Lysis methods

The saponin lysis method can be performed on either EDT A- or citrate-anticoagulated blood. Saponin solution to lyse erythrocytes is available in most laboratories for use with automated hematology instruments. 

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. 

Kulski JK, Pryce T. Preparation of mycobacterial DNA from blood culture fluids by simple alkali wash and heat lysis method for PCR detection. J. Clin. Microbiol. 1996 34 1985-1991. 

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. 

The Triton X-100 lysis method [9] is relatively simple and is a cost-effective method. To 10 mL of blood, 90 mL of cold (4°C) RBC lysis buffer (0.32 M sucrose, 10 mM Tris-Cl, pH 7.5, 5 mM MgCL, 1% Triton X-100) is added and mixed by inverting the tube a few times. This step releases cellular contents, and the crude nuclear fraction containing the DNA is then centrifuged at 3500 x g at 4°C for 30 minutes. The supernatant is decanted and the pellet is resuspended in a small volume of cold RBC buffer by vor-texing and brought upto 40 mL with the same buffer and recentrifuged as... 

Table 8.2. Comparison of Various Cell Lysis Methods...

Lysis Method Instrument Requirements Mode of Lysis Mechanism... 

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). 

Fig. 4.2. Isolation of environmental DNA from soils and sediments by the direct lysis approach. The DNA isolation of soil and sediment samples is based on the direct lysis method of Zhou et al. [16]. 50 g of each environmental sample are mixed with 135 ml of DNA extraction buffer (100 mM Tris/HCI, pH 8.0,100 mM sodium EDTA, 100 mM sodium phosphate, 1.5 M NaCI, 1 % (w/v) CTAB) and 1 ml of proteinase K (10 mg/ml) in GS3 tubes by horizontal shaking for 30 min at 37°C. Subsequently 15 ml of 20 % (w/v), SDS is added and the samples are incubated in a 65 °C water bath for 2 h with gentle end-over-end inversion every 15 to 20 min. After centrifugation at 6000 x g for 10 min at room temperature the resulting supernatants are transferred into new GS3 tubes. The remaining pellets are extracted two more times by suspending them in 45 ml of extraction...

In addition to the foregoing more general concerns are questions concerning the localization of an enzyme activity. The location of an enzyme can determine the type of cell lysis, since it could be more advantageous to lyse the cell completely or in such a manner that the organelles are left intact. For example, some lysis methods such as sonication completely disrupt mitochondria, nuclei, and Golgi systems. If an activity is localized in an organelle such as a mitochondrion, it would seem sensible to adopt a method that leaves these structures intact, to facilitate their separation from the rest of the cellular debris. Thus, for the isolation of mitochondrial enzymes, sonication is not the method of choice for cell lysis. 

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... 

Using an in situ lysis method, Gutierrez et al. [20] electrophoretically surveyed sixty-five halobacterial strains for the presence of large plasmids. Three quarters of the strains had plasmids visible by this method, the majority containing three or four [20]. Due to the limitations of the electrophoretic technique used, this analysis no doubt missed some of the largest plasmids (such as the largest Haloferax volcanii plasmid [15]). 

In the assay described here cells are permeablized using a detergent-based lysis method. Care must be taken to inhibit the membrane-bound enzyme, y glutamyl-transferase (y-GT), which metabolizes glutathione released from the cell (12). This is achieved by the use of an acidified lysis buffer (8). The lysate is then assayed for total protein and glutathione content. 

Ana lysis Method Formaldehyde Determi nat ion In AIr, Photometr i c Method, and lodometric Method," Federation of European Particleboard Manufacturers, Giessen, Germany, 1975. 

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

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. 

Several lysis methods can be used, depending on the facilities available. Sonica-... 

A variety of lysis methods, including chemical lysis [18, 19], thermal lysis [20], and lysis by mechanical forces [21, 22], or electrical pulse [23-26], have been successfully demonstrated in microfluidic devices. 

To release proteins encased in cells for analysis, the cells, including the cellular and nuclear membranes, must be destroyed through lysis. Cell lysis can be carried out in an appropriate solubilization solution [23]. Lysis methods includes two categories, gentle lysis or vigorous lysis. 

Gentle lysis methods are generally employed when the sample of interest consists of easily lysed cells (such as tissue culture cells, blood cells and some microorganisms). Gentle lysis methods can also be employed when only one... 

Vigorous lysis methods are employed when cells are less easily disrupted, for example, cells in solid tissues. These methods include sonication [31-33], French pressure [29, 30, 34], grinding [32, 35-37], mechanical homogenization [24, 25, 38], and glass bead homogenization [29, 30]. Vigorous lysis methods usually result in complete disruption of the cell membranes and some organelles. 

In terms of cell lysis on-chip, Li and Harrison [39] were the first to report cell lysis through combination of a chemical method using SDS and electrical techniques. Since then, other on-chip lysis methods have appeared, including mechanical [40], thermal [41], ultrasonic [42], and electrophoretic [43, 44]. 

All described strategies are based on the alkaline-lysis method. 

No plasmids were detected in the four non-pathogenic strains examined. The fact that the lysis method employed here is capable of detecting plasmids at least as large as 190 Mdal (Figure 2) indicates that no plasmids helow this size are found in the non-pathogens. 

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