Abstract
An efficient PCR cloning method is indispensable in modern molecular biology, as it can greatly improve the efficiency of DNA cloning processes. Here, I describe the development of three vectors for TA cloning and blunt-end cloning. Specifically, pCRT and pCRZeroT were designed to improve the efficiency of TA cloning. pCRZeroT can also be used with pCRZero to facilitate blunt-end cloning using the ccdB gene. Using pCRZero and pCRZeroT and applying the Golden Gate reaction, I developed a direct PCR cloning protocol with non-digested circular vectors and PCR products. This direct PCR cloning protocol yielded colony-formation rates and cloning efficiencies that are comparable with those obtained by conventional PCR cloning with pre-digested vectors and PCR products. The three plasmids I designed are available from Addgene (https://www.addgene.org/).
Highlights
Polymerase chain reaction (PCR) is an indispensable tool for amplification of genomic DNA and transcripts to analyze their functions[1]
Vectors that have a T-overhang at the 3′-end were developed as so-called ‘T-vectors’ for cloning of deoxyadenosine triphosphate (dA)-tailed PCR products amplified by Taq DNA polymerase[7,8]
In pCRT (2,728 bp) for TA cloning, T-overhang vector for cloning of dA-tailed PCR products amplified by Taq DNA polymerase can be prepared with one step digestion by a type IIS restriction enzyme, XcmI (Fig. 1a)
Summary
Polymerase chain reaction (PCR) is an indispensable tool for amplification of genomic DNA and transcripts to analyze their functions[1]. The availability of commercially available ready-to-use T-vectors has obviated the need for very specific and complicated procedures; while the vectors are generally very convenient, they are expensive Another T-vector preparation-method uses XcmI (a well-known type IIS restriction enzyme that recognizes an asymmetric nucleotide sequence) to digest outside of this recognition sequence and generate a T-overhang for TA cloning[8]. The linearization of T-overhang of pKILPCRs by type IIS restriction enzyme AspEI (Eam1105, AhdI) lacked adequate reproducibility and the vector showed low cloning efficiency owing to the contaminant exonuclease activity in the AspEI, which led to the blunting of DNA fragments
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
