基于退火缓冲液的SLiCE无缝克隆方法的改良

doi:10.13560/j.cnki.biotech.bull.1985.2019-0468

生物技术通报 ›› 2020, Vol. 36 ›› Issue (2): 214-222.doi: 10.13560/j.cnki.biotech.bull.1985.2019-0468

基于退火缓冲液的SLiCE无缝克隆方法的改良

杨东成, 蔡松, 赵筱, 王金华, 王永泽

湖北工业大学生物工程与食品学院,武汉 430068

收稿日期:2019-05-29 出版日期:2020-02-26 发布日期:2020-02-23
作者简介:杨东成,男,硕士,研究方向：工业微生物代谢工程;E-mail：yang_bio@126.com
基金资助:
国家科技支撑计划（2012BAD27B03）,湖北省自然科学基金项目（2015CFB637）,湖北工业大学博士科研启动基金项目（BSQD12143,BSQD13005）

Improvement of SLiCE Seamless Cloning Method Based on Annealing Buffer

YANG Dong-cheng, CAI Song, ZHAO Xiao, WANG Jin-hua, WANG Yong-ze

School of Food and Biological Engineering,Hubei University of Technology,Wuhan 430068

Received:2019-05-29 Published:2020-02-26 Online:2020-02-23

摘要/Abstract

摘要： 旨在建立一种在采用低感受态细胞转化的条件下完成Seamless ligation cloning extract（SLiCE）无缝克隆的方法。通过在SLiCE反应中引入变性和复性步骤来提高无缝克隆的效率。结果显示,当DNA片段经过SLiCE处理后再进行变性和复性处理,其产生的克隆子比对照多7倍左右,经过菌落PCR验证SLiCE无缝克隆的重组效率高达100%,在此条件下,采用1.7×10⁶ CFU/μg转化效率的感受态细胞转化SLiCE反应产物,不仅能够得到重组成功的克隆子,且经验证重组效率仍高达100%。采用引入变性和复性步骤的SLiCE方法,使用常用的CaCl₂方法制备的普通感受态细胞,就能得到阳性克隆子,实现无缝克隆,表明本方法能提升SLiCE技术的克隆效率和实用性。

关键词: SLiCE, 无缝克隆, 退火缓冲液

Abstract: The objective of this work is to establish a method to fulfill the Seamless Ligation Cloning Extract（SLiCE）under the condition of low efficiency competent cells. The step of denaturation and renaturation was introduced into SLiCE reaction to improve the efficiency of recombination. Results showed that denaturation and renaturation of DNA fragments using annealing buffer after SLiCE reaction resulted in about 7 times more clones than the control,and the recombination efficiency of SLiCE achieved as high as 100% by using colony PCR verification. Under this condition,the recombinant clones were obtained by transforming the SLiCE products into competent cells with 1.7×10⁶ CFU/μg transformation efficiency,and the recombination efficiency was still up to 100%. In sum,positive clones could be obtained using competent cells prepared by the routine CaCl₂ method if above optimized SLiCE method with denaturation and renaturation steps were performed. This indicates that the seamless cloning efficiency and practicability of SLiCE may be improved via the optimized method.

Key words: SLiCE, seamless cloning method, annealing buffer

杨东成, 蔡松, 赵筱, 王金华, 王永泽. 基于退火缓冲液的SLiCE无缝克隆方法的改良[J]. 生物技术通报, 2020, 36(2): 214-222.

YANG Dong-cheng, CAI Song, ZHAO Xiao, WANG Jin-hua, WANG Yong-ze. Improvement of SLiCE Seamless Cloning Method Based on Annealing Buffer[J]. Biotechnology Bulletin, 2020, 36(2): 214-222.

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基于退火缓冲液的SLiCE无缝克隆方法的改良

Improvement of SLiCE Seamless Cloning Method Based on Annealing Buffer

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