Comparison of different methods for the isolation and purification of total community DNA from soil.
The efficiency and reproducibility of DNA extraction from soil was tested for variations in lytic and purification treatments and their effect on yield and purity of DNA. The extraction yield was improved by increasing the concentration of EDTA or monovalent ions in isolation buffers, by the introduction of mechanical lysis treatments, and by the use of ethanol precipitation in place of PEG precipitation. Purity was improved using buffers with decreasing concentration of EDTA or by reducing the ionic strength of the buffer, and by all mechanical treatments. No lytic treatment was efficient on its own, the highest purity was achieved using Crombach buffer and a combination of bead-beating with lysozyme and SDS lysis followed by potassium acetate and PEG precipitation, phenol/chloroform purification, isopropanol precipitation, and spermine-HCl precipitation. Sonication sheared the DNA more than bead-beating. Lysozyme and SDS lysis without any mechanical treatments allowed isolation of larger fragments (40-90 kb). Denaturing gradient gel electrophoresis analysis of DNA isolated using a range of lytic treatments revealed alterations in band patterns which might reflect differences in the efficiency of lytic treatments.
Krsek
M
M
Department of Biological Sciences, University of Warwick, Coventry, UK.
Wellington
E M
EM
D004247:DNA / Q000032:analysis / Q000302:isolation & purification*; D017753:Ecosystem; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D014644:Genetic Variation; D009113:Muramidase; D016133:Polymerase Chain Reaction; D012967:Sodium Dodecyl Sulfate; D012987:Soil / Q000032:analysis*; D012988:Soil Microbiology
D012987:Soil; D012967:Sodium Dodecyl Sulfate; D004247:DNA; D009113:Muramidase