Method for remaking multiple cloning sites of known vector

Abstract

The invention discloses a method for remaking multiple cloning sites of a known vector, namely an adapter primer-PCR (Polymerase Chain Reaction) method. The method comprises the following steps of: amplifying the known DNA (Deoxyribonucleic Acid) fragment of any species by applying an adapter primer which respectively adds multiple restriction enzyme cutting sites to a 5' tail end; carrying out double enzyme cutting by using restriction enzymes positioned on both ends of the amplified fragment, and connecting to the multiple cloning sites of a vector to be remade by using a T4DNA joining enzyme; transforming a connected product into competent escherichia coli; and selecting positive clone, extracting plasmids, verifying whether the remade vector is carried with new enzyme cutting sites or not through enzyme cutting, and retaining a correct plasmid vector. The method disclosed by the invention can meet the requirements of a specific experiment by fast and conveniently adding the new enzyme cutting sites to the multiple cloning sites of the known vector, is low in cost and high in flexibility and can be applied to remaking the multiple cloning sites of the known vector.

Description

technical field [0001] The invention belongs to the technical field of cloning, and in particular relates to a method for transforming multiple cloning sites of known vectors. Background technique [0002] The directional cloning of the target sequence by double enzyme digestion is the most commonly used method in the vector construction process. This method can greatly reduce the self-ligation phenomenon of the vector itself during the recombination process and improve the construction efficiency. However, in many cases, especially when the target DNA fragment to be cloned is long, it is difficult to find two suitable restriction enzymes in the multiple cloning sites (MCS) of known vectors. location. In addition, during the double digestion process, the combination of some restriction enzymes will seriously reduce the digestion efficiency of each other or one of them, and ultimately affect the construction efficiency. Therefore, modifying the multiple cloning site of the...

AI Technical Summary

Problems solved by technology

However, because the multiple cloning site sequence in the vector is usually short, about 100bp, the yield of the digested fragments in the gel recovery is usually not high, resulting in low construction efficiency

Niu Lin et al. proposed to insert a 238bp auxiliary fragment into the MCS of the vector, then use this MCS to replace the MCS of the vector to be transformed, and finally excise the inserted auxiliary fragment to complete the transformation of the vector, but this method requires the original MCS Template sequence, resulting in less operational flexibility

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