A quantitative analysis of DNA extraction and purification from compost

A quantitative analysis of DNA extraction and purification from compost We quantified both DNA and humic acid concentrations during the extraction and purification of DNA from compost. The DNA extraction method consisted of bead-beating with SDS for cell lysis, poly(ethylene glycol)-8000 precipitation for preliminary DNA purification, and chromatography on a 10-ml Sephadex G-200 column for final DNA purification. Direct microscopic observation of pre- and post-lysis samples revealed that 95.3±2.3% of native cells was lysed. Sixty-three percent of the original DNA was lost during purification, resulting in a final DNA yield of 18.2±3.8 μg DNA/g of wet compost. The humic acid content was reduced by 97% during the purification steps resulting in a final humic acid concentration of 27±4.7 ng humic acid/μl. The purified DNA fragments were up to 14 kbp in size and were sufficiently free of contaminants to allow both restriction enzyme digestion by four different enzymes and PCR amplification of 16S rDNA. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal Of Microbiological Methods Elsevier

A quantitative analysis of DNA extraction and purification from compost

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Publisher
Elsevier
Copyright
Copyright © 2003 Elsevier Science B.V.
ISSN
0167-7012
eISSN
1872-8359
D.O.I.
10.1016/S0167-7012(03)00006-X
Publisher site
See Article on Publisher Site

Abstract

We quantified both DNA and humic acid concentrations during the extraction and purification of DNA from compost. The DNA extraction method consisted of bead-beating with SDS for cell lysis, poly(ethylene glycol)-8000 precipitation for preliminary DNA purification, and chromatography on a 10-ml Sephadex G-200 column for final DNA purification. Direct microscopic observation of pre- and post-lysis samples revealed that 95.3±2.3% of native cells was lysed. Sixty-three percent of the original DNA was lost during purification, resulting in a final DNA yield of 18.2±3.8 μg DNA/g of wet compost. The humic acid content was reduced by 97% during the purification steps resulting in a final humic acid concentration of 27±4.7 ng humic acid/μl. The purified DNA fragments were up to 14 kbp in size and were sufficiently free of contaminants to allow both restriction enzyme digestion by four different enzymes and PCR amplification of 16S rDNA.

Journal

Journal Of Microbiological MethodsElsevier

Published: Jul 1, 2003

References

  • Effects of humic substances on fluorometric DNA quantification and DNA hybridization
    Bachoon, D.S; Otero, E; Hodson, R.E
  • Direct extraction of microbial community DNA from humified upland soils
    Clegg, C.D; Ritz, K; Griffiths, B.S
  • Extraction of ribosomal RNA and genomic DNA from soil for studying the diversity of the indigenous bacterial community
    Duarte, G.F; Rosado, A.S; Seldin, L; Keijzer-Wolters, A.C; van Elsas, J.D
  • Rapid method for fluorometric quantification of DNA in soil
    Sandaa, R.A; Enger, O; Torsvik, V
  • Pre-PCR DNA quantitation of soil and sediment samples: method development and instrument design
    Stark, P.C; Mullen, K.I; Banton, K; Russotti, R; Soran, D; Kuske, C.R

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