A rapid microscale technique for isolation of recombinant plasmid DNA suitable for restriction enzyme analysis
1980; Elsevier BV; Volume: 3; Issue: 1 Linguagem: Inglês
10.1016/s0147-619x(80)90037-2
ISSN1095-9890
AutoresRonald D. Klein, Erik Selsing, Robert D. Wells,
Tópico(s)Advanced biosensing and bioanalysis techniques
ResumoA simple and rapid microscale technique is described for the isolation of plasmid DNA which involves cell lysis with phenol, centrifugation, phenol extraction, ethanol precipitation, and RNase digestion. The plasmid DNA is of suitable purity and quantity for multiple restriction endonuclease digestions and bacterial transformations. This “miniprep” procedure is applicable for a variety of types of plasmids ranging in size from 2900 to 18,400 base pairs (bp) and for a number of Escherichia coli strains. The plasmids are rapidly cleaved by all restriction enzymes (total of 14) tested to date. Recombinant clones have been screened for insertions as small as 10 bp and as large as 5000 bp. The procedure takes ~3 h and has been routinely used to simultaneously analyze 24 candidate clones. This procedure is reliable and useful for rapid screening of recombinant DNA candidates where analysis by restriction endonuclease digestion is necessary.
Referência(s)