Rapid Purification of Plasmid DNAs by Hydroxyapatite Chromatography (original) (raw)
Related papers
Preparation and purification of DNA from bacterial cells; characterization of plasmid DNA
The use of genetic material to deliver genes for therapeutic purposes has been practiced for many years. With the advancement in genetic engineering, foreign genes of industrial applications can be inserted into cloning vector for mass production in various host cells. Escherichia coli is an extremely important model organism in modern biological engineering, the suitable growth media is essential for the optimal expression of the genes in E. coli. The present study aims at isolation and purification of genomic DNA from E. coli, the characterization of pBR322 plasmid DNA. Bacterial culture conditions were optimized in shake – flask cultures based on optimal temperature, inoculum size and medium composition. Solutions and methods are disclosed for the effective, simple isolation of DNA from bacterial cells. High bioprocess recovery and product quality were primarily associated with the complete removal of total cellular RNA impurity. The process was demonstrated without the use of animal-derived RNase. High-molecular-weight (HMW) RNA and other impurities were removed by selective precipitation using calcium chloride at an optimal concentration. The optimal conditions for the growth of Escherichia coli were shown maximum absorbance as 7.5 at 370C temperature, 1% inoculum size using TB medium composition. The purified genomic DNA had concentration as 73.5 µg/ml and purity 1.8. The 0.5M CaCl2 was optimal concentration for removal of RNA. The plasmid DNA pBR322 was confirmed by comparing the band to 4.36 Kb, purity of plasmid was 1.85 and it contains 96.8% of super coiled DNA. The contaminants like chromosomal DNA, RNA, host cell proteins and mycoplasma were absent in the plasmid DNA.
Vol 4 Issue 3, 2010 Modification of alkaline lysis method for rapid plasmid DNA isolation
Isolation of quality plasmid DNA from bacterial cells is critical in molecular biology experiments. Although efficient protocol for plasmid isolation are developed and standardized, rapid and cost effective protocols are desirable while handling a large number of samples. In present study we are reporting a simple procedure for extracting naked plasmid DNA which can be used for a large number of samples with sufficient purity. The method involves denaturation of high molecular weight chromosomal DNA using strong alkali, during neutralization process, chromosomal DNA renatures, and proteins denature to form an insoluble clot in the presence of choatropic agent guanidine hydrochloride, leaving plasmid DNA in the supernatant. The modified protocol which is less expensive compared to the commercial plasmid DNA isolation kit available, yields quality plasmid DNA. We tested the yield and quality of different kinds of plasmid extracted from bacteria. The results suggest that the protocol c...
A rapid alkaline extraction procedure for screening recombinant plasmid DNA
Nucleic Acids Research, 1979
A procedure for extracting plasmid DNA from bacterial cells 1s described. The method 1s simple enough to permit the analysis by gel electrophoresis of 100 or more clones per day yet yields plasmid DNA which is pure enough to be digestible by restriction enzymes. The principle of the method is selective alkaline denaturation of high molecular weight chromosomal DNA while covalently closed circular DNA remains double-stranded. Adequate pH control is accomplished without using a pH meter. Upon neutralization, chromosomal DNA renatures to form an insoluble clot, leaving plasmid DNA in the supernatant. Large and small plasmid DNAs have been extracted by this method.
Plasmid DNA small-scale preparation based on nuclease inactivation by diethylpyrocarbonate
Technical Tips Online, 1997
▼For several years now, we have been using a modification of the rapid boiling method (Ref. 1) as a small-scale plasmid preparation procedure. Our method requires a short exposure of bacterial cells to lysozyme to weaken their cell walls, irreversible inactivation of nucleases by diethylpyrocarbonate (Fluka), heating at 70 • C to partially denature genomic bacterial DNA (and proteins) and elimination by sedimentation of the insoluble cloud that is formed upon cooling. Plasmid DNA remains in solution and is recovered by ethanol precipitation. The yield of plasmid DNA is within the range of that obtained using alternative methods (Ref. 1, 2): 10−30 µg of plasmid DNA [pBluescript SK + plasmid (Stratagene), 25 ± 3 µg DNA; pUC, 30 ± 5 µg DNA; and pUR, 10 ± 1 µg DNA] is obtained from 1.5 ml of Escherichia coli overnight culture in Luria Broth (LB) medium.
Evaluation of several enrichment procedures for the isolation of recombinant plasmid DNA
Molecular Biology Reports, 1978
A number of methods for the selective enrichment of recombinant plasmids were examined; these include alkaline phosphatase treatment of the restricted pBR322 vector, as well as a combination of this and S1 nuclease treatment of the ligated mixture of pBR322 and pCR1 plasmids or S. griseus DNA followed by D-cycloserine treatment to enrich for cells carrying recombinant molecules. The relative efficiencies of these methods were compared.
Large scale purification of linear plasmid DNA for efficient high throughput cloning
Biotechnology Journal, 2010
In this report we describe a rapid, simple, and efficient method for large 2 scale purification of linear plasmid DNA to answer demand from high throughput 3 gene cloning. The process is based on the separation of the linear vector from 4 small DNA fragments by anion exchange chromatography. Gene cloning 5 experiments by restriction/ligation or the In-Fusion™ technique confirmed the 6 high quality of the linearized vector as 100% of the genes were successfully 7 cloned. Plasmid DNA purification [8] has been the subject of many studies, 20 allowing large scale and high grade plasmid preparation [9-13], including the use 21 of anion exchange chromatography [14-17]. The separation of multiple small 22 restriction fragments by anion exchange chromatography on FPLC columns was 23 This article describes the purification of linearized expression vectors in 20 sufficient quantity and quality to efficiently perform hundreds of gene cloning in 21
Analytical Biochemistry, 1985
A procedure for extracting RNA-free plasmid DNA from bacterial cells is described. The method is simple and rapid enough to obtain pure plasmid DNA in 8 to 10 h after plasmid amplification. The protocol uses the alkaline extraction procedure described by Doly (1979, Nucl. Acid Res. 7, 15 13-1523). Plasmid DNA is then separated from highmolecular-weight RNA by ammonium acetate precipitation and from low-molecular-weight RNA contaminants by Ultrogel A2 column chromatography. The plasmid DNA obtained by this inexpensive technique is sufficiently pure to be used for restriction endonuclease analysis, S-end labeling, Sl mapping, DNA sequencing, and colony hydridization. o 1985 Academic Pres, Inc.
Plasmid DNA purification using a multimodal chromatography resin
Journal of Molecular Recognition, 2014
Multimodal chromatography is widely used for isolation of proteins because it often results in improved selectivity compared to conventional separation resins. The binding potential and chromatographic behavior of plasmid DNA have here been examined on a Capto Adhere resin. Capto Adhere is a recent multimodal chromatography material allowing molecular recognition between the ligand and target molecule, which is based on combined ionic and aromatic interactions. Capto Adhere proved to offer a very strong binding of nucleic acids. This property could be used to isolate plasmid DNA from a crude Escherichia coli extract. Using a stepwise NaCl gradient, pure plasmid DNA could be obtained without protein and endotoxin contaminations. The RNA fraction bound most strongly to the resin and could be eluted only at very high salt concentrations (2.0 M NaCl). The chromatographic separation behavior was very robust between pH values 6 and 9, and the dynamic binding capacity was estimated to 60 μg/ml resin.