共查询到20条相似文献,搜索用时 15 毫秒
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A rapid method for preparation of bacterial plasmids 总被引:7,自引:0,他引:7
A method for isolating plasmids from Escherichia coli which requires less than 8 h from cell pellet to purified plasmid essentially free of protein, RNA, and chromosomal DNA is presented. By this procedure, amplified plasmid pBR322 was isolated from E. coli strain RR1. The final product had no detectable protein or RNA, and plasmid comprised approximately 99% of the total DNA. The procedure includes lysozyme treatment in hypertonic solution followed by lysis with a mild detergent in the presence of high salt and an RNase inhibitor--conditions which prevent unfolding of the bacterial nucleoid. After centrifuging out the nucleoid and cell debris, the nucleic acids are selectively precipitated with a neutral solution of sodium trichloroacetate and ethanol. RNA is degraded with RNase and the degradation products and RNase are eliminated through a second trichloroacetate/ethanol precipitation. Finally, the plasmid is resuspended and passed through a nitrocellulose filter to remove aggregates and any residual protein and single-stranded DNA--giving a plasmid preparation suitable for electrophoretic fractionation or cleavage with restriction nucleases. 相似文献
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A rapid boiling method for the preparation of bacterial plasmids 总被引:67,自引:0,他引:67
Compounds separated on polyamide thin-layers and located in an appropriate manner can be introduced directly into the ion source of the mass spectrometer together with the chromatographic polyamide adsorbent. In spite of the large excess of polyamide, excellent mass spectra are obtained exhibiting low backgrounds. In this way, organic compounds can be rationally identified on a nanogram scale not requiring reference substances. The application of the procedure is described for selected compounds of different classes of substances, e.g., phenols, steroids, nucleosides, biogenic amines, and amino acids. 相似文献
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一种快速提取丝状真菌染色体DNA的方法 总被引:5,自引:0,他引:5
介绍了一种适用于丝状真菌染色体DNA大片段的快速提取方法,该方法以(100mM Tris,100mM NaGl,50mM EDTA-Na2 2%SDS,pH值9.0)为提取液,经石英砂研磨破壁.应用该方法成功地提取了粗糙脉胞菌(Neurospora crassa)、米曲霉(Aspergillus oryzae)、产黄青霉(Penicillium chrysogenum)和头孢霉菌(Cep- halosporium sp.)等4种不同丝状真菌的染色体DNA大片段,且所提DNA片段均大于20kb,可直接用于限制性酶切、PCR等分子生物学研究. 相似文献
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A method for the extraction of genomic DNA from the industrial yeastCandida utilis is described. The method is rapid, simple and produces DNA that is sufficiently pure for restriction analysis and should
be suitable for Southern blotting and the construction of gene libraries. 相似文献
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Direct lysis method for the rapid preparation of plasmid DNA. 总被引:2,自引:0,他引:2
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Josef Flossdorf 《Journal of microbiological methods》1983,1(5):305-311
A rapid method to evaluate the (G+C)-content of bacterial DNA is described. About 2 × 108 cells are lysed by the combined action of detergents and enzymes and put into a CsCl-gradient without any purification. Up to five samples may be run at once. The high background absorption of cell-derived material other than DNA is overcome by novel collimating optics. 相似文献
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C D Reymond 《Nucleic acids research》1987,15(19):8118
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Kook JK Kim MK Seong JH Kim DK Kim BO Park JC Kim KK Choe SJ Min BM 《FEMS microbiology letters》2003,219(1):121-127
A simple assay for the rapid screening of bacterial species- or subspecies-specific DNA probes for the random cloning method is presented, involving the use of genomic DNAs as probes and recombinant plasmid DNAs containing genomic DNA digested with HindIII as targets. The optimal amount of target DNAs and the concentration of digoxigenin-labeled genomic DNA probes were 20 ng and 100 ng ml(-1) (or 10 ng and 200 ng ml(-1)), respectively. The method was applied to the development of Fusobacterium nucleatum subspecies-specific probes. Our results showed that four out of 96 probes were F. nucleatum subspecies-specific, which was confirmed by Southern blot analysis. Our results indicate that the new method can be used for the rapid screening of species- or subspecies-specific probes. 相似文献
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《Gene Analysis Techniques》1989,6(5):93-96
A streamlined, reproducible boiling method for preparing plasmid as well as phage replicative form DNA is described. Both quantity and quality of DNA purified are sufficient for restriction enzyme analysis and sequencing. A small loopful of bacteria will provide enough DNA for several experiments, and multiple samples can be processed and prepared for digestion or sequencing in under 20 minutes. DNA preparations are stable for at least 6 months at −20°C. 相似文献
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