Pre-T carrier, preparation thereof and applications

Abstract

The invention provides a pre-T-vector, which is prepared by the following method. (1) Plasmids of resistant genes of sensitive antibiotics of escherichia coli without containing ampicillin are taken as templates to conduct PCR amplification; XcmI restriction enzyme sites are introduced into terminals of a primer 5`; and DNA fragments are obtained by amplification which contain the resistant genesof the antibiotics, and the upstream and the downstream of the DNA fragment express regulation sequences, and the two terminals of the DNA fragment contain the XcmI restriction enzyme sites. (2) The DNA fragments obtained in procedure (1) are connected with a pUCmT vector by a joining enzyme T4DNA. (3) The connected product obtained in procedure (2) is transformed into escherichia coli DH5 Alpha or JM109; and positive clones are screened, and then the pre-T-vector can be obtained. The pre-T-vector has the following advantages that vectors prepared by the pre-T-vector can be used to clone the product PCR quickly, and X-gal is not needed in the process of screening the positive clones with low negative rate.

Description

(1) Technical field [0001] The invention relates to a pre-T vector which can be used for rapid cloning of PCR products and its preparation and application. (2) Background technology [0002] PCR technology is a basic technology in molecular biology and genetic engineering research, and it is widely used in vitro to amplify known or unknown specific DNA fragments. Rapid and effective cloning of PCR products is the only way to carry out downstream experiments such as hybridization analysis, high-quality sequencing, and separation of target fragments from complex PCR products, so as to realize multiple uses of PCR products. T-vector is a new type of vector developed in recent years for direct cloning of PCR products. T-carriers are generally linear, with a 3'dT (deoxythymidine) protrusion at each end of the molecule. Using the terminal transferase activity of Taq DNA polymerase, a nucleotide, usually deoxyadenylic acid (dA), is added to the 3' end of the amplified DNA in a te...

AI Technical Summary

Problems solved by technology

This method has obvious disadvantages: first, it cannot guarantee that 100% of the ends of the carrier molecules can be added with dT, and second, it is difficult to control exactly 1 dT to be added.

This method requires the use of expensive X-ga1, and it is troublesome to prepare a blue-white screening plate

Second, if the spatial positions of the two XcmI restriction sites are relatively close, the restriction efficiency of the carrier will be affected. Even if the purity of the carrier DNA is high and the quality of the enzyme is also good, there will be a "knife" of the restriction carrier. , this linear vector cannot be ligated with PCR products, but the vector is self-ligated, resulting in the generation of negative clones