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 microg 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/microl. 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.
Department of Biological and Environmental Engineering, Cornell University, Ithaca, NY 14853, USA.
Michael
M
Howeler
William C
WC
Ghiorse
Larry P
LP
Walker
D001419:Bacteria / Q000235:genetics* / Q000302:isolation & purification; D015169:Colony Count, Microbial; D004269:DNA, Bacterial / Q000032:analysis* / Q000302:isolation & purification*; D004275:DNA, Ribosomal / Q000032:analysis; D004783:Environmental Microbiology; D006812:Humic Substances / Q000032:analysis; D016133:Polymerase Chain Reaction; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012037:Refuse Disposal
D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D006812:Humic Substances; D012336:RNA, Ribosomal, 16S