RESUMEN
A novel density barrier material, Phase Lock Gel, has been formulated and optimized for use in enhancing the separation between aqueous and organic phases during organic extraction/ centrifugation of nucleic acids. Phase Lock Gel is inert, stable to heating, compatible with most enzymatic reactions of nucleic acids without sacrificing subsequent downstream applications, and can be easily and conveniently used with most plasmid DNA, genomic DNA, phage DNA and RNA organic extraction protocols. Use of Phase Lock Gel as an aqueous/organic phase barrier material can decrease processing time and improve nucleic acid recovery from organic extraction purifications by as much as 30%, all while minimizing user exposure to volatile organics.
Asunto(s)
ADN/aislamiento & purificación , ARN/aislamiento & purificación , Células 3T3 , Animales , RatonesRESUMEN
The performance characteristics of a potent new RNase inhibitor have been evaluated, as have applications for its use in molecular biology. PRIME Inhibitor is a protein, not related to the commonly available human placental RNase inhibitors (HPRI). It has a high specific activity, enhanced temperature stability, broad reaction pH range and significantly greater cost-effectiveness than commercial HPRI. PRIME Inhibitor is suitable for use in in vitro transcription, in vitro translation, first- and second-strand cDNA synthesis, preparation of RNA and mRNA, and reverse transcription-polymerase chain reaction.
Asunto(s)
ARN/aislamiento & purificación , Ribonucleasas/antagonistas & inhibidores , Animales , Células CHO/enzimología , Células Cultivadas/enzimología , Costos y Análisis de Costo , Cricetinae , ADN Complementario , Reacción en Cadena de la Polimerasa/métodos , Biosíntesis de Proteínas , Sensibilidad y Especificidad , Transcripción GenéticaRESUMEN
An extremely rapid method, INSTA-PREP, has been developed to prepare plasmid DNA from 1 to 3 mL miniprep Escherichia coli bacterial cultures. Direct extraction of plasmid DNA from E. coli bacterial cells is achieved by a two-phase solution consisting of phenol-chloroform-isoamyl alcohol and water or buffer with efficient separation of the phases by centrifugation in the presence of the INSTA-PREP gel barrier material. Processing time, from E. coli culture to usable plasmid DNA, is two minutes or less per sample. Supercoiled plasmid DNA yields ranged from 3 to 10 micrograms per mL of culture depending on plasmid copy number. Plasmid DNAs prepared by INSTA-PREP were analyzed and are suitable for use in molecular biology procedures including restriction digestion, ligation with T4 DNA ligase, bacterial transformation, PCR, cultured cell transfection and T7 DNA polymerase or thermostable DNA polymerase-mediated dideoxynucleotide sequencing.
Asunto(s)
ADN Bacteriano/aislamiento & purificación , Plásmidos , Animales , Células CHO , Cricetinae , Enzimas de Restricción del ADN , ADN Superhelicoidal/aislamiento & purificación , ADN Polimerasa Dirigida por ADN , Escherichia coli/genética , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADN , Factores de Tiempo , Transfección , Transformación BacterianaRESUMEN
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.
Asunto(s)
Cromatografía/métodos , ADN/aislamiento & purificación , Plásmidos , Animales , Línea Celular , ADN Bacteriano/aislamiento & purificación , Escherichia coli/análisis , Escherichia coli/genética , Estudios de Evaluación como Asunto , Transfección , Transformación Genética , UltracentrifugaciónRESUMEN
Incubation in a HeLa whole-cell extract converted a plasmid DNA containing the adenovirus type 2 major late promoter into ordered nucleoprotein complexes similar to those described for simian virus DNA [Sinha, S. N., Hellwig, R. J., Allison, D. P. & Niyogi, S. K. (1982) Nucleic Acids Res. 10, 5533-5552]. Purified nucleoprotein complexes containing the plasmid DNA were able to serve as template for accurate transcription in vitro. Use of such nucleoprotein complexes eliminated the need for addition of nontemplate DNA (poly[d(I-C)]) to transcription reaction mixtures with template concentrations too low to yield a detectable specific-transcription signal from "naked" DNA. Of the four fractions resulting from phosphocellulose column chromatography of the whole cell extract, only the fraction that contained the RNA polymerase II activity was needed to accurately transcribe these nucleoprotein complexes in the presence of human placental ribonuclease inhibitor. In contrast, specific transcription of naked template DNA under these conditions required at least one additional fraction containing specificity factors. These results show that the nucleoprotein complexes contain factor(s) needed for specific initiation of transcription and that these complexes may be useful in the purification and analysis of these factors.
Asunto(s)
Adenovirus Humanos/genética , Nucleoproteínas/metabolismo , Regiones Promotoras Genéticas , ARN Polimerasa II/metabolismo , Transcripción Genética , Células HeLa/enzimología , Humanos , Nucleoproteínas/aislamiento & purificación , Plásmidos , Polidesoxirribonucleótidos/farmacología , Moldes Genéticos , Transcripción Genética/efectos de los fármacosRESUMEN
Interaction of SV40 DNA with three different HeLa cell extracts capable of directing correct initiation of transcription leads to the formation of ordered nucleoprotein complexes that are structurally similar to SV40 minichromosomes and eukaryotic chromatin. These nucleoprotein complexes can be conveniently purified by band sedimentation or gel filtration. Their sedimentation and elution properties resemble those of SV40 minichromosomes. Electron microscopy of purified complexes shows beaded structures that are sensitive to proteases, resulting in recovery of naked, largely undegraded DNA. Contour lengths and compaction ratios of these nucleoprotein complexes are similar to those of authentic SV40 minichromosomes. Their digestion patterns with micrococcal nuclease and pancreatic DNase I resemble those of SV40 minichromosomes. Such nucleosome-like structures can also be obtained with linear SV40 DNA. Unlike nucleosomes, no histones could be detected in the purified nucleoprotein complexes. Non-histone chromosomal protein fractions (high mol. wt. and free of high mobility group proteins) prepared from the HeLa cell extracts can also generate similar ordered structures. We conclude that ordered nucleoprotein structures with certain common characteristics can be formed by interaction of DNA with non-histone chromosomal proteins as well as with histones. Only the former structures are generated in currently used cell-free transcription systems. It appears that only those purified nucleoprotein complexes containing the promoter can be actively transcribed in the presence of additional cell-free extract, suggesting that such structures and their protein components may be important in transcription.