Plasmid and chromosomal DNA recovery by electroextraction of cyanobacteria.
High voltage electroporation has been investigated as a method for rapid recovery of plasmid and chromosomal DNA from the cyanobacteria Nostoc PCC 7121, Synechococcus PCC 7002, and Anabaena PCC 7120. Pulses of 18 kV/cm and higher applied to concentrated Nostoc cells carrying a shuttle plasmid (pRL25) resulted in copious release of nucleic acids and phycobiliproteins into the suspending medium. Small portions of these supernatants, when electroporated with Escherichia coli, gave rise to hundreds of E. coli transformants which contained pRL25. Electroporation of Synechococcus carrying plasmid pAQE19 did not cause detectable release of macromolecules but did reveal a low-level, voltage independent 'leakage' of pAQE19 into the medium. Electroextraction of Nostoc or Anabaena followed by addition of E. coli and delivery of a second high-voltage pulse permitted direct, one-cuvette transfer of shuttle plasmids from these cyanobacteria into E. coli. Electroextraction of single cyanobacterial colonies, as shown for Nostoc, also released sufficient chromosomal DNA for amplification of specific sequences by the polymerase chain reaction.
Moser
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D
Department of Biology and Microbiology, University of Wisconsin-Oshkosh, USA.
Zarka
D
D
Hedman
C
C
Kallas
T
T
D017033:Anabaena / Q000235:genetics; D001433:Bacteriolysis; D001483:Base Sequence; D002876:Chromosomes, Bacterial; D000458:Cyanobacteria / Q000235:genetics*; D004269:DNA, Bacterial / Q000302:isolation & purification*; D018274:Electroporation; D004926:Escherichia coli / Q000235:genetics; D007613:Kanamycin Resistance / Q000235:genetics; D008969:Molecular Sequence Data; D010957:Plasmids / Q000302:isolation & purification; D014170:Transformation, Genetic
D004269:DNA, Bacterial