NucleoSpin® 96 Trace - MACHEREY
Contents
1. 5 3 NucleoSpin 96 Trace use of the NucleoSpin Trace Filter Plate For hardware requirements refer to section 2 3 Before starting the preparation Check if Buffer B5 and Proteinase K were prepared according to section 3 1 Place the NucleoSpin Trace Filter Plate onto a square well block Add forensic material e g buccal swabs to the wells of the NucleoSpin Trace Filter Plate Premix 25 uL Proteinase K and the minimum volume of Buffer FLB necessary to soak the material completely to the sample Incubate several hours or overnight at room temperature 2 After incubation separate the lysate containing DNA from the forensic material by centrifugation 5 min 5 600 6 000 x g Proceed with step 2 of the general procedure section 5 1 or 5 2 addition of isopropanol 20 MACHEREY NAGEL 04 2014 Rev 03 Genomic DNA from forensic samples 6 Appendix 6 1 Troubleshooting Problem Possible cause and suggestions Poor DNA quality or yield Reagents not applied or prepared properly Reagents were not properly prepared Add the indicated volume of Proteinase Buffer PB to the Proteinase K vial and 96 100 ethanol to Buffer Concentrate B5 and mix Kit storage Store aliquots of the reconstituted Proteinase K at 20 C Store other kit components at room temperature Storage at low temperatures may cause salt precipitation Keep bottles tightly closed in order to prevent evaporation or2. 6 2 Ordering information Product REF Pack of NucleoSpin 96 Trace 740726 2 2 x 96 preps 740726 4 4 x 96 preps NucleoSpin 8 Trace 740722 1 12 x 8 preps 740722 5 60 x 8 preps NucleoSpin Trace Filter Plate 740677 20 NucleoSpin Forensic Filters 740988 10 50 250 10 50 250 pieces NucleoSpin Forensic Filters 740988 50B 250B 1000B 50 250 1000 pieces Bulk Buffer FLB 740322 500 500 mL Buffer BW 740922 100 mL Buffer B5 Concentrate 740322 500 500 mL for 100 mL Buffer B5 Proteinase K 740506 100 mg MN Wash Plate 740675 1 Rack of Tube Strips 740477 4 sets set consists of 1 Rack 740477 24 24 sets 12 Tube Strips with 8 tubes each and 12 Cap Strips 22 MACHEREY NAGEL 04 2014 Rev 03 Genomic DNA from forensic samples Product REF Pack of MN Square well Block 740476 4 sets 740476 24 24 sets NucleoVac 96 Vacuum 740681 1 Manifold NucleoVac Vacuum Regulator 740641 1 Self adhering PE Foil 740676 50 Visit www mn net com for more detailed product information 6 3 Product use restriction warranty NucleoSpin 8 Trace 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
3. Genomic DNA from forensic samples User manual NucleoSpin 96 Trace April 2014 Rev 03 MACHEREY NAGEL www mn net com Genomic DNA from forensic samples Table of contents 1 Components 1 1 Kit contents 1 2 Reagent to be supplied by user 4 2 Product description 5 2 1 The basic principle 5 2 2 Kit specifications 5 2 3 Required hardware 6 3 Storage conditions and preparation of working solutions 8 4 Safety instructions 9 5 Protocols 11 5 1 NucleoSpin 96 Trace vacuum processing 11 5 2 NucleoSpin 96 Trace centrifuge processing 17 5 3 NucleoSpin 96 Trace use of the NucleoSpin Trace Filter Plate 20 6 Appendix 21 6 1 Troubleshooting 21 6 2 Ordering information 22 6 3 Product use restriction warranty 23 MACHEREY NAGEL 04 2014 Rev 03 3 Genomic DNA from forensic samples 1 Components 1 1 Kit contents NucleoSpin 96 Trace 2 x 96 preps 4x 96 preps REF 740726 2 740726 4 Lysis Buffer FLB 200 mL 2 x 200 mL Wash Buffer B5 Concentrate 100 mL 2x 100 mL Elution Buffer BE 125 mL 2x 125 mL Proteinase K lyophilized 2x 33 mg 4x33 mg Proteinase Buffer PB 8 mL 15 mL NucleoSpin Trace Binding 2 4 Plates gray rings MN Wash Plates including six 2 4 paper sheets MN Square well Bocks 2 4 Rack of Tube Strips 2 4 User manual 1 1 1 2 Reagent to be supplied by user 96 100 ethanol For more detailed information regarding special hardware required for centrifuge or vacuum
4. H 334 May cause allergy or asthma symptoms or breathing difficulties if inhaled Kann bei Einatmen Allergie asthmaartige Symptome oder Atembeschwerden verursachen H 335 May cause respiratory irritation Kann die Atemwege reizen Precaution phrases P 261 Avoid breathing dust Einatmen von Staub vermeiden P 280 Wear protective gloves eye protection Schutzhandschuhe Augenschutz tragen P 302 352 IF ON SKIN Wash with plenty of water BEI KONTAKT MIT DER HAUT Mit viel Wasser waschen MACHEREY NAGEL 04 2014 Rev 03 9 Genomic DNA from forensic samples Precaution phrases P 304 340 P 305 351 338 P 312 P 332 313 P 337 313 P 342 311 P 403 233 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 If skin irritation occurs Get medical advice attention Bei Hautreizung Arztlichen Rat einholen rztliche Hilfe hinzuziehen Get medical advice attention Bei anh
5. INCLUDING WITHOUT LIMITATION AS TO THE SUITABILITY REPRODUCTIVITY DURABILITY FITNESS FOR A PARTICULAR PURPOSE OR USE MERCHANTABILITY CONDITION OR ANY OTHER MATTER WITH RESPECT TO MACHEREY NAGEL PRODUCTS In no event shall MACHEREY NAGEL be liable for claims for any other damages whether direct indirect incidental compensatory foreseeable consequential or special including but not limited to loss of use revenue or profit whether based upon warranty contract tort including negligence or strict liability arising in connection with the sale or the failure of MACHEREY NAGEL products to perform in accordance with the stated specifications This warranty is exclusive and MACHEREY NAGEL makes no other warranty expressed or implied The warranty provided herein and the data specifications and descriptions of this MACHEREY NAGEL product appearing in MACHEREY NAGEL published catalogues and product literature are MACHEREY NAGEL s sole representations concerning the product and warranty No other statements or representations written or oral by MACHEREY NAGEL s employees agent or representatives except written statements signed by a duly authorized officer of MACHEREY NAGEL are authorized they should not be relied upon by the customer and are not a part of the contract of sale or of this warranty Product claims are subject to change Therefore please contact our Technical Service Team for the most up to date information on MACH
6. This centrifuge must be able to accomodate the NucleoSpin Trace 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 MACHEREY NAGEL 04 2014 Rev 03 Genomic DNA from forensic samples 3 Storage conditions and preparation of working solutions Attention Buffer FLB contains chaotropic salts Wear gloves and goggles CAUTION Buffer FLB contains guanidine hydrochloride which can form highly reactive compounds when combined with bleach sodium hypochlorite DO NOT add bleach or acidic solutions directly to the sample preparation waste All kit components can be stored at room temperature 18 25 C and are stable for at least one year Upon storage especially at low temperatures a white precipitate may form in Lysis Buffer FLB Such precipitates have to be dissolved by incubating at 45 50 C for 10 min before use Before starting any NucleoSpin 96 Trace protocol prepare the following Wash Buffer B5 Add the indicated volume of ethanol 96 100 to Buffer B5 Concentrate Mark the label of the bottle to indicate that ethanol was added Store Wash Buffer B5 at room temperature 18 25 C for at least one year Before first use of the kit add the indicated volume see table below or on the bottle of Proteinase Buffer PB to dissolve lyophilized Proteinase K Proteinase K solution is stable at 20 C for at least 6 months Nucle
7. contamination Suboptimal elution Elution efficiencies decrease dramatically if elution is performed with buffers with pH lt 7 0 Use slightly alkaline elution buffer like BE pH 8 5 Be sure that all of the elution buffer gets into contact with the silica membrane No drops should stick to the walls of the columns Suboptimal performance of DNA in downstream experiments Carry over of ethanol Be sure to remove all of the ethanolic Buffer B5 after the final washing step Dry the NucleoSpin Trace Binding Plate for at least 10 min with maximum vacuum Insufficient Vacuum pressure is not sufficient vacuum Check if the vacuum manifold lid fits tightly to the manifold pressure base if vacuum is turned on Buffer volumes are not enough Insufficient Buffers are delivered in sufficient but limited amounts buffer volumes Calculate the needed buffer volumes and pour an additional amount of 10 into the reservoirs MACHEREY NAGEL 04 2014 Rev 03 21 Genomic DNA from forensic samples Problem Possible cause and suggestions Insufficient Do not fill back unused buffer from reservoir to the flask to buffer volumes avoid contaminations Ask technical service for extended continued buffer volumes Cross contamination during transfer of lysate Be sure that no liquid drops out of the tips while moving the tips with samples above the NucleoSpin Trace Binding Plate Cross contamination
8. manifold and place the NucleoSpin Trace Binding Plate on top Dispense 50 200 uL Buffer BE directly to the bottom of each well Incubate for 5 min at room temperature Apply vacuum for elution 0 6 bar 1 min Release vacuum 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 Note Elution with a centrifuge is recommended see section 5 2 Reduction of atmospheric pressure 16 MACHEREY NAGEL 04 2014 Rev 03 NucleoSpin 96 Trace centrifuge processing 5 2 NucleoSpin 96 Trace centrifuge processing Protocol at a glance For hardware requirements refer to section 2 3 For detailed information on each step see page 19 Before starting the preparation Check if Buffer B5 and Proteinase K were prepared according to section 3 1 Lyse samples 125 600 pL FLB 25 uL Proteinase K Mix RT several hours or overnight 2 Adjust DNA binding conditions 1 vol isopropanol per 2 vol lysate Mix 3 Transfer lysates to NucleoSpin Trace Binding Plate 4 Bind DNA to silica membrane of the 5 600 6 000 x g NucleoSpin Trace Binding Plate 3 min 5 Wash silica membrane 900 uL B5 5 600 6 000 x g 2 min 900 uL B5 5 600 6 000 x g 10 min 6 Dry silica membrane Not necessary see prolonged centrifugation at step 5 2 4 wash step 7 Elute DNA 50 200 uL BE 5 600 6 000 x g 3
9. K and at least 125 600 uL Buffer FLB and pipette it to the sample Incubate several hours or overnight at room temperature Optional Separate lysate from sample material See section 5 3 for use of the NucleoSpin Trace Filter Plate see ordering information 2 Adjust DNA binding conditions Add 1 vol e g 300 uL isopropanol to 2 vol e g 600 uL lysate mix 3 times and transfer to NucleoSpin Trace Binding Plate 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 Place the NucleoSpin Trace Binding Plate on top of the manifold 3 Transfer lysates Transfer the lysates resulting from step 2 carefully into the wells of the NucleoSpin Trace Binding Plate Do not moisten the rims of the individual wells while dispensing the samples moistened rims may cause cross contamination 4 Bind DNA to silica membrane Apply vacuum until all lysates have passed through the wells of the NucleoSpin Trace Binding Plate 0 2 bar 2 min Release the vacuum 14 MACHEREY NAGEL 04 2014 Rev 03 NucleoSpin 96 Trace vacuum processing Wash silica membrane Add 900 uL Buffer B5 to each well of the NucleoSpin Trace Binding Plate Apply vacuum 0 2 bar 1 min until all buffer has passed through the wells of the NucleoSpin Trace Binding Plate
10. Rev 03 23 Genomic DNA from forensic samples DNA RNA protein purification products of MACHEREY NAGEL for a well defined and specific application MACHEREY NAGEL shall only be responsible for the product specifications and the performance range of MN products according to the specifications of in house quality control product documentation and marketing material This MACHEREY NAGEL product is shipped with documentation stating specifications and other technical information MACHEREY NAGEL warrants to meet the stated specifications MACHEREY NAGEL s sole obligation and the customer s sole remedy is limited to replacement of products free of charge in the event products fail to perform as warranted Supplementary reference is made to the general business terms and conditions of MACHEREY NAGEL which are printed on the price list Please contact us if you wish to get an extra copy There is no warranty for and MACHEREY NAGEL is not liable for damages or defects arising in shipping and handling transport insurance for customers excluded or out of accident or improper or abnormal use of this product defects in products or 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
11. each well of the NucleoSpin Trace Binding Plate Seal plate with a new Self adhering PE Foil and centrifuge again at 5 600 6 000 x g for 2 min 18 MACHEREY NAGEL 04 2014 Rev 03 NucleoSpin 96 Trace centrifuge processing Remove the Self adhering PE Foil and add 900 uL Buffer B5 to each well of the NucleoSpin Trace Binding Plate Seal plate with a new Self adhering PE Foil and centrifuge again at 5 600 6 000 x g for 10 min During this step as much ethanolic Buffer B5 as possible is removed by centrifugation Dry silica membrane Residual washing buffer from the NucleoSpin Trace Binding Plate is removed by the prolonged centrifugation time of 10 min after adding the Buffer B5 as described in step 5 This prolonged time 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 Elute DNA For elution place the NucleoSpin Trace Binding Plate on the Rack of Tube Strips and pipette 50 200 uL Buffer BE directly to the bottom of each well Incubate 5 min at room temperature and centrifuge at 5 600 6 000 x g for 3 min Close the Tube Strips with Cap Strips for storage Be sure that all of the water gets into contact with the silica membrane no water drops should stick to the walls of the columns MACHEREY NAGEL 04 2014 Rev 03 19 NucleoSpin 96 Trace use of the NucleoSpin Trace Filter Plate
12. 2008 UV Gamma Eletron beam Ethylene oxide 30 27 3 13 87 100 ae 40 gt 70 Full profile Partial profile loadable WPartial profile unloadable iNo profile Figure 1 According to Shaw et al 2008 Comparison of the effects of sterilization techniques on subsequent DNA profiling Int J Legal Med 122 29 33 2 3 Required hardware Vacuum processing The NucleoSpin 96 Trace kit can be used with the NucleoVac 96 Vacuum Manifold see ordering information When using NucleoSpin 96 Trace 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 Trace Binding Plate and thus guarantee proper vacuum Establish a reliable vacuum source for the NucleoVac 96 Vacuum Manifold The manifold may be used with a vacuum pump house vacuum or water aspirator We recommend a vacuum of 0 2 to 0 4 bar reduction of atmospheric pressure The use of the NucleoVac Vacuum Regulator see ordering information is recommended Alternatively adjust the vacuum so that during the purification the sample flows through the column with a rate of 1 2 drops per second Depending on the amount of sample being used the vacuum times may need to be increased for complete filtration 6 MACHEREY NAGEL 04 2014 Rev 03 Genomic DNA from forensic samples Centrifugation For centrifugation a microtiterplate centrifuge is required
13. EREY 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 24 MACHEREY NAGEL 04 2014 Rev 03 Genomic DNA from forensic samples Last updated 07 2010 Rev 03 Please contact MACHEREY NAGEL GmbH amp Co KG Tel 49 0 24 21 969 270 e mail tech bio mn net com MACHEREY NAGEL 04 2014 Rev 03 25
14. Release the vacuum Add 900 uL Buffer B5 to each well of the NucleoSpin Trace Binding Plate Apply vacuum 0 2 bar 1 min until all buffer has passed through the wells of the NucleoSpin Trace Binding Plate Release the vacuum Remove MN Wash Plate After the final washing step close the valve release the vacuum and remove the NucleoSpin Trace Binding Plate from the vacuum manifold Put it on a clean paper towel to remove residual EtOH containing wash buffer Remove manifold lid MN Wash Plate and waste container from the vacuum manifold Dry silica membrane Remove any residual washing buffer from the outlets of the NucleoSpin Trace Binding Plate If necessary tap the outlets onto a clean paper sheet supplied with the MN Wash Plate or soft tissue until no drops come out Insert the Column Holder A with the NucleoSpin Trace Binding Plate 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 MACHEREY NAGEL 04 2014 Rev 03 15 NucleoSpin 96 Trace vacuum processing 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
15. altender Hautreizung Arztlichen Rat einholen rztliche Hilfe hinzuziehen If experiencing respiratory symptoms Call a POISON CENTER doctor Bei Symptomen der Atemwege GIFTINFORMATIONSZENTRUM Arzt anrufen Store in a well ventilated place Keep container tightly closed Beh lter dicht verschlossen an einem gut bel fteten Ort augbewahren 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 10 MACHEREY NAGEL 04 2014 Rev 03 NucleoSpin 96 Trace vacuum processing 5 Protocols 5 1 NucleoSpin 96 Trace vacuum processing Protocol at a glance For hardware requirements refer to section 2 3 For detailed information regarding the vacuum manifold setup see page 14 For detailed information on each step see page 15 Before starting the preparation Check if Buffer B5 and Proteinase K were prepared according to section 3 1 Lyse samples 125 600 pL FLB 25 uL Proteinase K Mix RT several hours or overnight 2 Adjust DNA binding conditions 1 vol isopropanol per 2 vol lysate Mix Prepare the NucleoVac 96 Vacuum Manifold 3 Transfer lysates to NucleoSpin Trace Binding Plate 4 Bind DNA to silica membrane of the 0 2 bar NucleoSpin Trace Binding plate 2 min 5 Wash silica membrane 900 uL B5 0 2 bar 1 min Reduction of atmospheric pres
16. min MACHEREY NAGEL 04 2014 Rev 03 17 NucleoSpin 96 Trace centrifuge processing Detailed protocol For hardware requirements refer to section 2 3 Before starting the preparation Check if Buffer B5 and Proteinase K were prepared according to section 3 1 Lyse samples Premix 25 uL Proteinase K and at least 125 uL Buffer FLB and pipette it to the sample Incubate several hours or overnight at room temperature Optional Separate Iysate from sample material See section 5 3 for use of the NucleoSpin Trace Filter Plate see ordering information 2 Adjust DNA binding conditions Add 1 vol e g 300 uL isopropanol to 2 vol e g 600 uL lysate mix 3 times and transfer to NucleoSpin Trace Binding Plate 3 Transfer lysates Remove the first Cap Strip and transfer the lysates resulting from step 2 into the wells of the NucleoSpin Trace 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 during centrifugation After transfer seal the openings of the plate with Self adhering PE Foil 4 Bind DNA to silica membrane Place the MN Square well Blocks with NucleoSpin Trace Binding Plates onto the centrifuge carriers and insert them into the rotor buckets Centrifuge at 5 600 6 000 x g for 3 min 5 Wash silica membrane Remove the Self adhering PE Foil and add 900 pL Buffer B5 to
17. oSpin 96 Trace 2 x 96 preps 4x 96 preps REF 740726 2 740726 4 Wash Buffer B5 100 mL 2x 100 mL Concentrate Proteinase K Add 400 mL ethanol 2x33 mg Add 3 mL Proteinase Buffer to each vial Add 400 mL ethanol to each bottle 4x33 mg Add 3 mL Proteinase Buffer to each vial MACHEREY NAGEL 04 2014 Rev 03 Genomic DNA from forensic samples 4 Safety instructions The following components of the NucleoSpin 96 Trace kits contain hazardous contents Wear gloves and goggles and follow the safety instructions given in this section GHS classification Only harmful features do not need to be labeled with H and P phrases up to 125 mL or 125 g Mindergef hrliche Eigenschaften m ssen bis 125 mL oder 125 g nicht mit H und P S tzen gekennzeichnet werden Component Hazard contents GHS symbol Hazard Precaution phrases phrases Inhalt Gefahrstoff GHS Symbol H S tze P S tze FLB Guanidine hydrochloride Substance does not have to be specially labeled 1 10 as hazardous Guanidinhydrochlorid 1 10 Die Substanz muss nicht als gef hrlich gekennzeichnet werden Proteinase K Proteinase K Iyophilized Danger 315 319 261 280 Proteinase K Iyophilisiert Gefahr 334 335 302 352 304 340 305 351 338 312 332 313 337 313 342 311 403 233 Hazard phrases H 315 Causes skin irritation Verursacht Hautreizungen H 319 Causes serious eye irritation Verursacht schwere Augenreizung
18. 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 IN VITRO diagnostic use Please pay attention to the package of the product IN V TRO diagnostic products are expressly marked as IVD on the packaging IF THERE IS NO IVD SIGN THE PRODUCT SHALL NOT BE SUITABLE FOR IN VITRO DIAGNOSTIC USE ALL OTHER PRODUCTS NOT LABELED AS IVD ARE NOT SUITED FOR ANY CLINICAL USE INCLUDING BUT NOT LIMITED TO DIAGNOSTIC THERAPEUTIC AND OR PROGNOSTIC USE No claim or representations is intended for its use to identify any specific organism or for clinical use included but not limited to diagnostic prognostic therapeutic or blood banking It is rather in the responsibility of the user or in any case of resale of the products in the responsibility of the reseller to inspect and assure the use of the MACHEREY NAGEL 04 2014
19. processing please see section 2 3 1 For preparation of working solutions and storage conditions see section 3 2 Elution Buffer BE 5 mM Tris HCl pH 8 5 Set of 1 rack 12 strips with 8 tubes each Cap Strips included 4 MACHEREY NAGEL 04 2014 Rev 03 Genomic DNA from forensic samples 2 Product description 2 1 The basic principle With the NucleoSpin 96 Trace method genomic DNA is prepared from forensic samples Lysis is achieved by incubation of samples in a solution containing chaotropic ions in the presence of Proteinase K at room temperature Appropriate conditions for binding of DNA to the silica membrane in the NucleoSpin Trace Binding Plate are created by addition of isopropanol to the Iysate The binding process is reversible and specific to nucleic acids Contaminations are removed by two washing steps with ethanolic buffer Pure genomic DNA is finally eluted under low ionic strength conditions in a slightly alkaline elution buffer 2 2 Kit specifications NucleoSpin 96 Trace is designed for the rapid small scale preparation of highly pure genomic DNA from forensic samples The obtained DNA can be used directly as template for PCR Typically yields of 1 2 ug genomic DNA can be purified from buccal swabs The final concentration of eluted DNA is 10 20 ng uL depending on elution buffer volume Typically the A A ratio is 1 8 1 9 NucleoSpin 96 Trace can be processed under vacuum or in a cent
20. rifuge see section 2 3 Parameters NucleoSpin 96 Trace Technology Silica membrane technology Format 96 well plate Processing Manual or automated vacuum or centrifugation Sample material Forensic samples buccal swabs blood spots Fragment size 200 bp approx 50 kbp Typical yield Depending on sample amount A sea A0 1 8 1 9 Elution volume 50 100 uL Preparation time 70 min plate excl lysis Binding capacity 20 ug MACHEREY NAGEL 04 2014 Rev 03 5 Genomic DNA from forensic samples Forensic quality product NucleoSpin 96 Trace is certified as forensic quality product Consumables used in forensics need to be treated carefully to prevent DNA contamination MACHEREY NAGEL therefore has a stringently controlled production process to avoid DNA contamination of consumables Further MACHEREY NAGEL uses ethylene oxide EO treatment to remove amplifiable DNA which might still be introduced during the manufacturing process MACHEREY NAGEL products carrying the forensic quality seal contain plastic materials that are EO treated This means DNA of any kind which might still be introduced into plastic consumables during the production process is inactivated by means of the treatment with ethylene oxide in order to prevent the generation of accidental human profile by PCR amplification Ethylene oxide treatment has been shown to be the method of choice to prevent DNA profiles due to DNA contamination Shaw et al
21. sure MACHEREY NAGEL 04 2014 Rev 03 11 NucleoSpin 96 Trace vacuum processing 900 uL B5 0 2 bar 1 min Remove MN Wash Plate 6 Dry silica membrane 0 6 bar 10 min 7 Elute DNA 50 200 pL BE 0 4 bar 2 min Reduction of atmospheric pressure 12 MACHEREY NAGEL 04 2014 Rev 03 NucleoSpin 96 Trace vacuum processing Setup of vacuum manifold Binding Washing steps Step 4 Place the NucleoSpin Binding Plate on top of the manifold lid Step 3 Place the manifold lid on top of the manifold base Step 2 Place the MN Wash Plate in the manifold Step 1 Insert spacers MTP MULTI 96 PLATE and waste container in the manifold base Final setup Elution step Step 4 Place the NucleoSpin Binding Plate on top of the manifold lid Step 3 Place the manifold lid on top of the manifold base Step 2 Place the Rack of Tube Strips in the manifold Step 1 Insert spacers MICROTUBE RACK in the manifold base Final setup MACHEREY NAGEL 04 2014 Rev 03 13 NucleoSpin 96 Trace vacuum processing Detailed protocol For hardware requirements refer to section 2 3 For detailed information regarding the vacuum manifold setup see page 14 Before starting the preparation Check if Buffer B5 and Proteinase K were prepared according to section 3 1 Lyse samples Premix 25 uL Proteinase
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