为了获得完整、高纯度的竹林土壤微生物总DNA,采用改良的十二烷基硫酸钠-高盐抽提法对5种竹林土壤进行DNA提取,通过DNA凝胶回收试剂盒纯化粗提的DNA,利用细菌16 S rDNA基因的V3区引物对纯化的DNA进行了聚合酶链式反应-变性梯度凝胶电泳(...为了获得完整、高纯度的竹林土壤微生物总DNA,采用改良的十二烷基硫酸钠-高盐抽提法对5种竹林土壤进行DNA提取,通过DNA凝胶回收试剂盒纯化粗提的DNA,利用细菌16 S rDNA基因的V3区引物对纯化的DNA进行了聚合酶链式反应-变性梯度凝胶电泳(PCR-DGGE)验证。结果表明,该方法所需土壤样品少,抽提的DNA片段均在23kb以上,完整性好;采用DNA凝胶回收试剂盒纯化能够有效去除粗提DNA中大部分杂质,获得高质量的DNA;以此DNA为模板进行DGGE检测所反映的微生物信息量丰富。展开更多
To obtain pure DNA directly from some complex forest soils are still very difficulty at present,though many methods even commercial kits have been attempted.This paper reports an economic and efficient method for furt...To obtain pure DNA directly from some complex forest soils are still very difficulty at present,though many methods even commercial kits have been attempted.This paper reports an economic and efficient method for further purifying crude DNA extracted from forest soils with two steps.First,the crude DNA was dissolved using the extraction buffer,which removed the debris by chloroform-isoamyl alcohol,and then reprecipitated the DNA by isopropanol;second,the recovered DNA was further purified with silica spin column.Results show that 82-91% of the humic acids was removed by step one.The remaining humic acids could be completely effaced through the second step.The recovered DNA following this protocol was quite pure and ready for sensitive conventional PCR reactions.This is an economic,efficient,and timesaving method.Moreover,crude DNA extracted by other methods can be also further purified with this new way.展开更多
文摘为了获得完整、高纯度的竹林土壤微生物总DNA,采用改良的十二烷基硫酸钠-高盐抽提法对5种竹林土壤进行DNA提取,通过DNA凝胶回收试剂盒纯化粗提的DNA,利用细菌16 S rDNA基因的V3区引物对纯化的DNA进行了聚合酶链式反应-变性梯度凝胶电泳(PCR-DGGE)验证。结果表明,该方法所需土壤样品少,抽提的DNA片段均在23kb以上,完整性好;采用DNA凝胶回收试剂盒纯化能够有效去除粗提DNA中大部分杂质,获得高质量的DNA;以此DNA为模板进行DGGE检测所反映的微生物信息量丰富。
基金supported by Project of National Science Foundation of China (30870419, 40971151)the National Basic Research Program of China (2008CB418004)the Open Research Foundation of Key Laboratory for Modern Sylvicultural Technology of Zhejiang Province (200605), Zhejiang Forestry College,China
文摘To obtain pure DNA directly from some complex forest soils are still very difficulty at present,though many methods even commercial kits have been attempted.This paper reports an economic and efficient method for further purifying crude DNA extracted from forest soils with two steps.First,the crude DNA was dissolved using the extraction buffer,which removed the debris by chloroform-isoamyl alcohol,and then reprecipitated the DNA by isopropanol;second,the recovered DNA was further purified with silica spin column.Results show that 82-91% of the humic acids was removed by step one.The remaining humic acids could be completely effaced through the second step.The recovered DNA following this protocol was quite pure and ready for sensitive conventional PCR reactions.This is an economic,efficient,and timesaving method.Moreover,crude DNA extracted by other methods can be also further purified with this new way.
