1011204_QNews299_JDist.qxd 25.02.1999 15:23 Uhr Seite 23 EXTRAS QIAGEN Lysis of bacterial cells for 001 Practical plasmid purification Hints Effective lysis of bacterial cells is a key step in plasmid isolation as DNA yield and quality depend on the quality of cell lysate used for the purification. This article describes the principle behind bacterial lysis, in particular alkaline lysis, and gives hints on how to ensure that lysis proceeds optimally. Alkaline lysis Alkaline lysis is one of the most commonly used methods for 1. Resuspension lysing bacterial cells prior to plasmid purification (1, 2), and E. coli cell optimized alkaline lysis is part of all QIAGEN® plasmid purification protocols. Production of alkaline lysates involves four basic steps (Figure 1): 2. Lysis NaOH/SDS 1. Resuspension Harvested bacterial cells are resuspended in Tris·Cl–EDTA buffer containing RNase A. 3. Neutralization KAc Ensure that bacteria are resuspended completely Tip leaving no cell clumps in order to maximize the number of cells exposed to the lysis reagents. 4. Clearing of lysates Buffer volumes for alkaline lysis in QIAGEN Tip plasmid purification procedures are optimized for Centrifugation Filtration particular culture volumes in LB medium. Do not use a culture volume larger than recommended in the Supernatant KDS precipitate protocol as this will lead to inefficient lysis and containing containing plasmid DNA genomic DNA reduce the quality of the plasmid preparation. + proteins For large scale purification of low-copy plasmids, Filtrate for which larger cultures volumes are used, it may containing Tip Plasmid DNA plasmid DNA be beneficial to increase the lysis buffer volumes in Chromosomal DNA order to increase the efficiency of alkaline lysis and thereby the DNA yield (Figure 2). Figure 1 Principle of alkaline lysis 23 1011204_QNews299_JDist.qxd 25.02.1999 15:23 Uhr Seite 24 QIAGEN EXTRAS 002 2. Lysis High volumes of lysis buffers Cells are lysed with NaOH/SDS. Sodium dodecyl sulfate (SDS) solubilizes the phospholipid and protein components of the cell membrane, leading to lysis and Low volumes of lysis buffers release of the cell contents. NaOH denatures the chromosomal and plasmid DNAs, as well as proteins. The presence of RNase A ensures that liberated cellular Amount of plasmid DNA in cleared lysate RNA is digested during lysis. Culture volume Figure 2 The effect of lysis buffer volumes on the amount of low-copy plasmid DNA in the cleared lysate for large-scale plasmid preparations. Avoid vigorous stirring or vortexing of the lysate as this can shear the bacterial Tip chromosome, leaving free chromosomal fragments in the supernatant which will copurify with the plasmid DNA. The solution should be mixed gently but thoroughly by inverting the lysis vessel 4–6 times. Do not allow the lysis to proceed for longer than 5 minutes. A 5-minute incubation Tip allows maximum release of plasmid DNA from the cell, while minimizing the release of chromosomal DNA and reducing the exposure of the plasmid to denaturing conditions. 3. Neutralization The lysate is neutralized by the addition of acidic potassium acetate. The high salt concentration causes potassium dodecyl sulfate (KDS) to precipitate, and denatured proteins, chromosomal DNA, and cellular debris are coprecipitated in insoluble salt-detergent complexes. Plasmid DNA, being circular and covalently closed, renatures correctly and remains in solution. Precipitation is enhanced by using chilled neutralization buffer and incubating on ice. Tip (For many plasmid miniprep procedures, use of chilled buffer and incubation on ice are not necessary.) Mix the solution gently but thoroughly to ensure complete precipitation. Tip 24 1011204_QNews299_JDist.qxd 25.02.1999 15:23 Uhr Seite 25 EXTRAS QIAGEN 003 4. Clearing of lysates References 1. Birnboim, H.C., and Precipitated debris is removed by either high-speed centrifugation or filtration, producing Doly, J. (1979) A rapid alkaline lysis procedure cleared lysates. for screening recombinant plasmid DNA. Nucl. Acids. Res. 7, 1513–1522. Lysate filtration technologies developed by QIAGEN ensure removal of all precipitated 2. Birnboim, H.C. (1983) A material without centrifugation, reducing plasmid purification time considerably, rapid alkaline extraction Tip method for the isolation of particularly when large culture volumes or large numbers of samples are involved. plasmid DNA. Methods Enzymol. 100, 243–255. Specialized modules are available for different scales of plasmid preparation, as well 3. Sambrook, J., Fritsch, E.F., as for high-throughput plasmid minipreps. and Maniatis, T. (1989) Molecular cloning: A Lab- oratory manual. Cold Spring Harbor: Cold Other lysis methods Spring Harbor Laboratory. 4. Ausubel, F.M., et al. A number of other methods have been described for lysing bacterial cells (3, 4). Some of these (1987) Current Protocols in Molecular Biology. methods were developed for different applications and may not be suitable for plasmid New York: John Wiley and Sons. DNA isolation. Boiling lysis: Bacterial cells are treated with lysozyme to weaken the cell walls and then lysed by heating in a boiling water bath for ~1 minute. Lysis with detergent: Bacterial cells are lysed by treatment with an ionic detergent (e.g., SDS) or a nonionic detergent (e.g., Triton® X-100). Mechanical lysis: Bacterial cells are lysed by mechanical disruption, for example by sonification. Enzymatic digestion: Some lysis methods include treatment of bacteria with enzymes such as lysozyme which assist in weakening cell walls. Lysis of bacteria other than E. coli Isolation of plasmid DNA from bacteria other than E. coli usually requires modifications to the lysis procedure in order to optimize lysis conditions for the particular species. Detailed protocols for plasmid DNA isolation from a variety of bacteria using QIAGEN plasmid purification procedures are available on request from QIAGEN Technical Services or your local distributor. QIAGEN Practical Hints for daily laboratory practice will continue in future issues of QIAGEN News. If there is any other information you would like to see on these pages of QIAGEN News, please let us know by calling QIAGEN Technical services or your local distributor. Signature: Date: 25 Witness: Date:.