有酷友提出对土壤DNA提取方法的讨论，故特设此帖，欢迎大家提供自己使用比较成功的方法，经验，采用的试剂盒及其效果效率和性价比等。

如果大家手头有相关文献，亦可作为附件贴出，并介绍其主要内容。

曾经看过周集中教授有一篇关于土壤微生物DNA提取方法的论文，试过了，效果不错。

我现在正在做这个呢，我用的试剂盒提，可是做了6次都没有，我就奇怪的很啊！

sdj83酷友选择目前这个试剂盒的原因是什么呢？和 其他的比较过了么？
提取是完全按照使用手册么？样品的来源怎样？
这些都可以和大家分享一下

同问  ！
粘篇文章 是用DGGE法做土壤中微生物的分子生态学的

[ 本帖最后由 whengqiang 于 07-12-3 02:11 编辑 ]

我用周集中的方法能提出DNA但在氯仿纯化过程中出现一层比水还轻的黄色物质，不知是什么

先附上些文献，待随后整理。

A strategy for optimizing quality and quantity of DNA extracted from soil

Helmut Bu¨rgmann , Manuel Pesaro, Franco Widmer, Josef Zeyera
Swiss Federal Institute of Technology(ETH-Zu¨rich), Institute of Terrestrial Ecology, Soil Biology, Grabenstrasse 3,CH-8952 Schlieren, Switzerland
Swiss Federal Research Station for Agroecology and Agriculture(FAL Reckenholz), CH-8046 Zu¨rich, Switzerland

Abstract

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 reactionŽPCR.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 qualityŽfragment 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.

[ 本帖最后由 Jack 于 07-12-11 23:35 编辑 ]

Comparisons of extraction and purification methods of soil microorganism DNA from rhizosphere soil

JIA Xia, HAN Shi-jie, ZHAO Yong-hua, ZHOU Yu-mei2

1 Environmental Science and Engineering College, Chang’an University, Xi’an 710054, P. R. China
2Institute of Applied Ecology of the Chinese Academy of Sciences, Shenyang 110016, P. R. China
3 College of Earth Science and Land Resource Management, Chang’an University, Xi’an 710054, P. R. China

Abstract:
Microorganism DNA of rhizosphere soil from Pinus koraiensis and Pinus sylvestriformis were extracted by proteinase K based on SDS method, CTAB method, PVP (polyvinylpolypyrrolidone) method, and freezing and thawing method and the crude DNA from rhizosphere soil were purified by dialysis method, silver beads absorption method, and squeezing DNA gel method. The results of different extracting and purifying methods were compared and evaluated. Results indicated that the best method of extraction for microorganism DNA in rhizosphere soil was proteinse K based on SDS method with high salt concentration of 1.0% (w/v) NaCl, which could effectively eliminate humic acids and other impurities. The dialysis method was suitable to purify DNA from rhizosphere soil because of effectively removing brown matters and humic acids and the purified products were suited to PCR amplification. Squeezing DNA gel method was also
a good purification method with the advantage of inexpensive in cost and efficient in use.

Keywords: Extraction; Microorganism DNA; Pinus koraiensis; Pinus sylvestriformis; Purification; Rhizosphere soil

Diversity of Basidiomycetes in Michigan Agricultural Soils


Michael D. J. Lynch and R. Greg Thorn

Department of Biology, University of Western Ontario, London, Ontario, Canada N6A

We analyzed the communities of soil basidiomycetes in agroecosystems that differ in tillage history at the
Kellogg Biological Station Long-Term Ecological Research site near Battle Creek, Michigan. The approach
combined soil DNA extraction through a bead-beating method modified to increase recovery of fungal DNA,
PCR amplification with basidiomycete-specific primers, cloning and restriction fragment length polymorphism screening of mixed PCR products, and sequencing of unique clones. Much greater diversity was detected than was anticipated in this habitat on the basis of culture-based methods or surveys of fruiting bodies. With “species” defined as organisms yielding PCR products with >99% identity in the 5
650 bases of the nuclear large-subunit ribosomal DNA, 241 “species” were detected among 409 unique basidiomycete sequences recovered. Almost all major clades of basidiomycetes from basidiomycetous yeasts and other heterobasidiomycetes through polypores and euagarics (gilled mushrooms and relatives) were represented, with a majority from the latter clade. Only 24 of 241 “species” had 99% or greater sequence similarity to named reference sequences in GenBank, and several clades with multiple “species” could not be identified at the genus level by phylogenetic comparisons with named sequences. The total estimated “species” richness for this 11.2-ha site was 367 “species” of basidiomycetes. Since >99% of the study area has not been sampled, the accuracy of our diversity estimate is uncertain. Replication in time and space is required to detect additional diversity and the underlying community structure.

DNA Extraction from Soils: Old Bias for New Microbial Diversity Analysis Methods

F. MARTIN-LAURENT,* L. PHILIPPOT, S. HALLET, R. CHAUSSOD, J. C. GERMON, G. SOULAS, AND G. CATROUX

UMR INRA MS Geosol, CMSE-INRA, 21034 Dijon Cedex, France

The impact of three different soil DNA extraction methods on bacterial diversity was evaluated using
PCR-based 16S ribosomal DNA analysis. DNA extracted directly from three soils showing contrasting physicochemical properties was subjected to amplified ribosomal DNA restriction analysis and ribosomal intergenic spacer analysis (RISA). The obtained RISA patterns revealed clearly that both the phylotype abundance and the composition of the indigenous bacterial community are dependent on the DNA recovery method used.  In addition, this effect was also shown in the context of an experimental study aiming to estimate the impact on soil biodiversity of the application of farmyard manure or sewage sludge onto a monoculture of maize  for 15 years.