Genomic DNA from Forensic Samples
Contents
1. 5 Dry NucleoSpin Trace Binding Strips by ca 0 6 bar vacuum or centrifugation or 5 600 6 000 x g 10 min optional dry the outlets of the NucleoSpin Trace Binding Strips before the vacuum step by tapping it to a sheet of paper 6 Elute highly pure genomic DNA 50 200 ul BE incubate 3 min ca 0 6 bar or 3 min 5 600 6 000 x g Reduction of atmospheric pressure 8 MACHEREY NAGEL 11 2003 Rev 02 NucleoSpin 8 Trace 4 1 Standard protocol for manual purification of genomic DNA under vacuum Prepare buffer B5 by adding ethanol Prepare proteinase K by dissolving one vial of lyophilzed powder in 3 ml proteinase Buffer see section 3 for details If using less than 96 samples fill up column holder with dummy strips see ordering information 1 Premix 25 ul proteinase K and at least 125ul buffer FLB and pipet to the sample Incubate several hours or overnight at room temperature Optional Separate lysate from sample material See support protocol for using NucleoSpin Trace Filter Plate see ordering information Prepare the NucleoVac 96 vacuum manifold Insert spacers MTP Multi 96 plate into the NucleoVac 96 vacuum manifold s short sides Place the waste container inside the vacuum manifold and insert a MN Wash Plate into the notches of the spacers Close the manifold with the lid Place a NucleoSpin Trace Binding Strips inserted in Column Holder A into the r2. Genomic DNA from Forensic Samples User manual NucleoSpin 8 Trace November 2003 Rev 02 MACHEREY NAGEL Da Genomic DNA from Forensic Samples Table of contents 1 Kit contents 4 2 Product description 5 2 1 The basic principle 5 2 2 Kit specifications 5 2 3 Required hardware 6 2 4 Suitability with other common vacuum manifolds 6 3 Storage conditions preparation of working solutions safety precautions and setup of vacuum source 7 4 General procedure 8 4 1 Standard protocol for manual purification of genomic DNA under vacuum 9 4 2 Standard protocol for manual purification of genomic DNA under centrifugation 11 4 3 Support protocol for using NucleoSpin Trace Filter Plate 13 5 Appendix 14 5 1 Troubleshooting 14 5 2 Ordering information 16 5 3 References 16 5 4 Product Use Restriction Warranty 17 MACHEREY NAGEL 11 2003 Rev 02 3 Genomic DNA from Forensic Samples 1 Kit contents NucleoSpin 8 Trace 12 x 8 preps 60 x 8 preps Cat No 740 722 1 740 722 Buffer FLB 100 ml 500 ml Buffer B5 concentrate a0 209 ml Proteinase K lyophilized 33 mg 5 x 33 mg Proteinase Buffer 3 6 ml 35 ml Buffer BE 50 ml 250 ml NucleoSpin Trace Binding 42 60 Strips MN Wash Plate including 2 10 six paper sheets MN Square well Blocks 2 10 MN Tube Strips 1 5 Cap Strips 12 60 Self adhering PE Foil 2 10 Protocol 1 1 1 For preparation of working solutions and storage conditions see point 3 Set of 1 rack
3. ca 600 mbar pressure difference for at least 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 close the valve and ventilate the vacuum manifold 6 For elution insert spacers Microtube Rack into manifold and rest rack with MN Tube Strips on spacers Insert Column Holder A with NucleoSpin Trace Binding Strips into manifold lid Pipet 75 200 ul BE directly to the bottom of each well and incubate for 5 minutes at room temperature Apply vacuum of 400 mbar for 2 minutes Note Elution with centrifuge is recommended see section 4 2 10 MACHEREY NAGEL 11 2003 Rev 02 NucleoSpin 8 Trace 4 2 Standard protocol for manual purification of genomic DNA under centrifugation Prepare buffer B5 by adding ethanol Prepare proteinase K by dissolving one vial of lyophilzed powder in 3 ml proteinase Buffer see section 3 for details If using less than 48 samples fill up Column Holder C with Dummy Strips see ordering information 1 Premix 25 ul proteinase K and at least 125 ul buffer FLB and pipet to the sample Incubate several hours or overnight at room temperature Optional Separate lysate from sample material See support protocol for using NucleoSpin Trace Filter Plate see ordering information 2 Mix lysate with isopropanol
4. general business terms and conditions of MACHEREY NAGEL which are printed on the price list Please contact us if you wish an extra copy MACHEREY NAGEL does not warrant against 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 against defects in products or components not manufactured by MACHEREY NAGEL or against 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 PRODUCTIVITY 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 incidental 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 The warranty provided herein and the data specifications and descriptions of this MACHEREY NAGEL product appearing in MACHEREY
5. silica membrane no water drops should stick to the walls of the columns 12 MACHEREY NAGEL 11 2003 Rev 02 NucleoSpin 8 Trace 4 3 Support protocol for using NucleoSpin Trace Filter Plate Prepare buffer B5 by adding ethanol Prepare proteinase K by dissolving one vial of lyophilzed powder in 3 ml proteinase buffer see section 3 for details 1 Put 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 adding Isopropanol MACHEREY NAGEL 11 2003 Rev 02 13 Genomic DNA from Forensic Samples 5 Appendix 5 1 Troubleshooting Problem Possible cause and suggestions Reagents not applied or restored properly e Reagents not properly restored Add the indicated volume of proteinase buffer to the proteinase K vial and 96 100 ethanol to buffer concentrate B5 and mix Kit storage e Store aliquots of the reconstituted proteinase K at 4 C e Store other kit components at room temperature Storage at low Poor DNA temperatures may cause Salt precipitation aa
6. 12 strips with 8 tubes each 4 MACHEREY NAGEL 11 2003 Rev 02 Genomic DNA from Forensic Samples 2 Product description 2 1 The basic principle With the NucleoSpin 8 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 Strips are created by addition of isopropanol to the lysate 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 8 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 Azgo 280 ratio is 1 8 1 9 The kit is for use with the NucleoVac 96 vacuum manifold Cat No 740681 or similar suitable vacuum manifolds See section 2 3 If using a centrifuge with a swing out rotor capable to accommodate the NucleoSpin Trace Binding Strips MN Square well Block sa
7. 15 Genomic DNA from Forensic Samples 5 2 Ordering information Product Cat No Pack of NucleoSpin 8 Trace 740 722 1 12 x 8 preps NucleoSpin 8 Trace 740 722 60 x 8 preps NucleoSpin Trace Filter Plate 740 677 20 MN Wash Plate 740 675 20 NucleoSwing Z 513 740 610 1 NucleoSwing Z 513 K refrigerated 740 610K 1 5 3 References Vogelstein B and D Gillespie 1979 16 MACHEREY NAGEL 11 2003 Rev 02 Proc Natl Acad Sci USA 76 615 619 Genomic DNA from Forensic Samples 5 4 Product Use Restriction Warranty NucleoSpin 8 Trace kits components were developed designed and sold for research purposes only They are suitable for in vitro uses only Furthermore is no claim or representation intended for its use to identify any specific organism or for clinical use diagnostic prognostic therapeutic or blood banking It is rather in the responsibility of the user to verify the use of the NucleoSpin 8 Trace kits for a specific application range as the performance characteristic of this kit has not been verified to a specific organism This MACHEREY NAGEL product is shipped with documentation stating specifications and other technical information MACHEREY NAGEL guarantees 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
8. For 150 ul lysate add 75 ul isopropanol mix 3 times and transfer to NucleoSpin Trace Binding Strips Note Ratio for mixing isopropanol with sample lysates is 1 vol isopropanol 2 vol sample lysate 3 Bind genomic DNA to silica membrane Centrifuge at 5 600 6 000 x g for 3 min 4 Wash silica membrane 15t wash Add 900 ul B5 to each well of the NucleoSpin Trace Binding Strips Centrifuge at 5 600 6 000 x g for 2 min Empty square well block 2 wash Add 900 ul B5 to each well of the NucleoSpin Trace Binding Strips Centrifuge at 5 600 6 000 x g for 10 min MACHEREY NAGEL 11 2003 Rev 02 11 NucleoSpin 8 Trace 5 Dry NucleoSpin Trace Binding Strips Residual washing buffer from NucleoSpin Trace Binding Strips is removed by the prolonged centrifugation time of 10 min after adding the wash buffer B5 as described in step 4 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 6 Elute highly pure genomic DNA For elution place the Column Holder C with NucleoSpin Trace Binding Strips on a rack with MN Tube Strips and pipet 50 200 ul 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 MN Tube Strips with Cap Strips for storage Be sure that all of the water gets into contact with the
9. 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 MACHEREY NAGEL 11 2003 Rev 02 17 Genomic DNA from Forensic Samples applications have been tested in MACHEREY NAGEL laboratories using MACHEREY NAGEL products MACHEREY NAGEL does not warrant the correctness of any of those applications Please contact MACHEREY NAGEL Germany Tel 49 2421 969 270 and 275 e mail TECH BlIO mn net com 18 MACHEREY NAGEL 11 2003 Rev 02
10. d e Keep bottles tightly closed in order to prevent evaporation or contamination Suboptimal elution e Elution efficiencies decrease dramatically if elution is done with buffers with pH lt 7 0 Use slightly alkaline elution buffer like BE pH 8 5 e 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 Carryover of ethanol E e Be sure to remove all of ethanol buffer B5 after the final washing downsieam step Dry the NucleoSpin Trace Binding Strips for at least 1 aie experiments 0 min with maximum vacuum Vacuum pressure is not sufficient Vacuum ma ifold e Check if the vacuum manifold lid fits tightly to the manifold base if vacuum is turned on 14 MACHEREY NAGEL 11 2003 Rev 02 Genomic DNA from Forensic Samples Problem Possible cause and suggestions Buffer volumes are not enough e Buffers are delivered in sufficient but limited amounts Calculate the needed buffer volumes and pour an additional amount of 10 into the reservoirs Buffers e Do not fill back unused buffer from reservoir to the flask to avoid contaminations Ask technical service for extended buffer volumes Gios Cross contamination during transfer of lysate contamina e Be sure that no liquid drops out of the tips while moving the tips tion with samples above the NucleoSpin Trace Binding Strips MACHEREY NAGEL 11 2003 Rev 02
11. ld Suitability Additional equipment Qiagen QlAvac 96 yes MN Frame see ordering information BioRad Aurum vacuum no manifold Eppendorf Perfect VAC AG Manifold Millipore MultiScreen Ho In general is the QlAvac 96 suitable for the use with the NucleoSpin Trace Binding Strips Nevertheless it is recommended to use the MN Frame to adjust the proper height of the MN Wash Plate and Elution Plate U Bottom inside the QlAvac 96 in order to ensure best performance 6 MACHEREY NAGEL 11 2003 Rev 02 Genomic DNA from Forensic Samples 3 Storage conditions preparation of working solutions safety precautions and setup of vacuum source Attention Buffer FLB contains guanidinium hydrochloride Wear gloves and goggles Store lyophilized proteinase K at 4 C All other kit components are stable at room temperature Before starting any NucleoSpin 8 Trace protocol prepare the following Proteinase K Add 3 ml proteinase Buffer per vial to dissolve the lyophilized proteinase K Store the proteinase K solution at 4 C for up to 3 months Dividing the solution into small aliquots and storage at 20 C is recommended if the solution will not be used up during this period All other components of the NucleoSpin 8 Trace kit should be stored at room temperature for a maximum of 1 year Storage at lower temperatures may cause precipitation of salts If a salt precipitate is observed incubate the bottle at 30 40 C for some minu
12. ndwich bucket height 85mm e g Hermle Macherey Nagel NucleoSwing Z513 Qiagen Sigma 4 15c Jouan KR4i Kendro Heraeus Multifuge 3 3 R Highplate Beckman Coulter Allegra R no NucleoVac 96 vacuum manifold is necessary Kit specifications at a glance NucleoSpin 8 Trace Sample type buccal swabs Average yield 1 2 yg Elution volume 50 100 ul Binding capacity 20 ug Time 6 strips or one plate 70 min MACHEREY NAGEL 11 2003 Rev 02 5 Genomic DNA from Forensic Samples 2 3 Required hardware The NucleoSpin 8 Trace kit can be used manually with the NucleoVac 96 vacuum manifold Cat No 740681 by using the Starter Set A containing Column Holders A and Dummy Strips see ordering information For automation on laboratory platforms with standard 96 well plate vacuum chambers the use of the Starter Set A is also required If automation is desired on a Qiagen BioRobot 9604 3000 the Starter Kit B is required Use of the MN Frame see ordering information is strongly recommended Processing of the NucleoSpin 8 Trace kit under centrifugation is possible by using the Starter Set C see ordering information containing Column Holders C MN Square well Blocks Tube Strips For detailed information refer to the Starter Set C manual 2 4 Suitability with other common vacuum manifolds The NucleoSpin 8 Trace kit can be used with other common vacuum manifolds For further details see list below Vacuum manifo
13. tes and mix well until all of the precipitation is redissolved Establish a reliable vacuum source for the NucleoVac 96 vacuum manifold The manifold may be used with vacuum pump house vacuum or water aspirator We recommend a vacuum of 200 400 mbar pressure difference Alternatively adjust vacuum 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 used the vacuum times might have to be increased for complete filtration Add the indicated volume of 96 100 ethanol to B5 concentrate NucleoSpin 8 Trace 12 x 8 preps 60 x 8 preps Cat No 740 722 1 740 722 40 ml 200 ml Buffer B5 add 160 ml ethanol to add 800 ml ethanol to concentrate 40 ml buffer B5 200 ml buffer B5 concentrate before use concentrate before use MACHEREY NAGEL 11 2003 Rev 02 7 NucleoSpin 8 Trace 4 General procedure 1 Pipet 25 ul proteinase K and at least 125 ul buffer FLB to the sample Incubate several hours or overnight at room temperature Optional Separate lysate from sample material See support protocol for using NucleoSpin Trace Filter Plate see ordering information 2 Add 1 Vol Isopropanol to 2 Vol lysate mix 3 times and transfer to NucleoSpin Trace Binding Strips 3 Bind DNA to silica membrane by applying ca 0 2 bar vacuum 2 min 4 Wash silica membrane 900 ul BS 900 ul B5 ca 0 2 bar 1 min each step
14. ubber seal of the vacuum manifold s lid and apply the samples to the wells of the plate 2 Add 1 Vol e g 330 ul Isopropanol to 2 Vol e g 660 ul lysate mix 3 times and transfer to NucleoSpin Trace Binding Strips 3 Bind genomic DNA to silica membrane Apply vacuum until all lysates have passed through the columns 200 mbar 2 min 600 mbar 10s Ventilate the vacuum manifold MACHEREY NAGEL 11 2003 Rev 02 9 NucleoSpin 8 Trace 4 Wash silica membrane 15t wash Add 900 ul B5 to each well of the NucleoSpin Trace Binding Strips Apply vacuum 200 mbar 1 min until all buffer has passed through the columns Ventilate the vacuum manifold 2 wash Add 900 ul B5 to each well of the NucleoSpin Trace Binding Strips Apply vacuum 200 mbar 1 min until all buffer has passed through the columns Ventilate the vacuum manifold Remove MN Wash Plate After the final washing step close the valve ventilate the vacuum manifold and remove the wash plate and waste container from the vacuum manifold 5 Dry NucleoSpin Trace Binding Strips Remove any residual washing buffer from the NucleoSpin Trace Binding Strips If necessary tap the outlets of the NucleoSpin Trace Binding Strips onto a clean paper sheet supplied with the MN Wash Plate or soft tissue until no drops come out Insert the column holder with NucleoSpin Trace Binding Strips into the lid and close the manifold Apply maximum vacuum
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