目的:探讨DNA提取方法优化后对转基因小鼠基因分型结果的影响,并比较分析与业内常用试剂盒基因型鉴定结果的差异。方法:在团队前期专利基础上,改进了DNA裂解液的配方和实验流程,并以新型转录因子Musculin和增强型绿色荧光蛋白(enhanced ...目的:探讨DNA提取方法优化后对转基因小鼠基因分型结果的影响,并比较分析与业内常用试剂盒基因型鉴定结果的差异。方法:在团队前期专利基础上,改进了DNA裂解液的配方和实验流程,并以新型转录因子Musculin和增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)转基因小鼠为例,对基因分型结果进行了比较研究和验证。结果:DNA提取时现配裂解液配方为100μL 0.025 N NaOH工作液、160μL 0.5 M EDTA工作液和40 mL超纯水;每个样本加入180μL裂解液,100℃30 min。与相关领域常用的基因分型试剂盒相比,该DNA提取优化方案能够在确保鉴定准确的前提下有效缩短基因分型时间,而且裂解液配置方法简单,反应次数多,步骤更简便,可大幅降低试剂成本和工作量。结论:本研究提供1种适用于转基因小鼠基因分型的经济、简单、可靠的DNA提取技术,具有较好的应用和推广价值。展开更多
A new method CTAB-silica for DNA extraction and purification from the leaves and buds of Castanea mollissima and Ginkgo biloba was tested. The method is based on the silica-based purification protocol developed by Bo...A new method CTAB-silica for DNA extraction and purification from the leaves and buds of Castanea mollissima and Ginkgo biloba was tested. The method is based on the silica-based purification protocol developed by Boom et al. (1990). By modifying the protocol, plant genome DNA could be extracted easily from dormant buds, mature leaves, and other parts of plant. Our results showed that the purified DNA was of high purity and could be analyzed by PCR. Furthermore, this CTAB-silica method took much less time for a successful DNA purification process compared to the traditional methods (CTAB and SDS). By our method, the suitable DNA can be extracted and purified from over 10 plant samples by one person in an hour.展开更多
AIM: To develop a simple and convenient method for extracting genomic DNA from intestinal microflora for en- terobacterial repetitive intergenic consensus (ERIC)-PCR detection. METHODS: Five methods of extracting bact...AIM: To develop a simple and convenient method for extracting genomic DNA from intestinal microflora for en- terobacterial repetitive intergenic consensus (ERIC)-PCR detection. METHODS: Five methods of extracting bacterial DNA, including Tris-EDTA buffer, chelex-100, ultrapure water, 2% sodium dodecyl sulfate and 10% Triton-100 with and without sonication, were compared with the commercial fecal DNA extraction kit method, which is considered as the gold standard for DNA extraction. The comparison was based on the yield and purity of DNA and the indexes of the structure and property of micro-organisms that were reflected by ERIC-PCR. RESULTS: The yield and purity of DNA obtained by the chelex method was similar to that obtained with the fecal DNA kit. The ERIC-PCR results obtained for the DNA extracted by the chelex method and those obtained for DNA extracted with the fecal DNA kit were basically the same.CONCLUSION: The chelex method is recommended for ERIC-PCR experiments in view of its simplicity and cost- effectiveness; and it is suitable for extracting total DNA from intestinal micro-organisms, particularly for handling a large number of samples.展开更多
文摘目的:探讨DNA提取方法优化后对转基因小鼠基因分型结果的影响,并比较分析与业内常用试剂盒基因型鉴定结果的差异。方法:在团队前期专利基础上,改进了DNA裂解液的配方和实验流程,并以新型转录因子Musculin和增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)转基因小鼠为例,对基因分型结果进行了比较研究和验证。结果:DNA提取时现配裂解液配方为100μL 0.025 N NaOH工作液、160μL 0.5 M EDTA工作液和40 mL超纯水;每个样本加入180μL裂解液,100℃30 min。与相关领域常用的基因分型试剂盒相比,该DNA提取优化方案能够在确保鉴定准确的前提下有效缩短基因分型时间,而且裂解液配置方法简单,反应次数多,步骤更简便,可大幅降低试剂成本和工作量。结论:本研究提供1种适用于转基因小鼠基因分型的经济、简单、可靠的DNA提取技术,具有较好的应用和推广价值。
文摘A new method CTAB-silica for DNA extraction and purification from the leaves and buds of Castanea mollissima and Ginkgo biloba was tested. The method is based on the silica-based purification protocol developed by Boom et al. (1990). By modifying the protocol, plant genome DNA could be extracted easily from dormant buds, mature leaves, and other parts of plant. Our results showed that the purified DNA was of high purity and could be analyzed by PCR. Furthermore, this CTAB-silica method took much less time for a successful DNA purification process compared to the traditional methods (CTAB and SDS). By our method, the suitable DNA can be extracted and purified from over 10 plant samples by one person in an hour.
基金The National Science Technology Pillar Program, 2007Z06-017Program for New Century Outstanding Talents from Universities, NCET-04-0906/NCET-06-0818+1 种基金Sichuan Provincial Basic Research Program, 04JY029-006-1/04JY021-100/07JY029-017Program for Key Disciplines Construction of Sichuan Province, SZD0418
文摘AIM: To develop a simple and convenient method for extracting genomic DNA from intestinal microflora for en- terobacterial repetitive intergenic consensus (ERIC)-PCR detection. METHODS: Five methods of extracting bacterial DNA, including Tris-EDTA buffer, chelex-100, ultrapure water, 2% sodium dodecyl sulfate and 10% Triton-100 with and without sonication, were compared with the commercial fecal DNA extraction kit method, which is considered as the gold standard for DNA extraction. The comparison was based on the yield and purity of DNA and the indexes of the structure and property of micro-organisms that were reflected by ERIC-PCR. RESULTS: The yield and purity of DNA obtained by the chelex method was similar to that obtained with the fecal DNA kit. The ERIC-PCR results obtained for the DNA extracted by the chelex method and those obtained for DNA extracted with the fecal DNA kit were basically the same.CONCLUSION: The chelex method is recommended for ERIC-PCR experiments in view of its simplicity and cost- effectiveness; and it is suitable for extracting total DNA from intestinal micro-organisms, particularly for handling a large number of samples.
