High-yielding plasmid extraction method from acidophilic heterotrophic bacteria of the genus Acidiphilium.
Plasmid yield from Acidiphilium strains always had been poor following various standard methods. We adopted some simple modifications in the alkaline lysis procedure to get a better yield of plasmid from these bacteria. An approximately 10- to 20-fold increase in the plasmid yield was achieved when harvested Acidiphilium cells were preincubated 16-20 h at pH 6 in nitrogen-free medium. Another independent approach showed that freezing (-18 to -20 degrees C) of the harvested cells initially and at two subsequent steps in the alkaline lysis procedure of plasmid DNA extraction improved the yield further by 1.5- to 3-fold. The combination of these changes yielded at least 15- to 30-fold more plasmid from various Acidiphilium strains as compared with standard methods.
Singh
Samarendra K
SK
Indian Institute of Chemical Biology, Kolkata 700032, India.
Banerjee
Pataki C
PC
D041801:Acidiphilium / Q000235:genetics*; D001483:Base Sequence; D004587:Electrophoresis, Agar Gel / Q000379:methods; D005615:Freezing; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics / Q000302:isolation & purification*; D013696:Temperature