Improvement of SLiCE Seamless Cloning Method Based on Annealing Buffer

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

Abstract

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

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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