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Volume 49 Number 3




ScienceAsia 49 (2023): 305-309 |doi: 10.2306/scienceasia1513-1874.2023.007


An easy method to improve the recombinant efficiency of psi-CHECK2


XiuKai Caoa, Jie Chenga,b,c, Shan Wangc, Wei Suna,c,*

 
ABSTRACT:     The vector psi-CHECK2 is widely used to validate the interactions of microRNA with mRNA. Typical primers for recombinant psi-CHECK2 are usually designed as protective bases at 50 end of DNA (5 prime) followed by enzyme restriction sites with partial target sequences at 30 end of DNA (3 prime). However, the recombinant efficiency of psi-CHECK2 is usually not high enough to fulfill our molecular experiments. Here, we demonstrated that using ∼15 bases of each dangling ends of linearized psi-CHECK2 instead of traditional protective bases could easily improve the recombinant efficiency of psi-CHECK2. In this study, the T4 ligation products of linearized psi-CHECK2 and digested PCR products were transformed into DH5α. The results showed that compared with the conventional primers, our improved primers have better performance in the number of bacterial colonies, the positive rate of PCR amplifications of bacterial solutions of randomly selected colonies, and the recombinant efficiency confirmed by Sanger sequencing. We also co-transformed the linearized psi-CHECK2 and digested PCR products without T4 ligase treatment to estimate the effects of endogenous homologous recombination of DH5α. Indeed, endogenous homologous recombination could promote the recombinant efficiency of psi-CHECK2 but was significantly lower than that of our method (combination of improved primers and T4 ligase treatment). This method may be an alternative to the constructions of other recombinant vectors.

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a Institutes of Agricultural Science and Technology Development, Joint International Research Laboratory of Agriculture and Agri-Product Safety of Ministry of Education of China, Yangzhou University, Yangzhou 225000 China
b College of Animal Science and Technology, Northwest A&F University, Yangling 712100 China
c College of Animal Science and Technology, Yangzhou University, Yangzhou 225000 China

* Corresponding author, E-mail: dkxmsunwei@163.com

Received 24 Sep 2021, Accepted 9 Sep 2022