Abstract
Novel directional cloning and expression vectors were developed for blunt-end ligation of PCR products that are suitable for high-throughput cloning and simplifying the screening procedure. The PCR products, without further processing, are cloned into vectors digested with SchI and, following transformation, the desired recombinants give typical blue colonies on selectable plates. The principle of this selection strategy is that the construction also generates a full-length ideal lacO gene. To the best of our knowledge, this is the first time that this lacO reconstruction strategy has been applied in the selection of recombinants.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Aslanidis C, de Jong PJ (1990) Ligation-independent cloning of PCR products (LIC-PCR). Nucleic Acids Res 18:6069–6074
Cranenburgh RM, Lewis KS, Hanak JA (2004) Effect of plasmid copy number and lac operator sequence on antibiotic-free plasmid selection by operator-repressor titration in Escherichia coli. J Mol Microbiol Biotechnol 7:197–203
Datsenko KA, Wanner BL (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA 97:6640–6645
Guo B, Bi Y (2002) Cloning PCR products-an overview. Methods Mol Biol 192:111–119
Hilson P (2006) Cloned sequence repertoires for small- and large-scale biology. Trends Plant Sci 11:133–141
Inoue H, Nojima H, Okayama H (1990) High efficiency transformation of Escherichia coli with plasmids. Gene 96:23–28
James P, Quadroni M, Carafoli E, Gonnet G (1994) Protein identification in DNA databases by peptide mass fingerprinting. Protein Sci 3:1347–1350
Li MZ, Elledge SJ (2005) MAGIC, an in vivo genetic method for the rapid construction of recombinant DNA molecules. Nat Genet 37:311–319
Li J, Li C, Xiao W, Yuan D, Wan G, Ma L (2008) Site-directed mutagenesis by combination of homologous recombination and DpnI digestion of the plasmid template in Escherichia coli. Anal Biochem 373:389–391
Liu VW, Nagley P, Ngan HY (2000) One-step directional cloning of blunt-ended polymerase chain reaction products. Anal Biochem 287:349–352
Marchuk D, Drumm M, Saulino A, Collins FS (1991) Construction of T-vectors, a rapid and general system for direct cloning of unmodified PCR products. Nucleic Acids Res 19:1154
Shuman S (1994) Novel approach to molecular cloning and polynucleotide synthesis using vaccinia DNA topoisomerase. J Biol Chem 269:32678–32684
Studier FW (2005) Protein production by auto-induction in high density shaking cultures. Protein Expr Purif 41:207–234
Thao S, Zhao Q, Kimball T, Steffen E, Blommel PG, Riters M, Newman CS, Fox BG, Wrobel RL (2004) Results from high-throughput DNA cloning of Arabidopsis thaliana target genes using site-specific recombination. J Struct Funct Genomics 5:267–276
Acknowledgments
The authors thank BL Wanner for donating the plasmid pKD20. This work was supported by grants from the China National Human Liver Proteomics Project (2004BA711A19) and the China National High-Tech 863 Program (2006AA02A310).
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Wu, Q., Zhong, X., Zhai, C. et al. A series of novel directional cloning and expression vectors for blunt-end ligation of PCR products. Biotechnol Lett 32, 439–443 (2010). https://doi.org/10.1007/s10529-009-0166-3
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10529-009-0166-3