Large-scale purification of plasmid DNA by fast protein liquid chromatography using a Hi-Load Q Sepharose column.

The large-scale purification of plasmid DNA was achieved using fast protein liquid chromatography on a Hi-Load Q Sepharose column. This method allows for the purification of plasmids starting from crude plasmid DNA, prepared by a simple alkaline lysis procedure, to pure DNA in less than 5 h. In contrast to the previously described plasmid purification methods of CsCl gradient centrifugation or high-pressure liquid chromatography, this method does not require the use of any hazardous or expensive chemicals. More than 100 plasmids varying in size from 3 to 15 kb have been purified using this procedure. A Mono Q Sepharose column was initially used to purify plasmids smaller than 8.0 kb; however, a Hi-Load Q Sepharose column proved more effective with plasmids larger than 8 kb. The loading of plasmids larger than 8 kb on the Mono Q column resulted in a high back pressure and the plasmid DNA could not be eluted from the column. Thus, for routine purification we utilize the Hi-Load Q Sepharose column. Plasmids purified by this method had purity, yield, and transfection efficiency in mammalian cells similar to those of plasmids purified by CsCl density gradient centrifugation.     

Rapid purification of plasmid DNA by a single centrifugation in a two-step cesium chloride-ethidium bromide gradient

A procedure for rapid, preparative purification of plasmid DNA is described and compared with a conventional equilibrium centrifugation method. A discontinuous, two-step CsCl-ethidium bromide gradient is used, with the starting position of the plasmid-containing extract being at the bottom of the tube. During centrifugation in a fixed angle rotor, covalently closed circular plasmid DNA is separated from contaminating protein, RNA, and chromosomal DNA in 5 hr. Plasmids purified by this method are considerably less contaminated with RNA than when purified by a 48-hr equilibrium run in a homogeneous gradient, as determined by agarose gel electrophoresis and 5'-end-labeling studies. Plasmid DNA purified in two-step gradients can be used directly for restriction endonuclease analysis and DNA sequencing.     

A procedure for the large-scale isolation of highly purified plasmid DNA using alkaline extraction and binding to glass powder

A preparative procedure for obtaining highly purified plasmid DNA from bacterial cells is described. The method is adapted from our earlier procedure, which gave partially purified plasmid in a form suitable for rapid screening of a large number of samples. In the present method, all detectable RNA, chromosomal DNA, and protein are removed without the use of enzymes, phenol extraction, dialysis, or equilibrium centrifugation. Binding of plasmid DNA to glass powder in the presence of 6 m sodium perchlorate is used for the final purification step.     

Purification and separation of various plasmid forms by exclusion chromatography

A chromatographic method for the rapid isolation of preparative amounts of plasmid DNA without the use of cesium chloride centrifugation is described. The protocol uses the alkaline extraction procedure and an exclusion column of Fractogel TSK 75S. From a clear lysate it is possible to obtain plasmid DNA completely free of proteins, RNA, and chromosomal DNA. From partially purified plasmid the procedure allows the separation of the different forms. This technique was successfully applied to different plasmids ranging in size from 2.9 to 17.5 MDa. It is a preparative method yielding easily 500 micrograms of pBR322 from 1 liter of amplified culture. The plasmid is suitable for topoisomerase I, topoisomerase II, and EcoRI assays.     

Macromolecular Content of Inclusions Produced by a Canine Adenovirus

Early inclusions induced by a canine adenovirus in a canine cell line, appearing before the formation of infectious virus particles, were purified by differential centrifugation in sucrose followed by CsCl density gradient centrifugation. Chemical analysis of these inclusions revealed that they contained deoxyribonucleic acid (DNA), ribonucleic acid, and protein. On the basis of density gradient centrifugation, the DNA extracted from the inclusions was found to be viral DNA. Electron microscope autoradiography showed that these inclusions were the sites of DNA synthesis. In addition, association of DNA polymerase activity with the inclusions was detected by incorporation of radioactivity from (3)H-thymidine triphosphate into a DNA product. The in vitro product of the enzyme had a density equal to that of viral DNA rather than host DNA. The level of DNA polymerase activity in exponentially growing infected and uninfected whole cells was similar, but in cells in stationary phase the enzyme activity of infected cells was twice that in noninfected cells. Furthermore, nuclei isolated from infected cells showed a fourfold increase in DNA polymerase activity over the noninfected cells.     

A simple method for the preparation of the covalently closed circular form of plasmid DNA

We describe a simple, rapid, inexpensive method for isolation of covalently closed circular plasmid DNA. The method involves the electrophoresis of crude DNA preparations in an agarose gel, electrotransfer onto a dialysis membrane and elution of the highly purified circular covalently closed plasmid DNA. Native and recombinant plasmid DNA have been purified by this method and shown to be suitable for restriction enzyme digestion and transformation of bacteria. The yield of this rapid purification procedure makes it a good alternative method to standard centrifugation in cesium chloride ethidium bromide gradients.     

A continuous method for the large-scale extraction of plasmid DNA by modified boiling lysis

This protocol describes a streamlined method of plasmid DNA extraction by continual thermal lysis, a modification of the basic boiling lysis technique, to simplify the processing of large volumes of Escherichia coli cultures. Fermented bacteria are harvested using a hollow fiber-membrane module and pre-treated with lysozyme prior to passing through a thermal exchange coil set at 70 degrees C to lyse the cells, and into a juxtaposed cooling coil on ice. The lysed and cooled bacteria are subsequently separated from the lysate by centrifugation and plasmid DNA is precipitated from the supernatant for further purification. The use of peristaltic pumps and two heating coils at constant temperature without the use of centrifugation enable the lysis process to become constant and controllable, providing a flow-through protocol for cell lysis and plasmid DNA extraction. Large volumes of bacterial cultures (20 l) can be processed in 2 h, yielding approximately 100 mg plasmid DNA l(-1) culture, making this an attractive protocol for consistent and large-scale preparation of plasmid DNA.     

A novel method for rapid isolation of plasmid DNA

A new disposable chromatographic column, pZ523, has been developed for separating plasmid DNA from bacterial chromosomal DNA. Use of pZ523 spun columns eliminates the need for ethidium bromide-cesium chloride density gradients which require long centrifugation times. pZ523 purified plasmids have been shown to be of purity suitable for restriction analysis, ligation, transfection of mammalian cells and transformation of bacteria. Unlike the traditional ultracentrifugation method, pZ523 offers an extremely rapid alternative method for purifying large amounts of plasmid DNA (2.5 mg to 4.5 mg) from cleared bacterial lysates in only 25 minutes.     

Physiological Studies of Bacillus subtilis Minicells

Minicells produced by Bacillus subtilis strains carrying the div IV-B1 mutation, (CU 403 div IV-B1 and CU 403 div IV-B1, tag-1), were purified by a procedure which destroys parental cells with ultrasound, but spares minicells. Such preparations generally contain 10(9) or more minicells/ml and less than 10(4) colony-forming units/ml. Purified minicells are resistant to autolysis in tris(hydroxymethyl)aminomethane buffer, pH 7.5, at 30 C, conditions which result in total lysis of parental cells. Minicells are not completely devoid of autolytic activity, however. The medium in which minicells are produced, the temperature at which purified minicells are incubated, and the genotype of cells from which the minicells are derived all influence the rate of autolysis of purified minicells. These parameters are demonstrated by using minicells obtained from div IV-B1 and div IV-B1, tag-1 strains. Ultrastructural differences have been observed in the products of autolysis of these two minicell strains. Minicells are sensitive to low levels of lysozyme and yield miniprotoplasts when the wall is removed in an osmotically protective environment. Although minicells are unable to grow, they can maintain their integrity over long periods of time, which suggests functional energy metabolism in minicells. Direct measurements of adenosine 5'-triphosphate (ATP) levels by the luciferase assay indicated that minicells can produce ATP. Oxygen consumption, measured by standard respirometry techniques, also indicates functional metabolism in minicells. These findings demonstrate that minicells purified by ultrasound are suitable material for study of physiological processes in anucleate cells.     

Large-scale isolation of plasmid DNA using cetyltrimethylammonium bromide

A rapid procedure for the large-scale isolation of plasmid DNA is described. The method utilizes cetyltrimethylammonium bromide to precipitate the plasmid following extraction of DNA by lysozyme digestion and boiling. The plasmid is then purified by passing through the spin column pZ523. The purity and yield of the plasmid obtained with this method is similar to that isolated by cesium chloride-ethidium bromide gradient centrifugation. The method does not involve any phenol-chloroform extractions and takes five to six hours for completion after growth of the bacterial cells. The plasmid obtained is amenable to digestion with various restriction endonucleases, can be used for cloning with high efficiency and is also suitable as template for dideoxy sequencing.     

Simple agarose gel electrophoretic method for the identification and characterization of plasmid deoxyribonucleic acid.

Agarose gel electrophoresis may be employed effectively for the detection and preliminary characterization of plasmid deoxyribonucleic acid (DNA) present in clinical isolates and laboratory strains of gram-negative microorganisms. The method is sensitive and does not require radioisotopes or ultracentrifugation. The estimation of plasmid mass from the extent of DNA migration in gels compares favorably with results obtained by electron microscopy of plasmid DNA purified by equilibrium density centrifugation. The method has proved to be a useful tool for survey work and the epidemiological investigation of plasmid dissemination, as well as an important adjunct to the genetic analysis of plasmids.     

A plasmid in the archaebacterium Methanobacterium thermoautotrophicum

The archaebacterium Methanobacterium thermoautotrophicum Marburg (DSM 2133) was found to contain a plasmid (pME2001) in covalently closed circular form. It was isolated by CsCl gradient centrifugation of total DNA in the presence of ethidium bromide. Multimers up to the hexamer were observed upon agarose gel electrophoresis and electron microscopy of a purified plasmid preparation. A restriction map was constructed. The length of plasmid pME2001 was determined to be approximately 4,500 bp. Southern hybridization of plasmid DNA to DNA extracted from Methanobacterium thermoautotrophicum delta H (DSM1053) revealed the presence of a plasmid with homologous sequences in the delta H strain.     

Improvement of transfection efficiency in cultured chicken primordial germ cells by percoll density gradient centrifugation

Chicken primordial germ cells (PGCs) differentiate into germ cells in gonads. Because PGCs can be cloned and cultured maintaining germline competency, they are a good means of modifying the chicken genome, but the efficiency of plasmid transfection into PGCs is very low. In this study, I attempted to improve the efficiency of PGC transfection. Cultured PGCs were purified by Percoll density gradient centrifugation, and were then transfected with plasmid DNA. For transient transfection, the transfection efficiency increased more than 7-fold by the Percoll method. The efficiency of stable transfection of PGCs also increased significantly. The stable transfectants that were isolated by this method accumulated in the developing gonads after microinjection into bloodstream of chick embryos, indicating that gene transfection by Percoll purification did not alter the function of PGCs in vivo.     

Study of the influence of plasmids on the arbitrary primer polymerase chain reaction fingerprint of Escherichia coli strains

Abstract To study the effect of plasmids on the arbitrary primer-polymerase chain reaction fingerprint of bacterial strains, the Escherichia coli strains DH5, Top10, and W3110 were transformed with plasmids of different sizes: respectively, pUC19, pCEP and two clinically important plasmids carrying resistance to several antibiotics. Total DNA, i.e. both chromosomal and plasmid DNA, was prepared from transformed cells by boiling the cell suspensions and by phenol-chloroform extraction; chromosomal DNA was prepared by the same methods from the non-transformed, plasmid-free strains; plasmid DNA of pUC19 was purchased; plasmid DNA of pCEP was purified from the transformed strains by caesium chloride density gradient centrifugation. Arbitrarily primed polymerase chain reaction was carried out for all of these preparations. Amplification carried out independently with three different primers resulted in similar patterns for the chromosomal preparations whether or not plasmid was present. Amplification of plasmid DNA gave different patterns, characterized by fragments larger than those obtained when total or chromosomal DNA were used as the target. These data illustrate that the plasmids studied here do not influence the chromosomal arbitrarily primed PCR fingerprint, although plasmids alone are amplified in the absence of chromosomal DNA. Experiments comparing different relative concentrations of plasmid and chromosomal DNA indicate that under natural conditions the amount of chromosomal DNA per cell is sufficient to inhibit observable amplification of the plasmid(s) present.     

Repair of UV damage in plasmid DNA by human fibroblasts

Summary Plasmid DNA from Bacillus subtilis was introduced into monolayers of human fibroblasts by means of a modification of the calcium phosphate coprecipitation technique, comprising centrifugation of the coprecipitate onto the cells and treatment with polyethyleneglycol. The amount of DNA resistant to removal from the monolayers ranged from 10% to 15% of the input DNA. By determination of the biological activity of the plasmid DNA, re-extracted after various periods following entry into the fibroblasts and subsequently used as donor for B. subtilis protoplasts, it was shown that the activity of the plasmid DNA was gradually lost. When ultraviolet light-inactivated plasmid DNA was used as donor, reactivation of the plasmid was observed, which was completed within 2 h. The dose-dependent incorporation of [¹⁴C]-thymidine suggests that DNA repair processes were involved in reactivation of the plasmid DNA.     

Improvement of Transfection Efficiency in Cultured Chicken Primordial Germ Cells by Percoll Density Gradient Centrifugation

Chicken primordial germ cells (PGCs) differentiate into germ cells in gonads. Because PGCs can be cloned and cultured maintaining germline competency, they are a good means of modifing the chicken genome, but the efficiency of plasmid transfection into PGCs is very low. In this study, I attempted to improve the efficiency of PGC transfection. Cultured PGCs were purified by Percoll density gradient centrifugation, and were then transfected with plasmid DNA. For transient transfection, the transfection efficiency increased more than 7-fold by the Percoll method. The efficiency of stable transfection of PGCs also increased significantly. The stable transfectants that were isolated by this method accumulated in the developing gonads after microinjection into bloodstream of chick embryos, indicating that gene transfection by Percoll purification did not alter the function of PGCs in vivo.     

A simplified method for the production of recombinant baculovirus

A simplified method for producing recombinant baculovirus for expression of foreign genes is described. The method utilizes insect cells infected with the wild-type virus before transfection with the plasmid transfer vector, instead of the standard procedure utilizing cotransfection with a plasmid and viral DNA. Recombinant virus is preselected by a limiting dilution dot-blot hybridization procedure, rather than by morphologic criteria alone. In addition, we have found that plasmid purification by anion-exchange chromatography is as efficacious for transfection as plasmid purified by cesium chloride density gradient centrifugation. These modifications allows for an efficient, rapid, inexpensive and more objective protocol for the selection of recombinant baculovirus compared to the conventional protocol.