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Arapid alkaline extraction procedure forscreening recombinant plasmid DNA
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TLDR
The method is 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, and achievesequate pH control without using a pH meter.Abstract:
Aprocedure forextracting plasmid DNAfrombacterial cellsis described. Themethodissimpleenough topermit theanalysis bygel electrophoresis of100ormoreclonesperdayyetyieldsplasmid DNAwhichis pureenough tobedigestible byrestriction enzymes.Theprinciple ofthe methodisselective alkaline denaturation ofhighmolecular weightchromosomal DNAwhilecovalently closedcircular DNAremains double-stranded. Adequate pHcontrol isaccomplished without usinga pHmeter.Uponneutralization, chromosomal DNArenatures toformaninsoluble clot,leaving plasmid DNAin thesupernatant. Largeandsmallplasmid DNAshavebeenextracted bythis method.read more
Citations
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References
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Journal ArticleDOI
Construction and characterization of new cloning vehicles. II. A multipurpose cloning system.
Francisco Bolívar,Raymond L. Rodriguez,Patricia J. Greene,Mary C. Betlach,Herbert L. Heyneker,Herbert W. Boyer,Jorge H. Crosa,Stanley Falkow +7 more
TL;DR: In vitro recombination techniques were used to construct a new cloning vehicle, pBR322, which is a relaxed replicating plasmid, does not produce and is sensitive to colicin E1, and carries resistance genes to the antibiotics ampicillin (Ap) and tetracycline (Tc).
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A complementation analysis of the restriction and modification of DNA in Escherichia coli.
TL;DR: Intercistronic complementation was observed between three classes of restriction and modification mutants of E. coli B, indicating that at least three cistron (the ram cistrons) are involved in the genetic control of the [restriction and modification of DNA].
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SUPERCOILED CIRCULAR DNA-PROTEIN COMPLEX IN Escherichia coli: PURIFICATION AND INDUCED CONVERSION TO AN OPEN CIRCULAR DNA FORM
TL;DR: Electron microscopic analyses indicate that the decrease in sedimentation rate of the ColE(1)-protein complex after treatment with these various agents is largely owing to an induced transition of ColE (1) DNA from the supercoiled to the open circular state.
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Nature of Col E 1 plasmid replication in Escherichia coli in the presence of the chloramphenicol.
TL;DR: The colicinogenic factor E(1) (Col E( 1)) in Escherichia coli continues to replicate by a semiconservative mechanism in the presence of chloramphenicol for 10 to 15 hr, long after chromosomal deoxyribonucleic acid (DNA) synthesis has terminated.
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A rapid method for the identification of plasmid desoxyribonucleic acid in bacteria.
TL;DR: A fast and very sensitive procedure is described for detecting plasmids in bacterial strains by agarose gel electrophoresis and the size of plasmid present in one or more copies per cell with a molecular mass ranging from 2 to over 150 megadaltons is identified.