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WO1998016653A1 - Compositions et procede d'isolement rapide d'adn plasmidique - Google Patents

Compositions et procede d'isolement rapide d'adn plasmidique Download PDF

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Publication number
WO1998016653A1
WO1998016653A1 PCT/US1997/018762 US9718762W WO9816653A1 WO 1998016653 A1 WO1998016653 A1 WO 1998016653A1 US 9718762 W US9718762 W US 9718762W WO 9816653 A1 WO9816653 A1 WO 9816653A1
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WO
WIPO (PCT)
Prior art keywords
plasmid dna
polyethylene glycol
effective amount
lysate
bacterial
Prior art date
Application number
PCT/US1997/018762
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English (en)
Inventor
Zhidong Chen
Duane Ruffner
Original Assignee
University Of Utah Research Foundation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University Of Utah Research Foundation filed Critical University Of Utah Research Foundation
Priority to CA002266853A priority Critical patent/CA2266853A1/fr
Priority to JP10518609A priority patent/JP2001502179A/ja
Priority to EP97912759A priority patent/EP0941360A1/fr
Publication of WO1998016653A1 publication Critical patent/WO1998016653A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/06—Lysis of microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor

Definitions

  • compositions and methods of use thereof for rapid isolation of plasmid DNA from bacterial cultures More particularly, in one preferred embodiment the invention relates to a composition that can be used for preparing a cleared lysate by precipitating cellular debris, chromosomal DNA, cellular RNA, and proteins, and then can be used for differentially precipitating plasmid DNA from the cleared lysate.
  • the invention relates to a composition comprising an enzyme for digesting cellular RNA, a stabilizer for protecting plasmid DNA from nuclease digestion, and a precipitating agent for precipitating plasmid DNA.
  • a method of using this composition comprises effecting simultaneous digestion of cellular RNA, protection of plasmid DNA from nuclease digestion, and precipitation of plasmid DNA from a cleared bacterial lysate.
  • compositions and methods of use thereof for rapid isolation of high yields of plasmid DNA from bacterial cultures wherein the plasmid DNA is uncontaminated with RNA and the method does not involve toxic chemicals or dedicated equipment would be a significant advancement in the art.
  • BRIEF SUMMARY OF THE INVENTION It is an object of the present invention to provide a composition for rapid isolation of highly pure plasmid DNA from bacterial cultures . It is also an object of the invention to provide a method of rapidly isolating highly pure plasmid DNA from bacterial cultures.
  • composition comprising at least about 3 M MgCl 2 and about 10% (w/v) of polyethylene glycol .
  • the polyethylene glycol has an average molecular weight of about 6000 to about 8000, and the MgCl 2 concentration is about 3 molar.
  • a method of isolating plasmid DNA from a bacterial culture comprises the steps of:
  • the bacterial cells are lysed by alkaline lysis.
  • the MgCl 2 and polyethylene glycol are contained in a composition comprising at least about 3 M MgCl 2 and about 10% (w/v) polyethylene glycol. More preferably, the composition comprises about 3 M MgCl 2 , and the polyethylene glycol has an average molecular weight of about 8000.
  • a method of making a cleared bacterial lysate from a bacterial culture comprises the steps of :
  • a composition for isolating plasmid DNA from bacterial cultures comprises an effective amount of an enzyme for digesting cellular RNA, an effective amount of a stabilizer for protecting plasmid DNA from nuclease digestion, and an effective amount of a precipitating agent for precipitating plasmid DNA.
  • the enzyme is ribonuclease and the effective amount of enzyme is in the range of about 1-100 ⁇ g/ml, more preferably 5-50 ⁇ g/ml .
  • the stabilizer is a bivalent cation chelating agent, more preferably ethylenediaminetetraacetic acid (EDTA) or salts thereof, and the effective amount thereof is in the range of about 1-100 mM, more preferably 5-50 mM.
  • the precipitating agent is polyethylene glycol and the effective amount of precipitating agent is in the range of about 5-25% by weight .
  • the polyethylene glycol preferably has an average molecular weight of about 6000-8000.
  • a method of isolating plasmid DNA from a bacterial culture comprises :
  • FIG. 1 shows a representation of minipreps of 1:1 serial dilutions of pUC19 prepared according to the present invention (lanes 1, 3, 5, and 7) and by ethanol precipitation (lanes 2, 4, 6, and 8) from the same cell lysate after separation in 1.0% agarose gel and visualization by ethidium bromide staining: lane M - BstEII/1 DNA molecular weight ladder.
  • FIG. 2 shows a representation of restriction endonuclease fragments of plasmid pBKBHIOS (11857 bp) digested with 32 restriction enzymes separated on 0.6% agarose gel and stained with ethidium bromide: 0-uncut; 1-BsaHI; 2-N ⁇ tI; 3-SmaI; 4-XbaI; 5-BamHI; 6-BpmI; 7- BspHI; 8-ClaI; 9-DrdI; 10-NdeI; 11-PstI; 12-SacI; 13- Sail; 14-SapI; 15-Xh ⁇ I; 16-EcoRI; 17-EcoRV; 18-BsgI; 19-
  • FIG. 3 shows a representation of plasmid DNA prepared in a microtiter plate procedure according to the present invention wherein the plasmid DNA from columns 9-12 was separated on 0.8% agarose gel and stained with ethidium bromide; columns 10 and 12 were not inoculated with bacteria, and no plasmid DNA was found in the uninoculated wells.
  • TE buffer comprises 10 mM Tris
  • alkaline lysis solution comprises 1% SDS in 0.2 N NaOH.
  • an effective amount means an amount sufficient to achieve a selected response without undue adverse effects.
  • an effective amount of an enzyme for digesting cellular RNA is an amount sufficient to digest such cellular RNA at a selected temperature within a selected period of time.
  • An effective amount of stabilizer is an amount sufficient to protect plasmid DNA from nuclease digestion at a selected temperature for a selected amount of time.
  • An effective amount of a precipitating agent is an amount sufficient to precipitate a selected plasmid at a selected temperature in a selected amount of time. According to the guidance provided herein and what is well known in the art, such effective amounts can be determined by a person skilled in the art without undue experimentation.
  • enzyme for digesting cellular RNA and similar terms mean a nuclease for digesting such cellular RNA.
  • nuclease is ribonuclease A.
  • ribonuclease A is contained in the claimed composition in an amount ranging from about 1 to about 100 ⁇ l/ml .
  • stabilizer means an agent for protecting plasmid DNA from nuclease digestion.
  • Preferred stabilizers are bivalent cation chelating agents, such as ethylenediaminetetraacetic acid (EDTA) and salts thereof.
  • Preferred amounts of stabilizers, such as EDTA or salts thereof, in the presently claimed composition are in the range of about 1 to about 100 mM.
  • precipitating agent means a polymer, such as polyethylene glycol or dextran, for precipitating plasmid DNA from solution. Polyethylene glycol is especially preferred, and is generally used in concentrations of about 5-50% by weight in the presently claimed composition.
  • polyethylene glycol (PEG) polymers that have a relatively low average molecular weight, such as PEG 6000 or PEG 8000.
  • PEG polyethylene glycol
  • One illustrative embodiment of the present composition comprises an aqueous solution of at least about 3 M MgCl 2 and about 10% (w/v) of polyethylene glycol, preferably having an average molecular weight of about 8000 (PEG8000) .
  • the concentration of the PEG8000 is fairly critical to the proper functioning of the composition, but the concentration of the MgCl 2 can vary.
  • the optimal concentration of MgCl 2 has been determined to be about 3 molar.
  • the MgCl 2 and polyethylene glycol can be added to the cell lysate to obtain a cleared lysate.
  • MgCl 2 and polyethylene glycol can be added to the cleared lysate to precipitate plasmid DNA.
  • the MgCl 2 and polyethylene glycol can be added either as a mixture or sequentially as separate solutions, but a mixture of the MgCl 2 and polyethylene glycol is preferred because of ease of preparation and use.
  • An illustrative method of using the present composition for differentially precipitating plasmid DNA from a bacterial culture comprises lysing the cells in a bacterial culture, preferably with alkaline lysis solution as is well known in the art; adding about 0.5 volume of the MgCl 2 /PEG8000 composition and mixing to precipitate cellular debris, RNA, chromosomal DNA, and protein and removing the precipitated cellular debris, RNA, chromosomal DNA, and protein to result in a cleared lysate; mixing about the same volume of the MgCl 2 /PEG8000 composition used in the previous step with the cleared lysate to differentially precipitate the plasmid DNA; and recovering the precipitated plasmid DNA.
  • a wash solution such as with 70% ethanol as is well known in the art.
  • the resulting plasmid DNA is pure enough for being digested with a restriction endonuclease or for nucleotide sequencing applications.
  • the present composition and method can be used for purifying plasmid DNA from cell lysates and also for the following applications: (1) as a pre-purification step in preparing ultra-pure plasmid DNA by anion-exchange or other chromatographic methods; (2) to isolate and purify other episomal DNA molecules, such as viral DNAs from various cells and tissues, such as from mammalian cells,- and (3) as a post-reaction cleanup step to remove or recover other molecules, such as RNA, single-stranded DNA, nucleotides, enzymes, and the like from double- stranded DNA.
  • the present invention is an improvement over a procedure by Toa Gosei Chemical Industrial Ltd., JP 61-
  • the only 96-well plate miniprep technology is available from Promega Corporation. D. Romanin et al . , Use of a 96 Well Format for Wizard Miniprep DNA Isolations, 49 Promega Notes 28-32 (1994) .
  • the Promega method is based on the binding of DNA to a silica resin, which involves the transfer of cell lysates to a set of mini-columns, addition of a DNA binding resin to the lysates, washing the samples in a manifold, and elution of the plasmid DNA from the columns into a 96-well plate.
  • the presently claimed method is not only based on a different DNA isolation mechanism, but also involves different DNA isolation procedures. Unlike Promega' s method, the present method does not require dedicated equipment or accessories, and all the steps are performed in the 96-well plates, from cell culture through isolating the plasmid DNA.
  • Example 1 An illustrative composition according to the present invention for use in isolating plasmid DNA from a bacterial culture was prepared by dissolving MgCl 2 and PEG8000 in deionized water to concentrations of 3 M MgCl 2 and 10% (w/v) PEG 8000.
  • Example 2 In this example, a procedure for using the composition according to Example 1 for isolating plasmid DNA is described:
  • Example 3 In this procedure, a mid-sized preparation, i.e. "midiprep,” procedure using the composition according to Example 1 is described:
  • Example 7 Add 2 ml of the solution of Example 1, mix by vortexing, and incubate the tube and its contents at 37°C for 5 minutes . 8. Centrifuge at 12,000 x g lor 10 minutes to pellet the plasmid DNA, and decant and discard the supernate .
  • Example 4 In this example, a large-scale miniprep procedure using 96-well microtiter plates is described:
  • a clone of pUT626 is prepared using this procedure.
  • the recoveries of plasmid DNA among the samples is relatively consistent, and no inter-well contamination is observed.
  • a clone of plasmid pHHC (2768 bp) is isolated according to the procedures of Example 2 and sequenced by the dideoxy method, F. Sanger et al . , DNA Sequencing with Chain-terminating Inhibitors, 74 Proc . Na ' 1 Acad. Sci. USA 5463 (1977) , hereby incorporated by reference, using 35 S- ⁇ -dATP and "SEQUENASE" version 2.
  • a DNA isolation solution has been developed that has been successfully used for the rapid isolation of plasmid DNA from bacterial cultures .
  • the composition comprises an effective amount of an enzyme for digesting cellular RNA, an effective amount of a stabilizer for protecting plasmid DNA from nuclease digestion, and an effective amount of a precipitating agent for precipitating plasmid DNA, as described above.
  • a method of using the solution is simple, fast, convenient, and cost effective. More than 95% of the plasmid DNA can be recovered from a cell lysate by this method.
  • the DNA obtained by this method is free of RNA contamination and is suitable for various manipulations in molecular cloning and DNA sequencing.
  • the method has been adapted to a 96-well microtiter plate format for high throughput minipreps, which is very useful for screening large numbers of clones .
  • the present method contains at least two distinct improvements over conventional methods that also involve the use of PEG for plasmid purification.
  • a bivalent cation chelating agent such as EDTA
  • plasmid DNA can be protected from degradation by cellular DNases during the 37°C incubation with RNase without first performing ethanol precipitation and the hazardous and tedious phenol-chloroform extraction.
  • EDTA and RNase in the PEG solution, three objectives are obtained simultaneously: (1) protecting the plasmid DNA, (2) digesting the RNA, and (3) precipitating the plasmid DNA. In conventional PEG methods, these objectives are realized in three or more separate steps .
  • the present method is simpler, faster, and can be adapted to large-scale miniprep procedures using a 96-well microtiter plate, as described below.
  • the plasmid isolation solution is composed of non- toxic components and is stable at 4°C for at least 6 months.
  • Other reagents required are TE buffer (10 mM Tris and 1 mM EDTA, pH 8.0), 1% SDS in 0.2 N NaOH, 3 M Na acetate (pH 5.0), and 70% ethanol.
  • 96-well (2 ml/well) microtiter plates e.g. Beckman #140504
  • aluminum lids e.g. Beckman #538619
  • Example 6 an illustrative composition according to the present invention is prepared by making a solution comprising 20 ⁇ g/ml of RNase A, 20 mM EDTA, and 20% PEG8000 in deionized water.
  • Example 7 An illustrative embodiment of the present method of isolating plasmid DNA comprises the steps of (a) preparing an alkaline lysate of bacterial cells, (b) mixing equal volumes of lysate and the composition prepared according to Example 6, (c) incubating the mixture at 37°C for a few minutes, and (d) then centrifuging incubated mixture to pellet the plasmid DNA. Preferably, the plasmid pellet is then washed to remove residual contaminants. The washed plasmid DNA is then dried and suspended in an appropriate buffer or water.
  • Example 8 In this example, a miniprep procedure using the composition according to Example 6 is described:
  • Plasmid DNA was also prepared by direct ethanol precipitation from half of the same cell lysate.
  • the purity of the plasmid DNA prepared according to the present invention was checked by agarose gel electrophoresis, and the yield (98.6%) was determined as the percent of plasmid DNA obtained by direct ethanol precipitation (FIG. 1) .
  • Example 9 In this example, a midiprep procedure using the composition of according to Example 6 is described:
  • plasmid pBKBHIOS (11857) was prepared from 50 ml of an overnight culture of bacterial strain STBL2. The plasmid was digested with 32 restriction enzymes at their respective optimal temperatures for 1 hour. The digested DNAs were then fractionated by electrophoresis on 0.6% agarose gel (FIG. 2) . The results show that the plasmid DNA purified with the solution of Example 6 was completely digested by the enzymes tested, and there was no RNA interfering with the detection of the plasmid bands.
  • Example 10 In this example, a large-scale miniprep procedure using 96-well microtiter plates and the composition according to Example 6 is described: 1. Aliquot 1 ml of antibiotic-containing LB medium, J. Miller, Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1972) , hereby incorporated by reference, into each of the 96 well using a repeat pipetter. 2. Inoculate one colony of bacteria into a well, cover the plate with an aluminum lid, and puncture holes on the lid with a needle to allow ventilation of the wells during incubation.
  • a clone of plasmid pUT626 (3185 bp) was prepared using the large-scale procedure. As shown in FIG. 3, the recoveries of the plasmid among the samples were relatively consistent, and no inter-well contamination occurred during the procedure, since the wells that were not inoculated with bacteria produced no plasmid DNA.
  • Example 11 A clone of plasmid pHHC (2768 bp) was isolated according the procedure of Example 8 and sequenced by the dideoxy method, F. Sanger et al . , DNA Sequencing with Chain-terminating Inhibitors, 74 Proc . Nat ' 1 Acad. Sci. USA 5463 (1977), hereby incorporated by reference, using 35S- -dATP and "SEQUENASE version 2.” The plasmid purified by this method was shown to be suitable for sequencing applications.
  • the solution and the plasmid isolation procedures described herein have many advantages over other conventional and commercially available methods .
  • the present method is simple.
  • one volume of solution is added to a bacterial cell lysate and, after a short incubation (2-5 minutes) , the plasmid DNA is obtained by centrifugation.
  • the procedure is also fast. Plasmid minipreps can be completed in 20-30 minutes. With the large-scale 96- well miniprep procedure, 96 minipreps can be obtained in about 90 minutes. Also, the procedure is safe.
  • the solution contains no toxic components, and no steps in the procedure require toxic organic solvents. Further, the procedure is convenient.
  • the SDS-NaOH lysis procedure is already routine in many laboratories .
  • the procedure is economical .
  • the reagent costs for the solution is estimated to be about 5 cents per miniprep. Only two microfuge tubes and a few pipette tips are required for each standard miniprep.
  • the solution is easy to prepare and is a single solution product. It accompanies no other specialized reagents and accessories.
  • the plasmid DNA obtained is clean and in good yield. There is no detectable RNA contamination, and the OD 260/280 ratio is about 1.8. More than 95% of the plasmid DNA in the cell lysate can be recovered.

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Abstract

Cette invention se rapporte à une composition utilisée pour isoler de l'ADN plasmidique dans une culture bactérienne. Cette composition contient au moins 3 moles environ de chlorure de magnésium et 10 % en poids environ de polyéthylène glycol. L'invention se rapporte à un procédé permettant d'obtenir une précipitation sélective D'ADN plasmidique à partir d'un lysat bactérien, qui consiste à lyser des cellules bactériennes de façon à produire un lysat bactérien; à mélanger au lysat du chlorure de magnésium et du polyéthylène glycol de façon à obtenir un mélange contenant au moins 1 mole environ de chlorure de magnésium et 3,3 % en poids environ de polyéthylène glycol, ce qui permet la précipitation de débris cellulaires, d'ADN chromosomique, d'ARN cellulaire et de protéines; à séparer et à extraire la matière précipitée de façon à obtenir un lysat transparent; à mélanger du chlorure de magnésium et du polyéthylène glycol au lysat transparent de manière à former un mélange contenant au moins 1,5 mole environ de chlorure de magnésium et 5 % en poids environ de polyéthylène glycol, ce qui permet la précipitation de l'ADN plasmidique; et à séparer du mélange l'ADN plasmidique précipité, ce qui permet d'obtenir l'ADN plasmidique isolé. Une autre composition conforme à l'invention contient un enzyme de digestion de l'ARN cellulaire, un stabilisateur servant à protéger l'ADN plasmidique de la digestion par des nucléases, et un agent de précipitation assurant la précipitation de l'ADN plasmidique. Un autre procédé d'isolement d'ADN plasmidique à partir d'une culture bactérienne consiste à assurer simultanément la digestion d'ARN cellulaire dans un lysat clarifié par un enzyme, la protection de l'ADN plasmidique vis à vis d'une digestion par des nucléases et la précipitation d'ADN plasmidique.
PCT/US1997/018762 1996-10-15 1997-10-15 Compositions et procede d'isolement rapide d'adn plasmidique WO1998016653A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
CA002266853A CA2266853A1 (fr) 1996-10-15 1997-10-15 Compositions et procede d'isolement rapide d'adn plasmidique
JP10518609A JP2001502179A (ja) 1996-10-15 1997-10-15 プラスミドｄｎａの迅速な単離のための組成物及び方法
EP97912759A EP0941360A1 (fr) 1996-10-15 1997-10-15 Compositions et procede d'isolement rapide d'adn plasmidique

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US2754296P 1996-10-15 1996-10-15
US60/027,542 1996-10-15
US4037597P 1997-03-10 1997-03-10
US60/040,375 1997-03-10

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999058664A1 (fr) * 1998-05-14 1999-11-18 Whitehead Institute For Biomedical Research Technique en phase solide pour isoler selectivement des acides nucleiques
KR20020029477A (ko) * 2000-10-13 2002-04-19 박제철 한 단계에 의한 유전자 추출방법
KR20020029476A (ko) * 2000-10-13 2002-04-19 박제철 한 단계에 의해 유전자를 추출하기 위한 라이시스버퍼시약
WO2002055739A3 (fr) * 2001-01-15 2003-04-03 Cytyc Corp Solution d'extraction d'acide nucleique et son utilisation
EP1374837A1 (fr) * 2002-06-26 2004-01-02 Wella Aktiengesellschaft Mousse aérosol pour traitement capillaire
US7527929B2 (en) 2004-07-30 2009-05-05 Agencourt Bioscience Corporation Methods of isolating nucleic acids using multifunctional group-coated solid phase carriers
EP1856262A4 (fr) * 2005-01-31 2009-11-04 Merck & Co Inc Procede de purification amont et aval pour production a large echelle d'adn plasmidique
US9017966B2 (en) 2007-05-23 2015-04-28 Nature Technology Corporation E. coli plasmid DNA production
CN113265395A (zh) * 2021-05-18 2021-08-17 苏州博腾生物制药有限公司 菌体裂解液澄清试剂以及在质粒提取工艺中的应用

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US5561064A (en) * 1994-02-01 1996-10-01 Vical Incorporated Production of pharmaceutical-grade plasmid DNA
US5705628A (en) * 1994-09-20 1998-01-06 Whitehead Institute For Biomedical Research DNA purification and isolation using magnetic particles

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US5561064A (en) * 1994-02-01 1996-10-01 Vical Incorporated Production of pharmaceutical-grade plasmid DNA
US5705628A (en) * 1994-09-20 1998-01-06 Whitehead Institute For Biomedical Research DNA purification and isolation using magnetic particles

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Title
ANALYTICAL BIOCHEMISTRY, February 1988, Volume 169, MATTHEWS et al., "REVIEW, Analytical Strategies for the Use of DNA Probes", pages 1-25. *
MANIATIS et al., Molecular Cloning, A Laboratory Manual, 1982, (Published by the COLD SPRING HARBOR LABORATORY, COLD SPRING HARBOR, NEW YORK, USA), pages 80-82. *

Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999058664A1 (fr) * 1998-05-14 1999-11-18 Whitehead Institute For Biomedical Research Technique en phase solide pour isoler selectivement des acides nucleiques
US6534262B1 (en) 1998-05-14 2003-03-18 Whitehead Institute For Biomedical Research Solid phase technique for selectively isolating nucleic acids
KR20020029477A (ko) * 2000-10-13 2002-04-19 박제철 한 단계에 의한 유전자 추출방법
KR20020029476A (ko) * 2000-10-13 2002-04-19 박제철 한 단계에 의해 유전자를 추출하기 위한 라이시스버퍼시약
WO2002055739A3 (fr) * 2001-01-15 2003-04-03 Cytyc Corp Solution d'extraction d'acide nucleique et son utilisation
US6939672B2 (en) 2001-01-15 2005-09-06 Cytyc Corporation Nucleic acid extraction solution and use thereof
EP1374837A1 (fr) * 2002-06-26 2004-01-02 Wella Aktiengesellschaft Mousse aérosol pour traitement capillaire
US7527929B2 (en) 2004-07-30 2009-05-05 Agencourt Bioscience Corporation Methods of isolating nucleic acids using multifunctional group-coated solid phase carriers
EP1856262A4 (fr) * 2005-01-31 2009-11-04 Merck & Co Inc Procede de purification amont et aval pour production a large echelle d'adn plasmidique
US7767399B2 (en) 2005-01-31 2010-08-03 Merck & Co., Inc. Purification process for plasmid DNA
AU2006211216B2 (en) * 2005-01-31 2011-02-03 Merck Sharp & Dohme Llc Purification process for plasmid DNA
CN101310022B (zh) * 2005-01-31 2013-01-16 默沙东公司 大规模生产质粒dna的上游和下游纯化方法
US9017966B2 (en) 2007-05-23 2015-04-28 Nature Technology Corporation E. coli plasmid DNA production
US9487788B2 (en) 2007-05-23 2016-11-08 Nature Technology Corporation E. coli plasmid DNA production
US9487789B2 (en) 2007-05-23 2016-11-08 Nature Technology Corporation E. coli plasmid DNA production
US9725725B2 (en) 2007-05-23 2017-08-08 Nature Technology Corporation E. coli plasmid DNA production
CN113265395A (zh) * 2021-05-18 2021-08-17 苏州博腾生物制药有限公司 菌体裂解液澄清试剂以及在质粒提取工艺中的应用

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CA2266853A1 (fr) 1998-04-23
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