PREPARATION OF DNA EXTRACTED FROM ENVIRONMENTAL WATER SAMPLES FOR PCRAMPLIFICATION

Sephadex G-200 spun columns have been used to purify DNA extracted fro m aquatic samples. Nucleic acid recovery using a previously-described protocol was only 10 to 15%. We optimized this method by employing a h igh salt (0.2 M NaCl) TE buffer (pH 8.0) and four slow-speed centrifug ation steps (130xg) in a swing-out centrifuge. DNA recovery improved t o approximately 75%. Purified DNA was a suitable substrate for PCR amp lification. Following PCR of DNA extracted from water samples amended with various concentrations of enterotoxigenic Escherichia coli (ETEC) , the ETEC heat-labile enterotoxin (LT) was detected in samples treate d with the Sephadex G-200 spun columns and not in untreated samples. T his method has the advantages of being inexpensive, simple and rapid. (C) 1998 Elsevier Science B.V.