Factors affecting the isolation of CCC DNA from Streptomyces lividans and Escherichia coli

Based on the results of a systematic study of factors affecting plasmid yield and purity, a procedure suitable for the rapid screening for and isolation of covalently closed circular DNA from Streptomyces lividans and Escherichia coli was developed. The method consists of lysis of lysozyme-treated bacteria combined with alkaline denaturation of DNA at high temperature. Renaturation of CCC DNA and precipitation of single-stranded DNA together with protein is achieved by the addition of a minimal amount of phenol/chloroform. The screening procedure uses only a single tube and the samples can be analyzed by agarose gel electrophoresis about 30 min after lysis. Removal of phenol and further purification of the plasmid preparation is achieved by consecutive precipitations with isopropanol and spermine, followed by extraction with ethanol, producing samples suitable for restriction endonuclease digestion, ligation, and transformation of S. lividans protoplasts or competent E. coli cells in about 2 h. All steps of the procedure are explained in detail with information about the effects of changing parameters. This should help the experimenter to obtain reproducible results and may be useful if the method has to be adapted to new strains or plasmids.     

A procedure for the isolation and purification of plasmid DNA from Rhizobium meliloti

A procedure is described for the isolation and purification of the DNA of plasmids that are indigenous to the agriculturally important nitrogen-fixing bacterium Rhizobium meliloti. The procedure involves the lysis of bacteria with an ionic detergent or a mixture of ionic and nonionic detergents, the extraction of total DNA from precipitated membrane-DNA complexes, the enrichment of supercoiled plasmid DNA by the selective alkaline denaturation of chromosomal DNA, and a further purification of plasmid DNA using cesium chloridepropidium diiodide gradients. This procedure yields pure plasmid DNA in amounts of 30 to 50 μg per liter of a culture of cell density of approximately one A550 unit. The DNA thus obtained has been found to be of sufficient purity to serve as substrate for the most commonly used restriction endonucleases.     

Simple and rapid method for isolating large plasmid DNA from lactic streptococci.

A procedure for the rapid isolation of plasmid DNA larger than 30 megadaltons from lactic streptococci is described. This protocol can be used on a preparative scale to isolate sufficient quantities of plasmid DNA required for restriction analysis, cloning, or transformation experiments. A scaled-down protocol is very useful for rapidly screening the plasmid content of streptococcal strains. With this methodology, previously undetected large plasmids were observed.     

In vivo recombination and the production of hybrid genes

In vivo recombination between homologous genes is increasingly being favoured as a means of generating proteins with altered and novel specificities. The typical procedure requires the cloning of two related genes on a single replicative plasmid of Escherichia coli and the selection or screening of recombinants. Up to now the recombination process between the cloned genes was generally thought to involve the recA function and the availability of free ends in the DNA molecule to be recombined. Our results show that neither is necessary. Recombinants are obtained by simply growing the bacteria that host the plasmid carrying the two cloned genes.     

Rapid isolation of high quality, multimeric plasmid DNA using zwitterionic detergent

Purification of plasmid DNA from bacteria is an essential tool in recombinant DNA technology and has become an essential task in laboratories to industries. Moreover, the recent progress of "Gene therapy" and "Genetic vaccination" also demands production of pharmaceutical grade plasmid DNA in 'kilogram' level. Despite existence of a number of purification protocols, all most all have been originated from a pioneering work [Birnboim, H.C., Doly, J., 1979. A rapid extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7, 1513-1523] and so suffer from one or more drawbacks, such as purification time, purity or quantity of isolated plasmid DNA. Here, we have reported an innovative approach for isolation of highly pure and functional plasmid DNA in significant amount, based on generation of "soft protein aggregate" with the help of zwitterionic detergents and alkali. Solibilized proteins and RNA could be removed by a simple and mild washing with Tris buffer of low ionic strength and multimeric plasmid DNA could be eluted in a single step from the protein aggregate. Additionally, isolated plasmid DNA could easily be digested by restriction enzymes and had high functionality in protein expression. Thus, considering both its remarkable simplicity and efficiency in producing sufficiently pure plasmid DNA, the new strategy would emerge a useful tool in modern recombinant technology and therapeutic applications.     

Rapid micropreps and minipreps of Ti plasmids and binary vectors fromAgrobacterium tumefaciens

A simple and versatile procedure has been developed for the isolation of both large helper/Ti plasmids and binary vectors fromAgrobacterium tumefaciens. Using a slightly modified alkaline lysis protocol, intact plasmid can be recovered from cultures grown in standard micro-centrifuge tubes or culture tubes in sufficient yield and purity to allow for restriction analysis on ethidium bromide stained gels of the >200 kb Ti plasmid DNA. Contamination by chromosomal DNA is minimal and there is thus no need for isopycnic gradient purification. This same procedure can be combined with a high temperature treatment (37°C) and antibiotic selection to generate preparations containing binary vector DNA that are virtually free of interfering Ti plasmid DNA. Restriction patterns produced from these binary vector DNA preparations are unambiguous and therefore preliminary screening by Southern hybridization can be eliminated.     

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.     

Electroporation-mediated transfection of mammalian cells with crude plasmid DNA preparations

We designed a simple and reproducible electroporation-mediated transfection procedure with which to screen mammalian expression vector-constructed cDNA libraries. Using a specific chamber composed of five parallel electrodes, the recipient cells can be electroporated separately with 40 plasmid DNA preparations in a single experiment. Over 300 crude plasmids prepared from E. coli (DH-5) carrying a pcD2neo-vector-derived cDNA library were tested. The efficiency of stable transfection by electroporation with crude plasmid DNA preparations was 10-times higher than with the CsCl-purified plasmid DNA. When the crude plasmids were digested with RNase, the efficiency of stable transfection markedly decreased, indicating that the contaminating bacterial RNA in the crude plasmid preparations has a strong carrier effect during the electroporation. Even when salmon sperm DNA or genomic DNA from the recipient cells was used as the carrier of the purified plasmid, the efficiency was not higher than that using the crude preparations. This procedure is useful not only for screening a number of cDNAs but also for routinely introducing biologically active foreign genes into cultured mammalian cells.     

Rapid Mini-Prep Isolation of High-Quality Plasmid DNA from Lactococcus and Lactobacillus spp

A simple, rapid plasmid mini-prep procedure for lactococci and lactobacilli which gives high yields and can be performed on overnight broth cultures is presented. Large plasmids were isolated from both lactococci and lactobacilli, including a 70-kb plasmid from Lactobacillus acidophilus C7. The purity of the resulting plasmid DNA makes it suitable for subsequent molecular manipulations. The convenience of the technique makes this rapid mini-prep procedure suitable for routine plasmid isolation from lactic acid bacteria.     

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 rapid microscale technique for isolation of recombinant plasmid DNA suitable for restriction enzyme analysis

A simple and rapid microscale technique is described for the isolation of plasmid DNA which involves cell lysis with phenol, centrifugation, phenol extraction, ethanol precipitation, and RNase digestion. The plasmid DNA is of suitable purity and quantity for multiple restriction endonuclease digestions and bacterial transformations. This “miniprep” procedure is applicable for a variety of types of plasmids ranging in size from 2900 to 18,400 base pairs (bp) and for a number of Escherichia coli strains. The plasmids are rapidly cleaved by all restriction enzymes (total of 14) tested to date. Recombinant clones have been screened for insertions as small as 10 bp and as large as 5000 bp. The procedure takes ~3 h and has been routinely used to simultaneously analyze 24 candidate clones. This procedure is reliable and useful for rapid screening of recombinant DNA candidates where analysis by restriction endonuclease digestion is necessary.     

A continuous process to extract plasmid DNA based on alkaline lysis

Rapid advances in the fields of DNA vaccines and gene therapy have produced increased demands for large quantities of recombinant plasmid DNA. The protocol presented here extracts plasmid DNA in a scalable continuous process based on an alkaline lysis protocol. In the process, harvested bacteria are passed through two mixing chambers at controlled speeds to effect lysis and control alkalinity. The resulting solution is passed through a series of filters to remove contaminants and then ethanol precipitated. This process replaces all the centrifugation steps before obtaining crude plasmid and can be easily scaled up to meet demands for larger quantities. Using this procedure, plasmid can be extracted and purified from 4 l of Escherichia coli culture at an OD 600 nm of 50 in <90 min. The plasmid yields are approximately 80-90 mg l(-1) culture.     

Large-scale high-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method

Here, we describe a Library screen transformation protocol using the lithium acetate/single-stranded carrier DNA/PEG method of transformation for Saccharomyces cerevisiae. This method is suitable for screening complex plasmid libraries such as those used for yeast two-hybrid analysis. This procedure takes up to 2.5 h to complete once the yeast culture has been grown.     

Site-directed mutagenesis of virtually any plasmid by eliminating a unique site.

We describe an efficient site-specific mutagenesis procedure that is effective with virtually any plasmid, requiring only that the target plasmid carry a unique, nonessential restriction site. The procedure employs two mutagenic oligonucleotide primers. One primer contains the desired mutation and the second contains a mutation in any unique, nonessential restriction site. The two primers are annealed to circular single-stranded DNA (produced by heating circular double-stranded DNA) and direct synthesis of a new second strand containing both primers. The resulting DNA is transformed into a mismatch repair defective (mut S) Escherichia coli strain, which increases the probability that the two mutations will cosegregate during the first round of DNA replication. Transformants are selected en masse in liquid medium containing an appropriate antibiotic and plasmid DNA is prepared, treated with the enzyme that recognizes the unique, nonessential restriction site, and retransformed into an appropriate host. Linearized parental molecules transform bacteria inefficiently. Plasmids with mutations in the unique restriction site are resistant to digestion, remain circular, and transform bacteria efficiently. By linking a selectable mutation in a unique restriction site to a nonselectable mutation, the latter can be recovered at frequencies of about 80%. Since most plasmids share common vector sequences, few primers, targeted to shared restriction sites, are needed for mutagenizing virtually any plasmid. The procedure employs simple procedures, common materials, and it can be performed in as little as 2 days.     

Rapid Mini-Prep Isolation of High-Quality Plasmid DNA from Lactococcus and Lactobacillus spp.

A simple, rapid plasmid mini-prep procedure for lactococci and lactobacilli which gives high yields and can be performed on overnight broth cultures is presented. Large plasmids were isolated from both lactococci and lactobacilli, including a 70-kb plasmid from Lactobacillus acidophilus C7. The purity of the resulting plasmid DNA makes it suitable for subsequent molecular manipulations. The convenience of the technique makes this rapid mini-prep procedure suitable for routine plasmid isolation from lactic acid bacteria.     

Rapid preparation of vector-free hybridization probes suitable for screening recombinant libraries

A procedure is described to rapidly prepare radioactively labeled DNA inserts from crude recombinant plasmid DNA preparations. These probes can subsequently be used to identify homologous nucleotide sequences in bacteria containing recombinant plasmids by colony hybridization. In a single procedure, crude recombinant plasmid DNA is both ³²P-labeled and fragmented by nick-translation in the presence of sufficient pancreatic DNase I to produce radioactive DNA of about 0.2–0.3-kb single-strand length. At this DNA fragment length the majority of the vector and insert sequences are on different DNA fragments. The insert DNA can then be separated from vector and contaminating Escherichia colt host chromosomal DNA by the following method. The DNA fragment population is first denatured and renatured under conditions such that the recombinant plasmid DNA reassociates but host DNA does not. The renatured plasmid DNA fragments are separated from the denatured host DNA by hydroxylapatite chromatography. The plasmid DNA fragments are then denatured and renatured with an excess of insert-free vector DNA. Conditions are chosen such that the insert DNA remains single-stranded while the vector DNA becomes double-stranded. The single-stranded insert DNA can be separated from the double-stranded vector DNA on hydroxylapatite and used directly for colony hybridization.     

A simple procedure for large-scale preparation of pure plasmid DNA free from chromosomal DNA from bacteria

A very simple, inexpensive procedure for preparing pure plasmid DNA from bacteria is described. In this method, lysozyme-induced spheroplasts are made in presence of 833 micrograms/ml of ethidium bromide which are then lysed by a mixture of Brij 58 and sodium deoxycholate, and the lysate is centrifuged at 48,000 g for 25 min whereby about 99.9% of total chromosomal DNA is pelleted. From the supernatant containing plasmid DNA, the proteins are removed by phenol extraction and the major part of RNA by CaCl2 precipitation, and finally the small amount of residual RNA is removed by RNase treatment. The average yield of pBR322 DNA from 1 liter of amplified culture by this procedure is 2 to 2.5 mg and the preparation is highly pure, containing only about 0.005% of total yield as chromosomal DNA contaminant. Moreover, the substrate activity and the transforming ability of the plasmid DNA prepared by this method remain unaffected.     

A simple and rapid method for screening transformant yeast colonies using PCR.

A simple and rapid procedure for screening transformant yeast colonies is described. In this method, a trace amount of plasmid DNA is isolated from a small amount of yeast cell mass; then, the presence of the exogenous DNA in each yeast colony is detected by PCR amplification.     

An automated process to extract plasmid DNA by alkaline lysis

With advances in the development of DNA vaccines and gene therapy, there is a growing need for plasmid DNA with high quality for fundamental research and clinical trials. In this report, a scalable automated process for large-scale preparation of plasmid is described. This process is based on alkaline lysis and can be easily scaled up to meet demands for larger quantities. In the process, harvested bacteria are passed through two mixing chambers at controlled speeds to affect lysis and control alkalinity. The resulting solution is passed through a series of filters to remove contaminants, and ethanol precipitated. System parameters are examined to maximize the quantity and quality of the prepared plasmid. Using this procedure, plasmid can be extracted and purified from 1 l of Escherichia coli cultures at an OD600 nm of 50 in less than 45 min. The plasmid yields are approximately 90 mg/l culture.     

不同培养时期的大肠杆菌和枯草芽孢杆菌间发生的跨属自然遗传转化

携带穿梭质粒的大肠杆菌与作为受体的枯草芽孢杆菌分别培养至不同生长阶段混合均匀后静置40min,涂布选择性平板,37℃培养30h后得到一定数目的转化子,DNaseⅠ敏感实验证实质粒是通过自然遗传转化而非其它形式发生转移。实验发现大肠杆菌可以在特定生长时期向胞外分泌DNA,并且在对数期具有最高的提供质粒的能力,而生长后期的细胞因为体系中DNase量的增加转化频率下降。进一步的研究发现枯草芽孢杆菌在营养丰富的LB培养基中也具有与基本培养基中相当的转化能力,并且在对数生长前期具有较高的转化频率。