An economical large scale procedure to purify E. coli amplifiable plasmids for DNA sequencing, in vitro transcription and in vitro mutagenesis.
A reproducible and economical procedure for obtaining a large and quantitative yield of highly purified covalently closed circular plasmid DNA is described. The procedure departs in several ways from more commonly used methods. These are a) avoidance of the use of CsCl, ethidium bromide and ultracentrifuge, b) enrichment of the plasmid DNA by selective denaturation of chromosomal DNA with an alkaline-SDS solution, c) enrichment of covalently closed circular plasmid DNA by extraction with acid-phenol, and d) removal of small degraded RNA fragments by molecular sieve chromatography after digestion with RNase A. The plasmid DNA prepared by this new procedure is free of contaminants and has been used for DNA sequencing, in vitro transcription, transformation and in vitro mutagenesis.
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Cannon
D001483:Base Sequence; D002498:Centrifugation; D011232:Chemical Precipitation; D002850:Chromatography, Gel; D004269:DNA, Bacterial / Q000302:isolation & purification*; D004270:DNA, Circular / Q000302:isolation & purification*; D004926:Escherichia coli / Q000235:genetics*; D005784:Gene Amplification; D008722:Methods; D009154:Mutation; D009691:Nucleic Acid Denaturation; D010957:Plasmids; D012329:RNA, Bacterial / Q000378:metabolism; D012259:Ribonuclease, Pancreatic / Q000378:metabolism; D014158:Transcription, Genetic
D004269:DNA, Bacterial; D004270:DNA, Circular; D012329:RNA, Bacterial; D012259:Ribonuclease, Pancreatic