生命经纬知识库 >>所属分类 >> DNA技术   

Thermal?Inactivation

标签: 暂无标签

顶[0] 发表评论(15) 编辑词条

A simple, reversible way to a stop restriction reaction is by adding EDTA, which chelates Mg2+ , thereby preventing catalysis. If further manipulations of the digested DNA are to be performed, the restriction endonuclease should be inactivated. Phenol/chloroform extraction and ethanol precipitation is an irreversible method for inactivation and removal of all restriction endonucleases; however, a more convenient method is thermal inactivation. Most restriction enzymes can be inactivated by incubation at 65℃ for 20min. Others remain active at 65℃ but lose their cleavage ability at a higher temperature. Even many thermophilic enzymes that show optimal activity at 50-55℃ can be inactivated at 80℃ in 20min. Information on the susceptibility of Fermentas restriction endonucleases to thermal inactivation and the temperature required to achieve this is presented in the table  "Reaction Conditions for Restriction Endonucleases ".

Conditions. 10-100 units of enzyme were incubated at optimal reaction conditions for 1 hour with 1µg of appropriate DNA substrate (usually plasmid DNA containing at least one recognition site for the restriction endonuclease tested). After incubation at 65℃ or 80℃ for 20min, 1µg of control DNA (lambda or Ad2 DNA used in standard unit determination reaction) was added to the reaction mixture and incubation was performed at optimal temperature for a further 60min. Reaction products were analyzed by agarose gel electrophoresis. The absence of subsequent substrate cleavage was interpreted as thermal inactivation of the restriction enzyme

附件列表


→如果您认为本词条还有待完善,请 编辑词条

上一篇DNA from sera-Molecular Biology 下一篇植物总DNA的快速少量抽提(CTAB法)

词条内容仅供参考,如果您需要解决具体问题
(尤其在法律、医学等领域),建议您咨询相关领域专业人士。
0

收藏到:  

词条信息

admin
admin
超级管理员
词条创建者 发短消息   

相关词条