RESUMO
DNA supercoiling plays critical roles in several essential DNA metabolic pathways, such as replication, transcription and recombination. Typically plasmid DNA molecules are used to measure DNA supercoiling status inside bacterial cells. In this chapter, we describe how to isolate plasmid DNA molecules from E. coli cells and determine DNA supercoiling density by 1% agarose gel electrophoresis containing chloroquine using plasmid pACYC184 as an example.
Assuntos
Escherichia coli/genética , Plasmídeos/genética , Cloroquina/metabolismo , Replicação do DNA , DNA Super-Helicoidal/química , DNA Super-Helicoidal/genética , Eletroforese em Gel de Ágar , Escherichia coli/crescimento & desenvolvimento , Conformação de Ácido Nucleico , Plasmídeos/química , Plasmídeos/metabolismoRESUMO
Due to asymmetrical charge distribution of the mammalian high mobility group protein A2 (HMGA2), which makes HMGA2 bind to both cation- and anion-exchange columns, we developed a rapid procedure for purifying HMGA2 in the milligram range. This purification procedure greatly facilitated biophysical studies, which require large amounts of the protein.