Protocol

Purification of Genomic DNA Extracted from Environmental Sources for Use in a Polymerase Chain Reaction

Department of Biological Sciences, Auburn University, Auburn, AL 36849, USA

¹Corresponding author (lilesma{at}auburn.edu).

INTRODUCTION

The ability to amplify genomic DNA in a polymerase chain reaction (PCR) is dependent upon the purity of the DNA template. Environmental genomic DNA often contains contaminants (e.g., polyphenols, humic acids, polysaccharides) that reduce template purity and can be difficult to remove, thereby inhibiting PCR amplification. There is thus a need for a method to purify extracted genomic DNA without reducing DNA concentration. In this protocol, extracted genomic DNA is embedded in agarose plugs and incubated in a formamide and salt (NaCl) solution to remove contaminants. The NaCl works to deproteinize and stabilize the DNA. The formamide serves to denature the DNA (which will subsequently be renatured within the agarose plug) and any contaminants that may be bound to the DNA. The purified DNA is extracted from the agarose plug using a standard commercial agarose extraction method, and the DNA may then be used as a template for PCR. Genomic DNA purified using this method has been shown to serve as an efficient template for PCR, without significant loss of DNA yield. An additional advantage of the method is that it allows the simultaneous processing of large numbers of samples at once.