Method for easily and rapidly evaluating efficiency of molecular cloning system

Abstract

The invention discloses a method for easily and rapidly evaluating the efficiency of a molecular cloning system. A DNA fragment is designed, at least two multiple cloning sites are arranged on the DNAfragment, restriction enzyme digestion sites of to-be-assayed restriction endonuclease are provided in the multiple cloning sites, and the restriction enzyme digestion sites only exist in the multiple cloning sites; sticky ends which are produced in the different multiple cloning sites by the same endonuclease can be complementarily connected; for the DNA fragment undergoes restriction enzyme digestion, electrophoresis is carried out after the restriction enzyme digestion product is connected by ligase, and the formed electrophoretic band is analyzed: if the electrophoretic band exists as a large fragment or a single complete vector fragment, then the endonuclease has lower or no activity; and if the electrophoretic band exists as a small fragment, then the ligase has lower or no activity. The provided method for evaluating the efficiency of a molecular cloning system can easily and rapidly test the activity of a main ingredient (endonuclease or ligase) in a restriction enzyme digestion connecting system.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a method for simply and quickly evaluating the efficiency of a molecular cloning system. Background technique [0002] Enzyme ligation is the most basic technical solution for molecular cloning and vector construction in the field of molecular biology. The enzymes used in the system are restriction endonucleases, which cut DNA fragments to generate complementary sticky ends, pair the sticky ends, and then use T4 ligase to connect the two DNA molecules into a complete molecule. The efficiency of the enzyme digestion-ligation system directly affects the success rate of molecular cloning, but in the laboratory environment, there are always occasional problems with low vector construction efficiency in stages, and whenever such problems occur, it is always necessary to find various reasons. Insufficient endonuclease or / and ligase activity is one of the reasons ...

AI Technical Summary

Problems solved by technology

The efficiency of the enzyme digestion-ligation system directly affects the success rate of molecular cloning, but in the laboratory environment, there are always occasional problems of low vector construction efficiency in stages, and whenever such problems occur, it is always necessary to find out the reasons

Insufficient endonuclease or / and ligase activity is one of the reasons for such problems, but molecular experiments are an experimental process invisible to the naked eye, and the root cause of the problem can only be checked through various tests

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