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Abstract

NUNCTM has developed a 96DeepWellTM DNA binding filter plateand method for the rapid purificationof high quality plasmid DNA. Bacterialcultures were incubated in 2 ml ster-ile DeepWell reception plates.Following standard alkaline lysis andneutralization, plasmid DNA was clari-fied using filter plates containing highretention, fast flow frits. Plasmid DNAwas then isolated using a 96DeepWell filter plate containing aplasmid DNA-binding filter matrix.Following optimization of the washbuffer components, up to 12.16±0.72µg/well of purified plasmid DNA wasrecovered. An A260/A280 ratio of 1.97confirmed the high purity of isolatedplasmid DNA. Well-to-well repro-ducibility was also demonstrated fol-lowing restriction digestion andagarose gel electrophoresis. The com-bination of NUNC 96 DeepWell DNAbinding plates along with optimized,standard reagents leads to a cost-effi-cient approach toward large scale,high purity plasmid DNA preparation.

Introduction

Plasmid purification using glass fiber isa popular, simple and efficient method,involving an initial alkaline lysis proce-dure (Birnboim and Doly, 1979;Sambrook et al, 1989; Engelstein et al,1998; Skowronski et al, 2000). Plasmidpurification kits, including 96 well-for-mat microplates and related solutions,have been developed by several suppli-ers. However, the price of these kitsmakes plasmid purification cost-inef-fective, particularly when processing alarge number of samples.

We have shown it is possible toimprove the plasmid purification pro-cedure and reduce the cost per prep.NUNC has developed a cost-efficientplasmid purification system, in which afritted plate is used for lysate clarifica-tion. A DNA purification procedure wasoptimized for both high quality andquantity of plasmid DNA. The amountof purified plasmid DNA obtained fromour glass fiber membrane is similar tothat from silica resin, but with a lowerrisk of mechanical shearing and at areduced cost.

Material and Methods

Bacterial culture and harvest:E. Coli strain HB101, containing pGEM-4plasmid with a histidine-rich calcium-binding protein cDNA insert, wasgrown overnight at 37°C with rotatingagitation in NUNC sterile 2 ml deepwellplate (NUNC #278743), and pelleted bycentrifugation (2000 x g, 5 min.).

Alkaline lysis: After removing thesupernatant, the bacterial pellet wasresuspended in 100 µl of GTE solution(50 mM glucose, 25 mM Tris-HCl, 10 mMEDTA, 100 µg/ml RNase A, pH 8.0), thentreated by 200 µl of lysis solution(0.2 N NaOH, 1% SDS), 200 µl ofneutralization buffer (3M potassium,5M acetate, pH 4.8) and 500 µl of 6Mguanidine hydrochloride, sequentially.

Clarification of plasmid DNA: Thesoluble plasmid DNA was clarified fromgenomic DNA and lysed cell debris bymeans of a fritted DeepWell filter plate(NUNC #278011). All buffers and plateswere maximized for yield and purity ofisolated DNA.

Cost-efficient plasmid DNA purification usingthe NUNCTM 96 DeepWellTM DNA binding filterplate

Volume 6 No. 39

Plasmid DNA binding: The plasmidDNA in clarified lysate was bound toglass fiber membranes in DeepWell fil-ter plates (NUNCTM #278010), optimizedfor yield and purity.

Wash and elute: After plasmid DNAbinding, any impurities were removedby an optimized wash buffer. Finally,the pure plasmid DNA was eluted fromthe filter with TE buffer.

Results

Following bacterial alkaline lysis, weoptimized conditions for lysate clarifi-cation, DNA binding, and DNA elution.For lysate clarification, several differentfrits and filter combinations wereassembled into DeepWell filtermicroplates and compared. The datareveal that fritted plates result in highlevels of purity and yield of plasmidDNA. Thus, the fritted plate was select-ed for plasmid DNA clarification. In theDNA binding plate, four different glassfiber membranes (various pore sizesand different sources) were examinedfor their DNA binding capability.A260/A280 data show that one glass fiber

STEPGrow E. Coli cells in sterile 2 ml DeepWell plate (Nunc #278743)

��Lyse bacteria and neutralize lysate, 15 min.

��

Clarify plasmid DNA with fritted filter plate (NUNC #278011), 1 min.

��

Bind plasmid DNA to NUNC glass fiber filter plate(NUNC #278010), 3 min.

��Wash and dry membrane, 8 min.

��

Elute pure plasmid DNA with TE into 1 ml reception plate

(NUNC #260252), 12 min.

Figure 1

membrane results in higher levels ofplasmid DNA in final eluent than otherglass fiber membranes, so this filtermembrane was employed in the NUNCDNA binding plate.

For the wash step, different wash solu-tions, containing varying concentrationof sodium and ethanol, various pHs(4.6-7.4) and other reagents were com-pared. The wash solutions containing80% ethanol yielded the highest levelsof plasmid DNA. Thus, 80% ethanolwith 10 mM Tris-HCl and 1 mM EDTA(pH 7.4) was selected. This buffered for-mulation with low concentration ofEDTA protects DNA in process and pro-vides favorable conditions for thedownstream applications.

Our optimized microplates and proto-col reproducibly yielded plasmid DNAof high quality and quantity. The aver-age ratio of A260/A280 OD units in 96wells is 1.97 ± 0.02 (means ± SD) (Fig.2). The average amount of purifiedplasmid DNA from 96 wells can be ashigh as 12.16 ± 0.72 µg/well from 5 mlof bacteria culture (Fig. 3). This meansthat the total capacity of the binding

filter exceeds what could realisticallybe expected from a standard“miniprep” procedure. Further demon-stration of reproducibility from well towell is obtained following agarose gelelectrophoresis, which shows equalintensity bands of plasmid DNA.

The purified plasmid DNA is suitablefor common downstream applicationssuch as restriction digestion andsequencing. Figure 4 shows equal sin-gle bands in agarose gel, after purifiedplasmid DNAs from different wells werecut by the restriction nuclease Hind IIIat one site. The purified plasmid DNA isalso suitable for sequencing (Fig. 5),(Hofmann et al, 1991).

Figure 3 Reproducibility of purified plasmid DNA from 96 wells. The average amount of plas-mid DNA from 96 wells is as high as 12.16 ± 0.72 µg/well (means ± SD) from (pooled) 5 ml ofbacteria culture using the NUNC plasmid purification procedure.

Figure 4 Restriction digestion of purified plasmid DNA. The puri-fied plasmid DNA is suitable for down-stream applications. The plas-mid DNA was digested by restriction nuclease Hind III prior toagarose gel electrophoresis.

Reproducibility of DNA Purity

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Figure 2 High quality plasmid DNA is confirmed by A260/A280 ratio determination.The average A260/A280 ratio is 1.97 ± 0.02 (means ± SD) across 96 wells.

Figure 5 Typical sequencing data. The purified plasmid DNA isalso suitable for sequencing.

3.8kb

Conclusion

High purity and yield of plasmid DNAwith low cost is critical for successfuldownstream applications. NUNCTM

Brand plasmid purificationmicroplates with the accompanyingdetailed optimized protocol provideexcellent tools for plasmid purifica-tion. Furthermore, NUNC plates arenot bundled with expensive reagents,thus providing more flexibility for theresearcher to formulate their own.These features are particularly benefi-cial for frequent and high volumeplasmid purification laboratories andcenters.

Credits

Wenxiao Lu1, Dan Dwyer1,Dan Schroen2 and Tom Cummins1 –Dept. of Research & Development1

and Dept. of Marketing2

ReferencesBirnboim, HC and J Doly, A rapid alkaline extraction procedure for screening recombinant plasmid DNA, NucleicAcid Res. 7:1513-23, 1979

Engelstein, M, TJ Aldredge, D Madan, JH Smith, JI Mao, DR Smith and PW Rice, An efficient, automatable tem-plate preparation for high throughout sequencing, Microb. Comp. Genomics 3:237-241, 1998

Hofmann SL, M Topham, CL Hsieh and U Francke, cDNA and genomic cloning of HRC, a human sacroplasmicreticulum protein, and localization of the gene to human chromosome 19 and mouse chromosome 7, Genomics9:656-669, 1991

Sambrook, J., EF Fritsch and T Maniatis, Molecular cloning: a laboratory manual, 2nd ed. Cold Spring HarborLaboratory Press, NY, page 1.25, 1989

Skowronski EW, N Armstrong, G Andersen, M Macht and PM McCready, Magnetic, microplate-format plasmidisolation protocol for high-yield, sequencing-grade DNA, Biotechniques 29:786-792, 2000

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