Optimizing alkaline lysis for DNA plasmid recovery.
Optimization of the alkaline lysis (P2) and neutralization (N3) steps in the recovery of DNA plasmids was pursued. Experiments were conducted at the test-tube and 5-litre scales with 3 kb (pUC18) and 20 kb (pQR150) plasmids. The scale and degree of mixing/shear did not affect the optimum yield of supercoiled plasmid during the P2 step, but did effect the time required for the optimum to be achieved. This optimum time for P2 at the large scale was longer (8-9 min), especially when a low-shear impeller was used. Also, when the yield of supercoiled plasmid reached a maximum during the P2 step, the purity (percentage of plasmids in the supercoiled form) simultaneously reached a minimum. As the duration of the N3 step increased from 1 to 6 min, the yield of the supercoiled plasmids remained fairly constant, provided that a lowshear impeller was used. The neutralized (post-N3) plasmid solution was shear-sensitive; however, mixing with a Rushton turbine in a tank (maximum energy dissipation rate in the mixing tank, epsilon (max), 12 m(2)/s(3); mixing-tank power consumption/volume of mixing tank, 2.0 W/m(3)) for 5-10 min resulted in a slight decrease in supercoiled plasmid and a notable increase in genomic DNA concentrations. The loss of the larger 20 kb plasmid (20%) was more than for the 3 kb plasmid. Finally, preparing the cells for alkaline lysis with lysozyme or low-pressure homogenization did not increase the plasmid yield. Furthermore, the homogenizer broke up the genomic DNA into fragments that followed through the entire Qiagen prep with the plasmids as impurities.
Clemson
Michael
M
Department of Chemical Engineering, Villanova University, Villanova, PA 19085, USA.
Kelly
William J
WJ
D000468:Alkalies / Q000494:pharmacology; D001433:Bacteriolysis / Q000187:drug effects*; D019149:Bioreactors; D018929:Cell Culture Techniques / Q000295:instrumentation / Q000379:methods*; D004249:DNA Damage; D004269:DNA, Bacterial / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D004926:Escherichia coli / Q000737:chemistry* / Q000187:drug effects*; D005818:Genetic Engineering / Q000379:methods; D006863:Hydrogen-Ion Concentration; D010865:Pilot Projects; D010957:Plasmids / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification*; D011786:Quality Control; D011995:Recombination, Genetic; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D012972:Sodium Hydroxide / Q000494:pharmacology; D013314:Stress, Mechanical; D014783:Viscosity
D000468:Alkalies; D004269:DNA, Bacterial; D012967:Sodium Dodecyl Sulfate; D012972:Sodium Hydroxide