Abstract
DNA extraction methods such as plasmid minipreps, gel, and PCR purifications, are indispensable techniques for genetic manipulations. There are numerous factors that contribute to the efficiency of these processes, which determine the success of complex downstream molecular analytics and diagnostic tests. To study and optimize these factors, we compared our own proprietary buffers to commercially available column-based kits, utilizing their spin columns and protocols. Through systematic substitution of the buffers in the kits with our own proprietary buffers, we selected the highest DNA yielding buffer recipes. Further analysis of the differences between the buffers showed that high concentrations and presence of certain chaotropic agents and cations are necessary for good plasmid miniprep, gel extraction, and PCR purification kits.
Highlights
The extraction and purification of nucleic acids are commonly used techniques to isolate genetic material from tissues, bacteria, plants, and viruses for important analytical, diagnostic and preparative downstream processes
We established a set of optimized (OPT) and completely "home-made" (HM) buffers for nucleic acid extraction and purification kits that are comparable to the two generic brands (A and B) in terms of plasmid yield and purity
Through the step-wise buffer substitution (Table 1), we found that P-P1, P-W2-1
Summary
The extraction and purification of nucleic acids are commonly used techniques to isolate genetic material from tissues, bacteria, plants, and viruses for important analytical, diagnostic and preparative downstream processes Amongst these methods, plasmid DNA extraction was the first to be reported [1] using the tedious alkaline extraction protocol. Sodium acetate is used to neutralize the alkaline pH, resulting in the formation of an insoluble network of denatured genomic DNA, protein-SDS complexes and high molecular weight RNA. These complexes were removed by high speed centrifugation, leaving the desired plasmid DNA in the supernatant [1]. Despite simplifying the process through the immobilization of plasmid DNA to the solid phase matrix (i.e. silica), plasmid extraction is still underlined by the need to disrupt bacterial cell walls, denaturation of nucleic acid binding proteins, inactivation of nucleases such as RNases, washing away of undesired contaminants, and elution of desired plasmid DNA
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