[An efficient method for DNA extraction from compost].
An efficient method for obtaining DNA from compost which contained high levels of organic matter was developed. The protocol consisted of washing with phosphate-EDTA before extraction, cell lysis with hot-SDS and enzymes (lysozyme, lywalzyme, proteinase K), removing humic acid and other inhibitors with PVPP and precipitation with PEG-8000. The compost total DNA was extracted from four different composts, the DNA yield was 63.54 +/- 12.08 to approximately 106.50 +/- 28.36 microg/g of dry compost. Molecular size of DNA obtained using this protocol was about 23kb and contained low protein and humic acid contamination with the A260/A280 ratios exceeding 1.6 and A260/A230 ratios reaching 1.8. Usually, additional purification steps such as agarose gel electrophoresis, gel permeation chromatography, or affinity chromatography were needed to get PCR-amplifiable DNA, but the DNA obtained using this protocol could directly be used to PCR-amplification and restriction enzyme digestion. Just like purity of DNA template, lower DNA yield also appears to introduce a bias towards lower community diversity. In this study compared the purified DNA the direct DNA reveals higher microbial community diversity assessed by denaturing gradient gel electrophoresis(DGGE) of amplified V3 region of 16S rDNA.
Department of Microbiology, College of Life Science, Nanjing Agricultural University, Nanjing 210095, China.
Li-hong
LH
He
Yong
Y
Zhao
Ming-jie
MJ
Chen
Ying-jie
YJ
Pan
D004269:DNA, Bacterial / Q000302:isolation & purification*; D005308:Fertilizers / Q000382:microbiology*; D006812:Humic Substances / Q000032:analysis; D016133:Polymerase Chain Reaction / Q000379:methods*; D012988:Soil Microbiology
D004269:DNA, Bacterial; D005308:Fertilizers; D006812:Humic Substances