Alkaline lysis

Klintschar M, Neuhuber F. Evaluation of an alkaline lysis method for the extraction of DNA from whole blood and forensic stains for STR analysis. J. Forensic Sci. 2000 45 669-673. 

Ion-exchange HPLC can also be useful in the separation of larger nucleic acid molecules. One such application is as an alternative to CsCl density gradient centrifugation in the preparation of plasmids. Plasmid molecules typically consist of between 1000 and 10 000 base pairs. The plasmid is first isolated from the bacterial cell by alkaline lysis and pure plasmid obtained from this crude extract by a one-step chromatographic separation. 

Extrachromosomal DNA molecules called plasmids are harbored in some strains of E. coli. The normal copy number of the plasmids is small, between 2 and 10 however, if these strains of E. coli are grown in the presence of chloramphenicol, up to 3000 copies may be replicated per cell. Plasmid DNA has been demonstrated to be a useful vehicle in molecular cloning. This experiment describes a method for the growth of E. coli and amplification of the ColEl plasmids. The plasmids will be isolated from E. coli cells by one of two methods, a large-scale boiling method or a microscale alkaline lysis method. The DNA plasmids will be measured for molecular size by agarose electrophoresis. 

Microscale Isolation of Plasmid DNA by Alkaline Lysis (Bimboim, 1983)... 

The cells were harvested and the plasmid was extracted according to the standard alkaline lysis with SDS method (16). 

Eppendorf-5 Prime Inc. (Boulder, Colorado) (with Zymark Corp, PerfectPrep-96 VAC Alkaline lysis and filtration method... 

Parodi, S., R.A. Mulivor, J.T. Martin, C. Nicolini, D.S.R. Sarma, and E. Farber. Alkaline lysis of mammalian cells for sedimentation analysis of nuclear DNA. Conformation of released DNA as monitored by physical, electron microscopic and enzymo-logical techniques. Biochim. Biophys. Acta 407 174-190, 1975. 

Clone a DNA fragment encoding a foreign protein into appropriate sites of pFastBacl vector in correct orientation with respect to the polyhedrin promotor. The fragment must contain its own initiator codon followed by an open reading frame and terminator codon. Prepare recombinant donor plasmid DNA from E. coli using the standard procedure (e. g., alkaline lysis method). 

M in ammonium acetate greatly improves template quality. Such a step should be examined as an amendment to any protocol that yields suboptimal template. The following protocol is a modified alkaline lysis protocol2 that in our experience routinely yields plasmid of reasonable template quality. All centrifugation steps are performed in an Eppendorf microcentrifuge (e.g., Model 5414) unless otherwise specified. The reader is referred to Sambrook et al.2 for media formulations. 

A protocol for in vitro transcription used in our laboratory is given below. Rapid plasmid minipreparations made by alkaline lysis without RNase treatment are usually sufficient to prepare DNA templates. DNA samples linearized by restriction digestion are extracted with chloroform, and precipitated with ethanol, and 0.2 to 0.5 /ig of DNA dissolved in 1 pil of distilled water is used per assay. Contamination with RNases must be avoided during the following steps. The reaction mixture for eight reactions is prepared at room temperature to avoid precipitates. 

After treatment they are washed with cold saline, and the cells are resuspended in cold distilled water at (2-4) x 105 cells in 0.1 ml and pipetted onto the surface of 1 ml alkaline lysis solution (5% sucrose containing 0.3 M NaOH, 0.5 M NaCl) in a glass vessel (2 cm i.d.) cooled on ice. 

Many procedures have been developed for DNA isolation, differing according to the scale of the operation, the nature of the biological source material, and the nature of the DNA, particularly whether plasmid or genomic (Ausubel et al. 1989 Maniatis et al. 1989 Harwood 1996). As a prototype procedure, we first describe the small scale isolation of plasmid DNA from E. coli by the alkaline lysis method. This miniprep procedure starts with E. coli cells from a few ml of culture which have... 

JETSTAR Plasmid Kit (GENOMED) plasmid isolation kit, based alkaline lysis of the bacterial cell. Chromosomal DNA sticks to the cell wall and coprecipitates with the cell debris, whereas plasmid DNA remains solubilized. An anion exchange resin, provided in columns, binds the plasmid DNA under high salt conditions, the DNA is washed and eluted in low salt conditions. The solutions are provided in the kit El (cell resuspension buffer 50 mM Tris-HCl, pH 8.0, 10 mMEDTA), E2 (cell lysis solution 200 mM NaOH, 1% SDS), E3 (neutralization buffer 3.2 M KOAc/HOAc, pH 5.5), E4 (column equilibration... 

Alkaline lysis solution II 0.2% NaOH, 1% SDS (make fresh). 

Add 600 pL of a 1 2 mixture of alkaline lysis plasmid prep solutions I and II (8.8 mL H20 1 mL 10% SDS 5 mL solution I 0.2 mL 10 /VNaOH). Tilt plate back and forth to distribute liquid over entire plate. Cells should lyse. Incubate on ice for 5 min. 

The protein-free preparation of the plasmid was purified following alkaline lysis using maxiprep columns (Qiagen, Hilden, Germany). 

Protocol 8 PI and BAC Miniprep Protocol with the Modified Alkaline Lysis Method... 

Prepare the PI and BAC DNAs by alkaline lysis using the Qiagen Plasmid Maxi kit (Qiagen). Resuspend cells in 16 ml of PI solution. Add 2 ml of 50 mg/ml lysozyme freshly made in PI solution. Mix and leave at room temperature for 15 min. 

ScFv into the culture media. Escherichia coli strain XLl/Blue was transformed with pEScFv 2G42D7 plasmid, and plasmid DNA was obtained by alkaline lysis (57) from the ampicillin-resistant line. 

G42D7. Plasmid DNA is obtained by standard alkaline lysis from an ampicil-lin-resistant line. Purified plasmid is checked by agarose electrophoresis. 

Plasmid is routinely recovered from bacteria by an alkaline lysis procedure, which lyses the bacterial cell while maintaining bacterial DNA attachment to the cell wall. This procedure enables subsequent precipitation of bacterial DNA and cellular debris, leaving a crude preparation enriched in plasmid. We routinely use a plasmid DNA purification kit provided by Qiagen that utilizes the alkaline lysis method for harvesting, and anion exchange column chromatography for rapid purification. We refer the reader to the detailed instructions provided in the kit by the manufacturer, which we have not found necessary to modify for purification of laboratory-use plasmid DNA. 

Alkaline lysis Crude, requires further purification (80,81)... 

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