Analytical Biochemistry 377 (2008) 218–222

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Analytical Biochemistry

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A non-alkaline method for isolating sequencing-ready plasmids

Bonnie Paul 1, Cheri Cloninger, Marilyn Felton 1, Ronik Khachatoorian, Stan Metzenberg *

Department of Biology, California State University, Northridge, CA 91330-8303, USA

a r t i c l e i n f o

Article history:Received 10 January 2008Available online 7 March 2008

Keywords:DNAPlasmidsIsolation and purificationNucleic acid denaturation

0003-2697/$ - see front matter � 2008 Elsevier Inc. Adoi:10.1016/j.ab.2008.03.005

* Corresponding author. Fax: +1 818 677 2034.E-mail address: stan.metzenberg@csun.edu (S. Me

1 Present address: Department of Natural Science,13356 Eldridge Avenue, Sylmar, CA 91342, USA.

a b s t r a c t

We describe a simple method of isolating plasmid DNA directly from Escherichia coli culture medium byaddition of lithium acetate and Sodium dodecyl sulphate, followed by centrifugation and alcohol precip-itation. The plasmid is sufficiently pure that it can be used in many enzyme-based reactions, includingDNA sequencing and restriction analysis. Chromosomal DNA contamination is significantly reduced bypretreatment of the culture with DNase I, suggesting that much of the contaminant is associated withpermeable dead cells. Chromosomal DNA contaminant can also be selectively denatured without damageto the supercoiled plasmid by alkaline denaturation in an arginine buffer or heat treatment in the pres-ence of urea or N,N-dimethylformamide.

� 2008 Elsevier Inc. All rights reserved.

The most widespread methods of bacterial plasmid preparationare based on detergent lysis of bacteria under alkaline conditionsfollowed by a high-salt acidification or zwitterionic detergent stepto precipitate cellular proteins and chromosomal DNA [1–3]. Cellu-lar RNA and some bacterial chromosomal DNA copurifies with theplasmid at this stage, and while the RNA can be readily degradedby RNase A treatment the separation of chromosomal DNA is moreproblematic [4]. Alkali-denatured chromosome is not removedfrom a plasmid sample by anion exchange resins or silica matricesand may be overlooked because it is not easily visible with gelstains such as ethidium bromide and SYBR green. While the con-taminant is insensitive to restriction endonucleases and is ‘‘unclon-able,” it may still interfere with PCR and determination of DNAconcentration by UV spectroscopy. Under ideal conditions the alka-line pH does not irreversibly denature the plasmid DNA in the cell;however, if a sodium-hydroxide-based lysis solution is added inexcess for a given cell mass, the pH can rise to the point wheresupercoiled plasmids are damaged [5–7]. This denatured super-coiled plasmid may have a slightly higher mobility on a gel thannative supercoiled plasmid and is resistant to restriction enzymes.

Plasmids prepared by alkaline detergent lysis or boiling are gen-erally not sufficiently pure to use in enzymatic reactions until addi-tional time-consuming steps are taken to remove impurities. Thesesteps may include affinity capture or chromatography [8–11], phe-nol and chloroform extraction [12–20], or CsCl gradient centrifuga-tion [21]. Nonalkaline methods of plasmid extraction that usephenol or detergents or heat treatment of cells have been devel-

ll rights reserved.

tzenberg).Los Angeles Mission College,

oped [13–15,18,19,22–37], but these also yield DNA that is margin-ally pure.

Here we describe a simple and novel nonalkaline method ofplasmid extraction from Escherichia coli that yields DNA of suffi-cient purity for restriction digestion, polymerase chain reaction,and DNA sequencing. Bacterial RNA is largely excluded withoutthe addition of RNase, which is an advantage if the plasmid is in-tended for use in RNA methods. We also describe a method ofreducing the amount of contaminating chromosomal DNA byDNase I treatment of bacteria prior to lysis and three additionalmethods of denaturing chromosomal DNA contamination afterlysis. These methods of mitigating chromosomal contaminationcan be applied to a plasmid prepared by any approach, includingalkaline lysis.

Materials and methods

Lysis of bacteria

In the simplest manifestation of this method, E. coli cells arelysed directly in their growth medium by the addition of 1 volumeof 6 M lithium acetate (pH 4.8), 0.5% (w/v) SDS, 2 mM EDTA. Fol-lowing addition, the solution is gently mixed, allowed to sit undis-turbed at room temperature for 5 min, and then subjected tocentrifugation at 10,000g for 10 min at room temperature. Thesupernatant is transferred to a new tube and mixed with 0.8 vol-ume of isopropanol, and the nucleic acid precipitate is collectedby centrifugation at 10,000g for 10 min at room temperature. Theresidual salts are washed from the precipitate and sides of the tubeby addition of 0.1–1 ml of 70% (v/v) ethanol, and this wash ethanolis collected and removed following a brief centrifugation. The pre-cipitate is dried in vacuo and dissolved in sterile water or TE buffer

Fig. 1. Agarose gel electrophoresis of pGEM3Zf+ plasmid isolated from four bact-erial strains by nonalkaline lysis. Each gel lane represents nucleic acids extractedfrom 0.75 ml of cell culture; bacterial strains XL-1 blue (lanes 1, 2), Top10 (lanes 3,4), Solopack Gold (lanes 5, 6), and DH5aF’ (lanes 7, 8). Lanes 2, 4, 6, and 8 includedtreatment of the culture with DNase I prior to lysis. The asterisk indicates themigration of chromosomal DNA and the arrow indicates the migration of super-coiled plasmid. Lane 10 is 0.4 lg k DNA digested with Hind III (fragment sizes 23.1,9.4, 6.5, 4.3, 2.3, 2.0, and 0.56 kb). Stained with ethidium bromide.

Nonalkaline isolation of plasmids / B. Paul et al. / Anal. Biochem. 377 (2008) 218–222 219

(10 mM Tris–Cl (pH 8.0), 1 mM EDTA) at 1/100 of the original cul-ture volume. The solution may be clarified by centrifugation at10,000g for 10 min to remove any insolubles.

This method takes approximately 30–40 min from the point ofliquid culture to having a purified DNA, and requires less han-dling time than a commercial kit because only a single lysis re-agent is used. The cost of extraction from 1–5 ml of bacterialculture is approximately 10–50% of the price of a single miniprepextraction using a commercial kit; however, the lysis solution canbe used more sparingly if large cultures are first concentrated bycentrifugation and resuspended in 0.1 volume of TE buffer. Theyield of supercoiled plasmid is approximately 100–500 ng/ml ofculture if the cells are grown to saturation in Luria–Bertanimedium.

While lithium acetate (pH 4.8) buffer at a final concentration of3 M is the recommended salt condition during lysis, we haveachieved success with a range of concentrations of 1.5–4 M. Below1.5 M lithium acetate the amount of contaminating chromosomalDNA increases dramatically. Lithium acetate may also be used atpH 8.0 in the context of a 20 mM Tris–Cl buffer, with the acetateacting as a salt instead of a buffering species, and we have hadsome success using high molarities (>2 M) of other salts such asLiCl, NaCl, or KCl. However, the pH 4.8 buffered lithium acetatecondition described here gives the most reproducible results. Werecommend a concentration of 0.25% (w/v) SDS during lysis; how-ever, concentrations of 0.2–2.0% are also successful. Periods of lysisbetween 30 s and 20 min yield similar results; however, we recom-mend a 5-min lysis period for consistent results. As little as 0.1%(w/v) SDS generates lysis at room temperature if the period ofincubation is at least 5 min. Lysis is slower if the cells are incubatedon ice and higher concentrations of SDS may need to be employedif low temperatures are used.

The SDS will gradually precipitate out of the lysis solution atroom temperature and the solution can be either rewarmed beforeuse or stored indefinitely in a warm incubator. Alternatively, a lith-ium acetate and EDTA solution can be prepared separately from a5% (w/v) SDS solution, and these will be stable at room tempera-ture without precipitation. In that case lysis of bacteria is inducedby adding the two solutions separately: 1 volume of 6 M lithiumacetate (pH 4.8), 2 mM EDTA, followed by 0.05 volume of 5% (w/v)SDS.

Optional pretreatment of bacterial cells with DNase I

A bacterial culture is brought to a concentration of 10 mMMgCl2 and 10 lg/ml DNase I (bovine pancreatic; Sigma–Aldrich)and either returned to a 37 �C shaker incubator or held at roomtemperature for at least 30 min.

Three optional methods for denaturing chromosomal DNAcontaminant

(1) Denaturation using arginine buffer. The purified plasmid isincubated in a 0.5 M arginine buffer (pH 11.7) at room temperaturefor at least 1 h; 1 volume of a 1 M arginine buffer can be added to aplasmid to generate this pH condition.

(2) Denaturation using urea. The purified plasmid is incubatedin 8 M urea at 65 �C for 5 min; 4 volumes of a 10 M urea solutioncan be added to a plasmid to generate this condition.

(3) Denaturation using dimethylformamide: The purified plas-mid is incubated in 50% (v/v) N,N-dimethylformamide at 65 �Cfor 5 min; 1 volume of deionized N,N-dimethylformamide can beadded to a plasmid to generate this condition.

In all three methods, the incubation is terminated by addition of1 volume of 0.5 M lithium acetate (pH 4.8), 1 mM EDTA, followedby precipitation using 0.8 volume isopropanol or 2.5 volumes eth-

anol. The DNA is collected by centrifugation at 10,000 g for 10 minat room temperature; the pellet is washed with 70% ethanol as de-scribed above and dried in vacuo.

Restriction analysis and DNA sequencing

Restriction endonucleases were obtained from Promega Corp.and used with the buffer stocks recommended and provided bythe company. Nonisotopic DNA sequencing was performed on anABI 3130 automated DNA sequencer.

Solutions and reagents

The lysis solution is prepared using lithium acetate (reagentgrade or SigmaUltra grade; Sigma–Aldrich), glacial acetic acid(ACS grade; Fisher Scientific), SDS (molecular biology grade orReagentPlus grade; Sigma–Aldrich), and EDTA (Molecular Biologygrade; Sigma–Aldrich). The arginine buffer (pH 11.7) is preparedusing L-arginine (reagent grade P98% (TLC); Sigma–Aldrich) andNaOH (ACS grade; Fisher Scientific) yielding a 6.31:1 molar ratioof zwitterion to anion.

A Wizard Plus MiniPreps DNA Purification System was obtainedfrom Promega Corp. and employed following the manufacturer’sinstructions. All other reagents were obtained from Sigma–Aldrichor Fisher Scientific.

Bacteriological strains

Top10, Top10F’, and DH5aF’ were obtained from Invitrogen. Sol-opack Gold, XL-1 blue, and BL21 were obtained from Stratagene.

Fig. 2. Selective denaturation of chromosomal DNA contaminant. (A) pHIL-S1 plasmid isolated from 10-fold-concentrated Top10 cells by nonalkaline lysis (lanes 1–5),Birnboim and Doly alkaline lysis (lane 7), or Wizard Plus Minipreps kit (lanes 8 and 9). The nonalkaline lysate DNA was untreated (lane 1) or treated with 0.5 M argininebuffer, pH 11.7, for 1 h (lane 2) or 25 h (lane 3) or with urea (lane 4) or N,N-dimethylformamide (lane 5). Lanes 7 and 8 represent crude lysates, and lane 8 represents WizardPlus Miniprep column-purified DNA. Lane 6 is k DNA digested with Hind III. (B). pGEM3Zf+ plasmid isolated from frozen, 10-fold-concentrated Top10 cells by nonalkaline lysisand untreated (lane 1) or treated with 0.5 M arginine buffer, pH 11.7, for 1 h (lane 2) or with urea (lane 3) or N,N-dimethylformamide (lane 4). Lane 5 is k DNA digested withHind III. Cultures were not pretreated with DNase I. The asterisks indicate the migration of chromosomal DNA and the arrows indicate the migration of supercoiled plasmid.

220 Nonalkaline isolation of plasmids / B. Paul et al. / Anal. Biochem. 377 (2008) 218–222

Recombinant materials were handled under the NIH Guidelines forResearch Involving Recombinant DNA Molecules.

Results

Direct isolation of plasmid from cells in culture medium

A method of nonalkaline extraction of the 3.2-kb plasmidpGEM3Zf+ (Promega) was tested in four bacterial strains asdescribed under Materials and methods. In brief, cells were lyseddirectly in their culture medium by addition of 1 volume of 6 Mlithium acetate (pH 4.8), 0.5% (w/v) SDS, 2 mM EDTA, and a clari-fied supernatant was collected by isopropanol precipitation. Asshown in Fig. 1 (lanes 1, 3, 5, and 7), supercoiled plasmid is readilyisolated from all four strains with minimal contamination of cellu-lar RNA. Some bacterial chromosomal DNA copurifies with theplasmid, and because the contaminant has not been denatured itis visible in a stained gel. However, if the cells are treated withDNase I prior to lysis (lanes 2, 4, 6, and 8), the levels of contaminat-ing chromosomal DNA drop dramatically. This effect suggests thatsome of the chromosomal DNA contamination in any plasmidpreparation may originate with cells permeable to DNase I (i.e.,dead cells). In the method presented here, only a small fractionof the chromosomal contaminant is attributable to DNA releasedfrom live cells during lysis.

Additional methods for mitigation of the chromosomal contaminant

Whereas DNase I pretreatment reduces the overall amount ofchromosomal DNA in a plasmid preparation, the following optional

methods may be advantageous if the plasmid DNA is to be used ingenetic engineeering work. Treatment of the 8.3-kb pHIL-S1 (Invit-rogen) plasmid with 0.5 M arginine (pH 11.7) for 1 h at room tem-perature selectively denatures the chromosomal DNA (lane 2 ofFigs. 2A and B), leading to the disappearance of the contaminantband. This selective denaturation is also demonstrated usingpGEM3Zf+ prepared from XL-1 blue cells (lanes 3 and 6 ofFig. 3C). We have found that the supercoiled plasmid is not subjectto gradual denaturation in the arginine buffer and that the plasmiddoes not change perceptibly between 1 and 25 h of incubation(lanes 2 and 3 of Fig. 2A). The longer incubation in alkali does resultin complete hydrolysis of the cellular RNA contaminant. Alterna-tively, a purified plasmid can be treated with 8 M urea at 65 �Cfor 5 min (lane 4 of Fig. 2A, and lane 3 of Fig. 2B) or 50% (v/v)N,N-dimethylformamide at 65 �C for 5 min (lane 5 of Fig. 2A andlane 4 of Fig. 2B), and either of these treatments will selectivelydenature the chromosomal contaminant and leave the plasmidundisturbed.

Comparison of alkaline and nonalkaline lysis methods

The Birnboim and Doly method of plasmid extraction with a0.2 N NaOH solution leads to a 5–30 times greater yield of plas-mid than the method described here and may be preferable if ahigh yield is important. However, as shown in lane 7 of Fig. 2A,the higher yield of pHIL-S1 using the Birnboim and Doly methodis also accompanied by extensive contamination with cellularRNA and chromosomal DNA, and the full extent of chromosomalcontamination may not be evident because much of it may havebeen denatured and rendered invisible to the gel stain. Commer-cial kits based on the Birnboim and Doly method often include

Fig. 3. Use of pGEM3Zf+ prepared by nonalkaline lysis for DNA sequencing and restriction digestion. (A) DNA sequence analysis of plasmid template prepared from bacterialstrain XL-1 blue (showing extension of the sequencing primer by 485 to 500 bp). (B) Same region of sequence using plasmid template prepared from Top10 cells. (C)Restriction digestion of pGEM3Zf+ using EcoRI (lanes 1, 4) or Hind III (lanes 2, 5). Lanes 3 and 6 show undigested DNA. Plasmid used in lanes 4–6 was pretreated with 0.5 Marginine buffer, pH 11.7, for 1 h to denature the chromosomal DNA. The asterisk indicates the migration of chromosomal DNA and the arrow indicates the migration ofsupercoiled plasmid.

Nonalkaline isolation of plasmids / B. Paul et al. / Anal. Biochem. 377 (2008) 218–222 221

RNase A as a solution component during lysis to degrade the cel-lular RNA contaminant. This elimination of RNA contamination isevident in a crude lysate of Top10 cells carrying pHIL-S1, pre-pared using solutions from a Wizard Plus MiniPreps DNA Purifi-cation System (lane 8 of Fig. 2A). Chromosomal DNAcontamination is visible in this crude lysate, although once againthe full extent cannot be determined by gel stain. The purity ofplasmid prepared by the simplest method described in this pa-per (lithium-acetate-based lysis followed by isopropanol precip-itation) appears comparable to kit-purified samples (comparelanes 1 and 9 of of Fig. 2A).

The plasmid is sufficiently pure for DNA sequencing and restrictiondigestion

The DNA samples shown in lanes 1, 3, 5, and 7 of Fig. 1 repre-sent the simplest form of purification of plasmid, without steps ta-ken to mitigate RNA and chromosomal DNA contamination, andthese samples were used for nonisotopic DNA sequencing. Thesesamples yielded sequencing ‘‘lengths of read” ranging 423–841nt using the M13 universal forward primer and an ABI 3130 auto-mated sequencer, and these are similar to results that we obtainwith kit-purified DNA samples. Fig. 3 shows the high quality of se-quence obtained after extending the primer 485 to 500 nt, usingplasmid prepared from XL-1 blue (Fig. 3A) or Top10 (Fig. 3B). Wehave used the lithium acetate-lysis method to isolate other plas-mids for immediate sequencing and from other bacterial strainssuch as Top10F’ and BL21.

Discussion

We describe a novel method for preparing plasmids that isinexpensive and generates plasmid that can be directly usedin DNA sequencing and other enzymatic methods. During thisnonalkaline lysis procedure the cell suspension remains turbidor even opaque, which is different from that in the alkaline ly-sis method where the cell suspension becomes transparentupon addition of NaOH and then becomes opaque after additionof a high salt neutralizing solution [1–3]. The pellet that we ob-serve after centrifugation of the nonalkaline lysate is usuallycompact, unlike the loose pellets obtained with the alkalinemethod that are sometimes difficult to separate from theirsupernatant. With our method the bacterial chromosome andcellular proteins are largely salted out of solution because thecells are made permeable by the detergent and the chromo-some is kept compact by the high ionic strength and absenceof denaturing conditions. The bacterial RNA is precipitated bythe lithium salts, so it is also largely eliminated during the firstcentrifugation step. Under these conditions about 10% of theplasmid escapes into the supernatant and can be collected byalcohol precipitation. It is sufficiently pure to be used directlyin enzymatic methods such as restriction digestion, DNAsequencing, and polymerase chain reaction. By comparison,plasmid prepared by alkaline lysis is often not sufficiently purefor these applications until it has been separated from inhibi-tors by organic extractions or binding to affinity matrices[8–11].

222 Nonalkaline isolation of plasmids / B. Paul et al. / Anal. Biochem. 377 (2008) 218–222

We observed that contaminating chromosomal DNA levels can-not be reduced by washing the cells in a high- or low-salt conditionor a mild nonionic detergent before lysis but could be eliminatedby treating the cells with DNase I. Just as with alkaline methods,chromosomal DNA can be released from cells if the nonalkaline ly-sate is shaken or vortexed and we suggest that lysis be conductedgently. The optional approaches of selective denaturation of chro-mosomal DNA could be applied, as needed, to any method of plas-mid purification.

Other nonalkaline methods of plasmid extraction have beendeveloped that use detergents [13,15], organic extraction[14,18,19,22–26], thermal lysis [27–32], osmotic shock [33–36],or proteinase K digestion [37]. The methods that we describe herefor plasmid purification provide some advantage in speed, simplic-ity, and generation of a plasmid product that is largely free of chro-mosomal DNA contamination and ready to use in enzymaticreactions.

Acknowledgments

Funding for research was provided by the Office of Research andSponsored Projects, California State University, Northridge. B.P. andM.F. were supported by Project TRAILS at Los Angeles MissionCollege.

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