一种采用阴离子交换柱快速纯化<em>Taq</em> DNA 聚合酶的方法

余彦; 李程勋; 陈树清; 叶长亮; 黄力坤; 许铁城; 林文雄

一种采用阴离子交换柱快速纯化Taq DNA 聚合酶的方法

福建农林大学生命科学学院农业生态研究所,福建福州 350002

基金项目:

国家自然科学基金项目(31271670)

详细信息

通讯作者:
林文雄(1957-),男,博士生导师,研究方向:作物生理与分子生态学(E-mail: lwx@fjau.edu.cn)

中图分类号: Q 784
计量
- 文章访问数: 2260
- HTML全文浏览量: 155
- PDF下载量: 293
出版历程
- 收稿日期: 2012-01-11
- 修回日期: 2012-05-18
- 刊出日期: 2012-07-29

Rapid Purification of Taq DNA Polymerase Using Anion-exchange Column

Institute of Agroecology/School of Life Sciences,Fujian Agriculture and Forestry University, Fuzhou,Fujian 350002,China

摘要

摘要: 通过37℃恒温振荡培养含有TaqDNA聚合酶基因的E.coli菌株,并用IPTG诱导该基因表达获得TaqDNA聚合酶蛋白,利用40%硫酸铵沉淀该蛋白后溶解于storage buffer中。此法获得的TaqDNA聚合酶带负电荷,因此采用阴离子交换柱纯化蛋白。试验结果表明,此法与传统的透析方法相比能快速地去除生物小分子杂质,同时去除透析方法无法除去的杂蛋白;既能保证TaqDNA聚合酶的生物学活性,同时能缩短纯化时间、提高TaqDNA聚合酶的纯度。以土壤微生物DNA、水稻cDNA为模板进行PCR扩增并对扩增产物进行测序,结果显示纯化后的TaqDNA聚合酶具有较高的扩增效率和保真性。
- Taq DNA聚合酶 /
- 硫酸铵 /
- 阴离子交换柱
Abstract: In this study, the Escherichia coli strain contains the gene encoding Taq DNA Polymerase was shaking-cultured in constant temperature incubator shaker at 37℃ for synthesizing of the Polymerase, using IPTG as an inducer.The enzyme was then precipitated by 40% ammonium sulfate.Taq DNA Polymerase obtained in this way was negatively charged, and anion-exchange column was used to purify the protein. It was found that this performance can remove the unneeded small biological molecules more quickly than the traditional method of dialysis; meanwhile, it not only keeps biological activities of Taq DNA Polymerase, but also shortens time of purification and improves pure degree of Taq DNA Polymerase. The rice cDNA,rice genomic DNA and soil microorganism DNA were used as template to PCR amplification and the PCR products were sequenced. The results showed that the polymerase had great activity and fidelity.
- Taq DNA polymerase /
- ammonium sulfate /
- anion-exchange column

HTML全文

参考文献(1)

BROCK T D.The value of basic research: discovery of Thermus aquaticus and other extreme thermophiles[J].Genetics,1997,146( 4) : 1207-1210.

施引文献

资源附件(0)

计量

文章访问数: 2260
HTML全文浏览量: 155
PDF下载量: 293
被引次数: 0

一种采用阴离子交换柱快速纯化Taq DNA 聚合酶的方法

通讯作者: 林文雄(1957-),男,博士生导师,研究方向:作物生理与分子生态学(E-mail: lwx@fjau.edu.cn)

计量

出版历程

Rapid Purification of Taq DNA Polymerase Using Anion-exchange Column

计量

出版历程

目录

通讯作者:
林文雄(1957-),男,博士生导师,研究方向:作物生理与分子生态学(E-mail: lwx@fjau.edu.cn)