Development of simple and efficient protocol for isolation of plasmids from mycobacteria using zirconia beads.
A two-step protocol has been developed for isolation of plasmids from recombinant mycobacteria via Escherichia coli. First either mycobacterial primary transformants or propagated cultures were lysed in a mini-bead beater using zirconia beads and the lysate thus obtained was used to transform E. coli recA mutant cells. Secondly, plasmid DNA was isolated from recombinant E. coli cells and analysed. Bead beating times of 2 min for Mycobacterium smegmatis, a rapid grower, and 4 min for M. bovis BCG, a slow grower, were found to be optimal for recovery of plasmid DNA. This protocol was also amenable to other mycobacterial species such as M. avium, M. fortuitum and M. tuberculosis H37Ra. Plasmid recovery from the recombinant M. bovis BCG using this protocol is approximately 300-fold higher than that reported for the electroduction method.
Madiraju
M V
MV
Department of Biochemistry, The University of Texas Health Center at Tyler, 75710, USA.
Qin
M H
MH
Rajagopalan
M
M
D015139:Blotting, Southern; D004926:Escherichia coli / Q000235:genetics; D008863:Microspheres; D009161:Mycobacterium / Q000235:genetics*; D009170:Nontuberculous Mycobacteria / Q000235:genetics*; D010957:Plasmids / Q000302:isolation & purification*; D013997:Time Factors; D014169:Transformation, Bacterial; D015040:Zirconium
D015040:Zirconium; C028541:zirconium oxide