic reactions like DNA sequencing Repurify the PCR products and elute with NE buffer or nuclease free water Alternatively the DNA may be precipitated with ethanol and redissolved in buffer NE buffer or nuclease free water Not enough DNA used in sequencing reactions Quantitate DNA by agarose gel electrophoresis before setting up sequencing reactions MACHEREY NAGEL 04 2014 Rev 03 21 PCR clean up Problem Possible cause and suggestions Suboptimal performance of PCR product in sequencing reactions problems with downstream applications continued Contamination of PCR product preparation with ethanol Insufficient drying after final washing step with Buffer NT3 Remaining ethanol may cause problems with downstream applications like DNA sequencing or loading of samples onto agarose gel Eluted DNA contains residual primers pr
17. move the Rack of Tube Strips or Elution Plate U bottom containing eluted DNA and seal them with the supplied Cap Strips for the tube strips or adhesive cover foil for the elution plate for further storage Reduction of atmospheric pressure MACHEREY NAGEL 04 2014 Rev 03 19 NucleoSpin 8 PCR Clean up elution of DNA using a centrifuge 5 2 NucleoSpin 8 PCR Clean up elution of DNA using a centrifuge Eluting the purified DNA in a centrifuge may be necessary when higher concentrations of the final DNA are required for downstream applications Using a centrifuge allows the dispensed volume to be reduced down to 50 75 uL giving a DNA concentration of about 70 200 ng uL depending on elution buffer volume and fragment length Required hardware For centrifugation a microtiterplate centrifuge that can accommodate the NucleoSpin PCR Clean up Binding Strips inserted into a Column Holder C stacked on a rack of Tube Strips is required It is also necessary that the centrifuge reaches accelerations of 5 600 6 000 x g bucket height 85 mm For processing the 8 well strips the Starter Set C see ordering information containing Column Holders C MN Square well Blocks and Rack of Tube Strips is required Stop the method after the final washing step with Buffer NT3 Remove the NucleoSpin PCR Clean up Binding Strips from the manifold s top and tap on a sheet of filter paper to remove residual wash buffer from th
18. n 8 well strips under centrifugation Visit www mn net com for more detailed product information 6 3 Reference Vogelstein B amp Gillespie D 1979 Proc Natl Acad Sci USA 76 615 619 6 4 Product use restriction warranty NucleoSpin 8 PCR Clean up Core Kit components are intended developed designed and sold FOR RESEARCH PURPOSES ONLY except however any other function of the product being expressiy 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 MACHEREY NAGEL 04 2014 Rev 03 23 PCR clean up 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 t
19. oSpin 8 PCR Clean up manual vacuum processing For hardware requirements refer to section 2 3 For detailed information regarding the vacuum manifold set up see page 17 For detailed information on each step see page 18 For use of the NucleoSpin 8 PCR Clean up Core Kit REF 740463 4 refer to section 2 4 regarding recommended accessories Before starting the preparation Check if Buffer NT3 was prepared according to section 3 Set up the vacuum manifold according to the sheme Protocol at a glance Adjust the volume of the reaction mixture For PCR samples lt 100 pL to 100 uL using Tris buffer pH 7 0 7 5 nuclease free water pH 7 0 7 5 or use Buffer NE 2 Dispense binding buffer to NucleoSpin 200 pL NT PCR Clean up Binding Strips 3 Transfer PCR samples to NucleoSpin 100 uL diluted PCR sample PCR Clean up Binding Strips and mix 4 Bind DNA to silica membrane of the 0 2 bar to 0 4 bar NucleoSpin PCR Clean up Binding 1 min Strips by applying vacuum 5 Wash silica membrane 2 x 900 pL NT3 0 2 bar to 0 4 bar 1 min 6 Remove MN Wash Plate Reduction of atmospheric pressure 14 MACHEREY NAGEL 04 2014 Rev 03 NucleoSpin 8 PCR Clean up manual vacuum processing 7 Dry NucleoSpin PCR Clean up Binding 0 3 to 0 4 bar Strips by applying vacuum 10 15 min Optional Dry the outlets of the NucleoSpin PCR Clean up Binding Strips by placing it on a sheet of filter p
20. p and by the improved shape of the outlets of the NucleoSpin PCR Clean up Binding Strips Drying the NucleoSpin PCR Clean up Binding Strips under vacuum is sufficient because the bottom of the strips is protected from buffer residues during the washing steps by the MN Wash Plate As a result we recommend trying to integrate the MN Wash Plate into the automated procedure 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 DNA 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 PCR Clean up kit formerly known as NucleoSpin 8 Extract Il on various liquid handling instruments can also be found at www mn net com at Bioanalysis Literature 2 6 Elution procedures Elution of purified PCR products The efficiency of the DNA elution depends on the pH of the elution buffer Elution is most effective at pH 8 0 8 5 When using nuclease free water for elution the pH value should be checked and if necessary adjusted to 8 0 8 5 Lower pH of the selected elution buffer may lead to lo
21. rdering information please refer to section 6 2 8 MACHEREY NAGEL 04 2014 Rev 03 PCR clean up Protocol step Adjustment of binding conditions Suitable consumables not supplied with the Core Kits 4 8x Round well Block per 48 x 8 preps Remarks Optional If a premix of sample and Binding Buffer NT is favored Binding of DNA to the membrane and wash steps 8x MN Wash Plate per 48 x 8 preps 2x MN Square well Block MN Wash Plate minimizes the risk of cross contamination vacuum processing only For waste collection during centrifugation reusable Elution 8x Rack of Tubes Strips with Cap Strips per 48x8 preps or 8x Round well Block with gt Cap Strips per 48x8 preps or 8x Elution Plate U bottom _ gt For vacuum processing only MACHEREY NAGEL 04 2014 Rev 03 PCR clean up 2 5 Automated processing on robotic platforms NucleoSpin 8 PCR Clean up can be used fully automated on many common laboratory workstations For the availability of scripts and general considerations about adapting NucleoSpin 8 PCR Clean up on a certain workstation please contact MN Full processing under vacuum enables complete automation without the need for centrifugation steps regarding drying of the binding membrane and elution step The risk of cross contamination is reduced by optimized vacuum settings during the elution ste
22. wer recoveries Yield of larger DNA fragments gt 5 10 kbp can be increased by using pre warmed 70 C elution buffer also see table below An elution volume of 75 125 uL Buffer NE as well as a 3 5 min incubation at room temperature 18 25 C of the elution buffer on the silica membrane are recommended See the following table for correlation between the dispensed elution buffer volume and typical recoveries following the standard protocol The recommended dispense volume of elution buffer is 125 uL Correlation between dispensed elution buffer volume and typical recovery Dispensed elution buffer 75 uL 100 uL 125 uL 150 uL 175 uL Recovered elution buffer containing PCR products 30 5 uL 55 5 uL 80 5 uL 10545 uL 1305 uL 10 MACHEREY NAGEL 04 2014 Rev 03 PCR clean up 9 o 9 787 bp product p100 gt on D 252 bp product 3 2 5 p p 80 120 lt T E lt ae zZ a 5 gt 100 D 4 g 2 6 3 g 3 gt o g 80 8 a 5 23 So 6 8 Concentration 40 40 2 60 80 100 120 140 160 180 200 Dispensed elution buffer uL r T T T T T T 1 20 40 60 80 100 120 140 160 Recovered elution buffer uL Figure 1 Recovery rate and concentration depend on elution volume Two different PCR products 252 bp and 787 bp have been purified with the NucleoSpin 8 PCR Clean up kit Average DNA recovery rate depends on the size of PCR product Size of PCR product Aver
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