A strategy for optimizing quality and quantity of DNA extracted from soil.
The efficiency of a bead beating method was studied in detail with regard to a variety of factors including beating time and speed, volume and temperature of the buffer, as well as amount and type of beads employed. The results presented here reveal that all of these parameters have a significant effect on yield and quality of DNA extracted from soils. Precise adjustment of extraction conditions allows for significantly higher yields of high quality DNA from soils than previously reported. We further evaluated the effect of the extraction conditions on the apparent soil microbial community structures, as observed by polymerase chain reaction (PCR) and RFLP. Differences in the fingerprints of DNA extracted under different conditions suggest that results could be biased when using gentle extraction procedures. Based on multiple subsequent extractions using very harsh extraction conditions, we propose a protocol for the quantification of the total DNA content in soils. Extractions from six soils of different texture and chemical characteristics with selected bead beating protocols revealed that the quality (fragment size and purity) of the extracted DNA was generally very good, but also depended on the soil characteristics. While a single, general protocol for optimal DNA recovery from all soils cannot be given, this study provides detailed guidelines on how to optimize the general method to obtain optimal DNA from individual soils.
Bürgmann
H
H
Swiss Federal Institute of Technology (ETH-Zürich), Institute of Terrestrial Ecology, Soil Biology, Grabenstrasse 3, CH-8952, Schlieren, Switzerland. buergmann@ito.umnw.ethz.ch
Pesaro
M
M
Widmer
F
F
Zeyer
J
J
D002021:Buffers; D004247:DNA / Q000032:analysis / Q000302:isolation & purification*; D004269:DNA, Bacterial / Q000032:analysis / Q000302:isolation & purification; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D012987:Soil / Q000032:analysis*; D012988:Soil Microbiology; D013696:Temperature
D002021:Buffers; D004269:DNA, Bacterial; D012987:Soil; D004247:DNA