Construction of a Zero-background T-vector for Cloning PCR Products

LÜ Xin; CHEN Li-hua; MA Zhi-peng; LI Xiao-lin; LI Yue-ren

doi:10.19303/j.issn.1008-0384.2017.03.016

Volume 32 Issue 3

May 2017

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Article Navigation > Fujian Journal of Agricultural Sciences > 2017 > 32(3): 312-316

LÜ Xin, CHEN Li-hua, MA Zhi-peng, LI Xiao-lin, LI Yue-ren. Construction of a Zero-background T-vector for Cloning PCR Products[J]. Fujian Journal of Agricultural Sciences, 2017, 32(3): 312-316. doi: 10.19303/j.issn.1008-0384.2017.03.016

Citation:

LÜ Xin, CHEN Li-hua, MA Zhi-peng, LI Xiao-lin, LI Yue-ren. Construction of a Zero-background T-vector for Cloning PCR Products[J]. Fujian Journal of Agricultural Sciences, 2017, 32(3): 312-316. doi: 10.19303/j.issn.1008-0384.2017.03.016

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Construction of a Zero-background T-vector for Cloning PCR Products

doi: 10.19303/j.issn.1008-0384.2017.03.016

LÜ Xin^{1, 2
,},
CHEN Li-hua^{1, 2},
MA Zhi-peng³,
LI Xiao-lin⁴,
LI Yue-ren^{1, 2
,
,}

1.
Fujian Provincial Key Laboratory for Precision Instrument Tests in Agricultural Fields, Fuzhou, Fujian 350003, China
2.
Institute of Agricultural Quality Standards and Testing Technology Research, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian 350003, China
3.
Technology Center of Xiamen Entry-exit Inspection and Quarantine Bureau, Xiamen, Fujian 361000, China
4.
Dongdu Entry-exit Inspection and Quarantine Bureau, Xiamen, Fujian 361000, China

Received Date: 2016-05-17
Rev Recd Date: 2016-10-24
Publish Date: 2017-03-28

Abstract

Abstract

This experiment aimed to improve the shortcomings of low ligation efficiency and weak positive selection in cloning PCR products with T-vector.The approach called for an addition of T-overhang to a blunt-end plasmid. Firstly, a 3'-T overhang was attached to the blunt-end pBluescript SK (+) plasmid utilizing a terminal transferase, Taq DNA polymerase. Secondly, thepBS vector was ligated by T4 DNA ligase to form thesupercoiled blunt-end pBS plasmid without a 3'-T overhang, whereas the plasmidwith a 3'-T overhang remained linear in appearance. Finally, the DNA fragment of 3 000 bp, which was pBST-vector for cloning PCR products, could be purified byagarose gel electrophoresis followed by usinga gel extraction kit. The 500 bp and 1 000 bp DNA fragments were cloned into pBS T-vector with a100% cloning efficiency. It appeared that the newly developed procedures provided a valid zero-background T-vector for cloning PCR products.
- cloning PCR products,
- zero-background,
- T-vector

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References(15)

References

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