Maximizing the Plasmid Yield in Laboratory-scale Maxiprep Preparations through a Modified Alkaline Lysis Approach

Abstract

Plasmid purification is an important step in molecular biology and good quality / quantity of plasmids are essential for cloning, production of therapeutic plasmid. Commercial kits for plasmid purification are costlier and the traditional methods are suitable for lesser culture volume. Hence, the study was aimed to enhance the yield of therapeutic grade plasmid in maxiprep preparation by modifying culturing conditions and the traditional alkaline lysis method. To increase the yield of bacterial cell mass carrying the plasmid of interest, LB broth supplemented with 1.9% yeast extract (LBY) was used. The dry pellet yield (g/ 250 ml of culture volume) of DH5α cells containing recombinant plasmid (pVAX1 -ILTV-gB) in LB broth and LBY broth after 18 hours of overnight culture were 1.142 ± 0.016 and 1.535 ± 0.016, respectively. Further, the traditional alkaline lysis method was improved by using calcium chloride, Sodium chloride and PEG6000 (40%) which boosted the yield of plasmid with increasing amount of supercoiled plasmid. The mean yield of plasmid purified from 250 ml of culture volume using the commercial kit and the modified method was 566 µg and 3530 µg, respectively. The modified alkaline lysis method yielded five times higher yield compared to purification by kit.  The plasmid purified using the modified method was pure, cost-effective, and ideal for cloning and transfection. This modified method of purification enhanced the yield of supercoiled plasmid which is essential for therapeutic purpose.