Preparation method of T vector for T-A cloning

Abstract

The invention discloses a method for preparing for T carrier used in T-A clone, which chooses a normal plasmid with high copy quantity as table to design four pairs of primer and uses point discontinuity to construct two types of differential discretion plasmid in the order advance sequence; it separately constructs TTTTAAA and TTTAAAA sequence box on the polyclonal point of the two types of discretion plasmid and leads two different enzyme cutting points BamHI and HindIII; it separately leads the discretion plasmids in the bacteria to augment and uses the inner cutting enzyme of the identifying TTTAAA sequence to cut, mix, degenerate and compound the augmented plasmid; it dose enzyme cutting to the reacted mixture, dispels the parental generation molecule to separate the T carrier. It adopts special biology compositing technique to produce the T carrier.

Description

technical field [0001] The invention relates to a method for constructing a plasmid vector in the field of genetic engineering, in particular to a method for preparing a T vector for T-A cloning by using a unique biosynthesis method. Background technique [0002] T-A cloning method (Clark, J.M.Novel non-templated nucleotide addition reactions catalyzed by procaryotic and eucaryotic DNA polymerases (new prokaryotic or eukaryotic DNA polymerase catalyzed non-template-dependent nucleic acid addition reactions). Nucleic Acids Res.1988, 16, 9677-9686; Marchuk, D., Drumm, M., Saulino, A. and Collins, F.S. Construction of T-vectors, a rapid and general system for direct cloning of unmodified PCR product (construction of T-vectors, a rapid and general A system for direct cloning of original PCR products). Nucleic Acids Res.1991, 19, 1154; Holton, T.A.and Graham, M.W.A simple and efficient method for direct cloning of PCR products using ddT-tailed vectors (a di...

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Problems solved by technology

Now due to the strict quality control of the manufacturer, its efficiency has also been greatly improved, and it can meet the routine cloning operation. However, due to its many operation steps, the quality is not easy to control. At present, it is only used in several common vectors, and it must be performed by a professional company. to produce, so the price is more expensive

The third method is currently also used in the preparation of T vectors, but there is a potential problem in the construction of T vectors with endonucleases. Partially digested pre-T vectors are mixed in T vectors, which will lead to excessive background of non-recombinant transformants. high

In addition, the T vector produced by this method seems to be natural, but it is not widely used in practice, nor is it adopted by many biological companies, except that the endonuclease is too expensive, or there is In addition to too many recognition sites, a more important reason may be that these enzymes are difficult to use for ligation after cleaving DNA. For example, the DNA fragments produced by Ahd I and Xcm I have a base protruding at the 3′ end, which is even larger than Flat ends are more difficult to connect (http: / / www.neb-china.com / cn)

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