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1. If the robot workstation does not allow the use of selected culture tubes transfer bacterial culture from the tubes into a suitable square well block available from MN see ordering information For this transfer 1 5 ml of the culture to each well of the square well block Harvest the cultures by centrifugation Discard supernatant Usually 1 5 ml of culture are sufficient for DNA preparation However if necessary add an additional 1 0 1 5 ml of the bacterial culture to each well of the square well block centrifuge again and discard the supernatant 10 MACHEREY NAGEL 08 2002 Rev 01 Automated Plasmid DNA Purification Do not use more than 5 ml LB culture or 3 ml rapid growing bacterial strain using 2 x YT or TB medium because lysis efficiency might be lower when using cell pellets which are too large MACHEREY NAGEL 08 2002 Rev 01 11 NucleoSpin Robot 96 Plasmid core kit 5 General Procedure 1 Cultivate and harvest LB bacterial cells 2x YT TB 10 min 1 000 xg 2 Resuspend bacterial cells 250 ul A1 mix or shake Coo 3 Lyse bacterial cells 250 pl A2 RT 2 5 min Coo optional shake 4 Neutralize 350 pl A3 optional mix or o shake 5 Transfer of crude lysates to NucleoSpin Plasmid Filter 7 Plate purple 12 MACHEREY NAGEL 08 2002 Rev 01 NucleoSpin Robot 96 Plasmid core kit 6 Clear crude lysates by ca 0 2 0 4 bar E EE ee vacuum filtration2. Rev 01 NucleoSpin Robot 96 Plasmid core kit 5 2 Support protocol Elution of DNA using a centrifuge Optional step Elution of purified DNA in a centrifuge may be necessary when higher concentrations of the final DNA are required for downstream applications Using a centrifuge allows reduction of the dispensed volume to 50 75 ul 1 Stop the method after the final washing step with buffer A4 Remove NucleoSpin Plasmid Binding Plate from the manifold s top and tap on a sheet of filter paper to remove residual wash buffer from the outlets 2 Cover NucleoSpin Plasmid Binding Plate with self adhering PE foil Place the plate on top of a square well block or round well block see ordering information and centrifuge for 10 min at maximum speed gt 4 000 x g optimal 5 800 x g Note We recommend to use a centrifuge e g Hermle MACHEREY NAGEL NucleoSwing Z513 Qiagen Sigma 4 15 Jouan KR4i Kendro Heraeus Multifuge 3 3 R Highplate rotor with a swing out rotor which is capable of accommodating the NucleoSpin Plasmid Binding Plate square well block sandwich bucket hight 85 mm Do not use a microtiter plate as a support for the NucleoSpin Plasmid Binding Plate Microtiter plates may crack when centrifuging at gt 2 500 x g 3 Insert the NucleoSpin Plasmid Binding Plate to a new square well or round well block Remove the self adhering PE foil and dispense elution buffer 50 75 pl directly onto the sil
3. e Use high copy number plasmid Incomplete lysis of bacterial cells e See Possible cause and suggestions above No ethanol added to buffer A4 concentrate ethanol evaporated e Add indicated volume of ethanol to buffer A4 concentrate and mix Keep bottle tighty closed to prevent evaporation of ethanol Replace buffer A4 in open trough reservoirs MACHEREY NAGEL 08 2002 Rev 01 Automated Plasmid DNA Purification Problem Possible cause and suggestions Elution conditions are not optimal Poor plasmid If possible use a slightly alkaline elution buffer like AE 5 mM yield Tris HCl pH 8 5 When using nuclease free water for elution continued make sure the pH value is within the range of pH 8 0 8 5 Elution efficiencies drop drastically with buffers lt pH 7 High level contamination with chromosomal DNA Excessive mixing steps after addition of lysis buffers A2 and A3 or before transfer of crude lysate to the NucleoSpin Plasmid Filter Plate Mixing will cause shearing of chromosomal DNA leading to a co purification during the preparation of plasmid DNA e Reduce number of mixing cycles reduce shaker action Culture volume was too high e Reduce culture volume if lysate is too viscous for gentle and complete mixing Bacterial culture overgrown e Overgrown bacterial cultures contain lysed cells and degraded DNA See suggestions in section 4 Growing of bacterial cultures Lysis was too lo
4. chromosomal DNA 4 2 Cultivation of bacteria in a square well block Use a suitable 96 well square well block for growing bacteria available from MN see ordering information Add 1 2 1 5 ml of selected medium with appropriate antibiotic e g 100 ug ml ampicillin to each well of the square well block To avoid cross contamination due to spillage during incubation do not exceed a total culture volume of 1 5 ml Inoculate each well with a single bacterial colony Cover the square well block with a suitable gas permeable see ordering information Grow the culture in a suitable incubator at 37 C for 16 24 h with vigorous shaking 200 400 rpm The square well block may be fixed to the shaker with large size flask clamps for 2 I flasks or tape Note The yield of plasmid DNA depends on growth conditions bacterial strain and cell density of the culture as well as on the size and copy number of the vector Use of high copy number plasmids such as pUC pBluescript or pGEM and E coli strains like DH5a or XL1 Blue are recommended Growth times of 16 24 h are usually sufficient However for poor growing bacteria prolonged incubation times of up to 30 h may be required 4 3 Cultivation of bacteria in tubes Use 1 5 ml of appropriate culture medium Depending on the bacterial strain and copy number of the plasmid up to 5 ml LB medium or 3 ml 2 x YT or 3 ml TB medium can be used Grow bacteria with vigorous shaking for 10 14 h Optional
5. directly 1 min to 5 min Se _at into the NucleoSpin Plasmid Binding Plate phi transparent lt S optional incubate 1 to 3 min before applying vacuum 7 Reassemble vacuum manifold Discard the NucleoSpin Plasmid Filter Plate 8 Bind DNA to silica ca 0 4 bar c D membrane of the 1 min GS r NucleoSpin Plasmid Binding Plate by applying vacuum 9 Wash silica membrane 2 x 900 ul A4 ca 0 4 bar 8 1 min lt 10 Remove MN Wash plate reduction of atmospheric pressure MACHEREY NAGEL 08 2002 Rev 01 13 NucleoSpin Robot 96 Plasmid core kit 11 Dry NucleoSpin Plasmid Binding Plate by applying vacuum optional dry the outlets of the NucleoSpin Plasmid Binding Plate by placing it on a Sheet of filter paper before applying vacuum 10 min 15 min maximum vacuum 12 Insert elution pale U bottom e g available from MN 13 Elute highly pure plasmid DNA optional incubate 1 3 min 75 150 pl AE ca 0 4 bar 1 min reduction of atmospheric pressure 14 MACHEREY NAGEL 08 2002 Rev 01 NucleoSpin Robot 96 Plasmid core kit 5 1 Standard protocol for automated purification of high copy plasmid DNA using common laboratory automation workstations Note The list numbers in this protocol do not correspond with the list numbers in section 5 General procedure 1 Centrifuge square well block containing the bact
6. with common sequencing chemistries MACHEREY NAGEL 08 2002 Rev 01 15 NucleoSpin Robot 96 Plasmid core kit 6 Select method or program for plasmid DNA purification Optional After transfer to the NucleoSpin Plasmid Filter Plate incubate crude lysates for 1 3 min This incubation allows the formation of a compact white precipitate This step is usually not required for culture volumes up to 1 5 ml Optional A washing step with buffer AW is recommended when using end host strains Buffer AW is not included in the NucleoSpin Robot 96 Plasmid Core kit See ordering information 7 Completely dry the NucleoSpin Plasmid plate by applying maximum vacuum IMPORTANT This step removes residual washing buffer A4 from the NucleoSpin Plasmid Binding Plate The removal is only effective when maximum vacuum is used allowing maximum airflow to go through the wells Residual ethanol from buffer A4 may inhibit subsequent enzymatic reactions 8 Elution of purified plasmid DNA Dispense between a minimum of 75 ul and a maximum of 150 ul see section 2 4 of elution buffer AE Lower volumes of elution buffer will cause inhomogeneous results For increased DNA concentration a minimum elution volume of 75 ul can be used By applying higher volumes of dispensed elution buffer the concentration of resulting eluted DNA will decrease but the efficiency of elution will increase cp Fig 1 16 MACHEREY NAGEL 08 2002
7. Automated Plasmid DNA Purification User manual NucleoSpin Robot 96 Plasmid Core kit August 2002 Rev 01 MACHEREY NAGEL Da Automated Plasmid DNA Purification Table of contents 1 Kit contents 2 Product description 2 1 The basic principle 2 2 Kit specifications 2 3 Elution procedure 2 4 Automation 3 Storage conditions and preparation of working solutions 4 Growing of bacterial cultures 4 1 Selection of culture medium 4 2 Cultivation of bacteria in a square well block 4 3 Cultivation of bacteria in tubes 5 General Procedure o woona e h a fs 5 1 Standard protocol for automated purification of high copy plasmid DNA using common laboratory automation workstations 5 2 Support protocol Elution of DNA using a centrifuge 6 Appendix 6 1 Troubleshooting 6 2 Ordering information 6 3 References 6 4 Product Use Restriction Warranty MACHEREY NAGEL 08 2002 Rev 01 15 17 18 18 21 21 22 Automated Plasmid DNA Purification 1 Kit contents 24 x 96 preps 740 616 24 Buffer A1 6 x 200 ml Buffer A2 6 x 200 ml Buffer A3 6 x 200 mi Buffer A4 concentrate 6 x 4 x 100 ml Buffer AE 6 x 2 x 50 ml RNase A 6 x 80 mg NucleoSpin Plasmid Binding Plate 24 transparent NucleoSpin Plasmid T Filter Plate purple MN Wash plate including six paper 24 sheets Protocol 6x1 1 The NucleoSpin Robot 96 Plasmid Core Kit consists of 6 boxes with 4 x 96 preps each For preparation
8. DES 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 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 produc
9. at e The kit is for use with common laboratory automation workstations see section 2 3 e This kit provides reagents and basic consumables NucleoSpin Filter Plate NucleoSpin Plasmid Binding Plate MN Wash Plate for purification of up to 15 20 ug of highly pure plasmid DNA suitable for direct use in standard molecular biology applications like automated fluorescent sequencing PCR or restriction analysis e Using the NucleoSpin Robot 96 Plasmid kit allows simultaneous processing of up to 96 samples typically within less than 90 minutes Actual processing time depends on configuration of workstation used e Typically yields of 5 15 ug plasmid DNA can be purified from 1 5 ml overnight cultures e Yield depends on copy number and size of plasmid lt 15 kbp selected culture medium and bacterial host strain e Membrane capacity is about 20 ug The final concentration of eluted DNA is 50 200 ng ul depending on elution buffer volume and bacterial culture MACHEREY NAGEL 08 2002 Rev 01 5 Automated Plasmid DNA Purification e Typically the A260280 ratio is gt 1 8 Eluted DNA is ready to use for e g automated fluorescent sequencing e g ABI 3700 3100 377 373 LICOR MegaBace ALF restriction analysis and PCR Kit specifications at a glance NucleoSpin Robot 96 Plasmid Culture volume 1 5 ml Average yield 5 15 ug Elution volume 75 150 ul Binding capacity 20 ug Vectors lt 15 kb Time prep 90 min 96 p
10. erial culture for 10 min at 1 000 x g It is strictly recommended to centrifuge the bacterial culture under these conditions Centrifugation at higher g forces may produce tight pellets which are more difficult to resuspend Optional If centrifugation at higher g forces is used a shaker integrated on the robot worktable will be necessary for complete resuspension of the bacterial pellet after addition of buffer A1 2 Discard supernatant Remove residual medium by placing the square well block upside down on clean paper sheet or soft tissue 3 Place square well culture block on a suitable vortexer to facilitate the complete resuspension of bacterial pellets with buffer A1 Place square well block in the desired position of the robot worktable 4 Prepare buffer A1 by adding RNase A Prepare buffer A4 by adding ethanol see section 3 for details 5 Add buffers to the reservoirs or place the buffer bottles in the corresponding positions of the robot worktable Place the plastic equipment like plates and the assembled vacuum manifold in the locations as specified in the individual robotic programs Optional The elution buffer AE 5 mM Tris HCl pH 8 5 may be substituted by nuclease free water check pH is 8 0 8 5 before use This is recommended if the eluted DNA has to be concentrated for downstream applications or Tris salts interfere with downstream applications A concentration of Tris higher than 10 mM can interfere
11. hing step with buffer A4 Remaining ethanol may cause problems with downstream applications like DNA sequencing or loading of samples onto agarose gel NucleoSpin Plasmid Filter Plate sticks to manifold ee e Clean gasket Remove any residual salt Clean manifold top with water and ethanol Do not use grease 20 MACHEREY NAGEL 08 2002 Rev 01 Automated Plasmid DNA Purification 6 2 Ordering information Product Cat No Pack of M ceo apin KOPOP ae racine 740616 24 24 x 96 preps NucleoSpin Robot 96 Plasmid 740708 2 2 x 96 preps NucleoSpin Robot 96 Plasmid 740708 4 4 x 96 preps NucleoSpin Robot 96 Plasmid 740708 24 24 x 96 preps Resuspension buffer A1 740911 1 11 without RNase A Lysis buffer A2 740912 1 11 Neutralisation buffer A3 740913 1 11 Wash buffer A4 concentrate 740914 1 200 ml for 1 buffer Wash buffer AW 740916 1 1 Elution buffer AE 740917 1 1 RNase A lyophilzed 740 505 100 mg RNase A lyophilized 740 505 50 50 mg Square well block 740670 20 Gas permeable foil 740674 50 Self adhering PE foil 740676 50 MN Frame 740680 1 6 3 References Birnboim H C amp Doly J 1979 Nucleic Acids Res 7 1513 1523 Vogelstein B amp Gillespie D 1979 Proc Natl Acad Sci USA 76 615 619 MACHEREY NAGEL 08 2002 Rev 01 21 Automated Plasmid DNA Purification 6 4 Product Use Restriction Warranty NucleoSpin Robot 96 Plasmid Core kits components were developed designed and
12. ica membrane Incubate for 1 3 min at room temperature Note Alternatively a 96 well thermocycler plate can be inserted into the square well block 4 Centrifuge for 2 min at maximum speed gt 4 000 x g optimal 5 800 x g to collect the DNA MACHEREY NAGEL 08 2002 Rev 01 17 Automated Plasmid DNA Purification 6 Appendix 6 1 Troubleshooting Problem Possible cause and suggestions Incomplete lysis of bacterial cells Cell pellet not properly resuspended e tis essential that the cell pellet is completely resuspended prior to lysis No cell clumps should be visible before addition of lysis buffer A2 If necessary increase number of mixing cycles or duration of shaking SDS in buffer A2 precipitated e SDS in buffer A2 may precipitate upon storage If this happens incubate A2 at 30 40 C for 5 min and mix well before use Too many bacterial cells used e Usage of LB as the growth medium is recommended When using rich media like TB cultures reach very high cell densities Reduce culture volume to 1 0 1 5 ml Poor plasmid yield No or not enough antibiotic used during cultivation e Cells harbouring the plasmid of interest may become overgrown by nontransformed cells Add appropriate amounts of freshly prepared stock solutions to all media solid and liquid Bacterial cultures are too old e See suggestions in section 4 Growing of bacterial cultures High copy number plasmid was not used
13. lved RNase A to the buffer A1 bottle and mix thoroughly Store buffer A1 containing RNase A at 4 C buffer A1 including RNase A is stable for up to 6 months Equilibrate buffer A1 to room temperature before starting plasmid DNA preparation e All other components of the NucleoSpin Robot 96 Plasmid kit should be stored at room temperature for a maximum of one year Storage of buffer A2 at temperatures below 20 C may cause precipitation of SDS If a salt precipitate is observed incubate the bottle at 30 40 C for some minutes and mix well until all of the precipitation is redissolved e Add indicated volume of 96 ethanol to buffer A4 concentrate before use NucleoSpin Robot 96 Plasmid Core kit 24 x 96 preps Cat No 740616 24 Buffer A4 24 x 100 ml concentrate add 400 ml ethanol to each bottle MACHEREY NAGEL 08 2002 Rev 01 9 Automated Plasmid DNA Purification 4 Growing of bacterial cultures 4 1 Selection of culture medium The cultivation of cells is recommended at 37 C in LB Luria Bertani medium at constant shaking 200 250 rpm Alternatively rich media like 2 x YT or TB Terrific Broth can be used By using 2 x YT or TB bacteria grow faster and reach the stationary phase much earlier than in LB medium lt 12 h This may lead to a higher percentage of dead or starving cells when starting the preparation The resulting plasmid DNA from overgrown cultures may be partially degraded or contaminated with
14. ng e Lysis step must not exceed 5 min Tips e Use widebore tips or disposable tips for transfer of crude lysate to the NucleoSpin Plasmid Filter Plate to prevent shearing of the chromosomal DNA RNA in the eluate RNA was not degraded completely e Ensure that RNase A is added to buffer A1 before use e Reduce culture volume if necessary MACHEREY NAGEL 08 2002 Rev 01 19 Automated Plasmid DNA Purification Problem Possible cause and suggestions Carryover of ethanol e Be sure to remove all of ethanolic buffer A4 after the final washing step Dry the NucleoSpin Plasmid Binding Plate for at least 10 min with maximum vacuum Elution of plasmid DNA with TE buffer e EDTA may inhibit enzymatic reactions like DNA sequencing Repurify the plasmid DNA and elute with AE buffer or nuclease free water Alternatively the plasmid DNA may be precipitated Suboptimal with ethanol and redissolved in AE buffer or nuclease free performance water of plasmid DNA in E coli strains with high endogenous nuclease levels are used as sequencing host PSA CHlOns Perform the washing step with buffer AW not supplied before problems with gt downstream washing with ethanolic buffer A4 applications Not enough DNA used for sequencing reactions e Quantitate DNA by agarose gel electrophoresis before setting up sequencing reactions Contamination of final plasmid preparation with ethanol e Insufficient drying after final was
15. of working solutions and storage conditions see section 3 4 MACHEREY NAGEL 08 2002 Rev 01 Automated Plasmid DNA Purification 2 Product description 2 1 The basic principle The NucleoSpin Robot 96 Plasmid procedure is a modified version of the Birnboim and Doly alkaline lysis plasmid miniprep protocol Bacterial cultures are harvested by an initial centrifugation step After resuspension of the pelleted bacteria buffer A1 and alkaline cell lysis buffer A2 a neutralization and binding buffer buffer A3 containing large amounts of chaotropic ions is added Resulting bacterial crude lysates are cleared by vacuum filtration with the NucleoSpin Plasmid Filter Plate The cleared lysates containing the plasmid DNA are collected into the NucleoSpin Plasmid Binding Plate The chaotropic salt leads to a reversible adsorption of the plasmid DNA to the NucleoSpin silica membrane during the second vacuum filtration step High purity of the final plasmid DNA preparation is achieved by complete removal of cellular contaminants salts detergents and other compounds in subsequent washing steps Highly pure plasmid DNA is finally eluted with elution buffer AE 5 mM Tris HCl pH 8 5 or water pH 8 0 8 5 and can be directly used for further applications 2 2 Kit specifications e NucleoSpin Robot 96 Plasmid is designed for the automated 96 well small scale purification of high copy plasmid DNA from E coli in the microtiter plate form
16. reereyerywrywryseyrry yey wryery wy wrery myer wre is MACHEREY NAGEL 08 2002 Rev 01 7 Automated Plasmid DNA Purification 2 4 Automation NucleoSpin Robot 96 Plasmid is designed for use on common laboratory automation workstations such as Robot Supplier Robot Beckman Coulter Biomek 2000 FX Cavro MiniPrep series Hamilton Microlab STAR MWG RoboSmart RoboPrep Perkin Elmer MultiPROBE II II HT Qiagen BioRobot 9600 3000 8000 Tecan Genesis RSP RWS Separation System series Zymark SciClone ALH Note As other laboratory automation workstations are currently under evaluation please contact MN directly if your workstation is not on this list Visit MN on the internet at www mn net com or contact your local MACHEREY NAGEL distributor for availability of ready to run scripts and for technical support regarding hardware software setup instructions and selection of the protocol All MN protocols can be downloaded from our website 8 MACHEREY NAGEL 08 2002 Rev 01 Automated Plasmid DNA Purification 3 Storage conditions and preparation of working solutions Attention Buffer A3 contains guanidinium hydrochloride which is an irritant Buffer A2 contains SDS and sodium hydroxide which are irritant and hazardous Wear gloves and goggles when handling them e Before first use of the kit add 1 ml of buffer A1 to the RNase A vial and vortex Transfer the whole amount of redisso
17. reps 2 3 Elution procedure See table for correlation between dispensed elution buffer volume and typical recoveries following the standard protocol The default volume of dispensed elution buffer in the available programs is 125 ul Dispensed elution buffer 75ul 100pl 125ul 150p 175 ul Recovered elution buffer containing plasmid DNA 30 5ul 5545 ul 8045 ul 10545 ul 13045 yl 6 MACHEREY NAGEL 08 2002 Rev 01 Automated Plasmid DNA Purification 250 100 80 200 a d 9 g 60 S S 8 fa 1504 6 S o 5 4 8 lt 7 m 5 z o a 100 20 concentration 50 60 80 100 120 140 160 180 200 dispensed elution buffer pl 20 40 60 80 100 120 140 160 recovered elution buffer pl Fig 1 Recovery rate and concentration depend on elution volume 10 ug of pBluescript were purified with NucleoSpin Robot 96 Plasmid and eluted with the indicated elution buffer volumes High recovery is achieved with 120 ul elution buffer dispensed as concentration drops A Fig 2 Purity and yields of pBluescript KS 2 96 Spee E ele O ne eater ani kbp A pUC 18 derivate 3 65 kbp B pcDNA3 1 B 8 6 kbp C and pCMV 7 2 kbp D using io NucleoSpin Robot 96 Plasmid 15 ul out of 125 pl h i ia d hea haah At et st hh Med Ne Nt Md Med han dan hanh et ded eld eluate were analyzed on a 1 agarose gel C AS ta hd a a a hd a bs s ah at D ferywryerrer
18. 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 Robot 96 Plasmid Core 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 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 EXCLU
19. ts 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 22 MACHEREY NAGEL 08 2002 Rev 01 Automated Plasmid DNA Purification 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 BIO mn net com MACHEREY NAGEL 08 2002 Rev 01 23
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