Constructing method of oligo-amino acid plasmid

Abstract

The invention discloses a constructing method of an oligo-amino acid plasmid, comprising the following steps of: A, designing and synthesizing two pairs of primers a, b and c, d; B, respectively performing polymerase chain reaction (PCR) on the two pairs of primers a, b and c, d with the same plasmid template; C, mixing the reaction liquids obtained in the step B, then performing denaturation and renaturation processes; D, after template digestion, directly converting into host cell escherichia coli BL21, i.e. digesting the PCR reaction product by DnpI enzyme in a reaction system of 17mL of PCR reaction product, 2mL of Tango buffer solution and 1mL of DnpI enzyme under the reaction condition of 37DEG C for 30 minutes, converting the competence of the escherichia coli BL21 by the reaction product, selecting monoclonal colonies, sequencing and identifying; and E, accurately sequencing. The method is simple, the materials and reagents are relatively cheap, and the connecting step of the blunt end ligase of a linear plasmid is omitted, thus obviously improving the connecting efficiency of plasmid oligo-amino acid mutation, and being favorable for industrial or medical scaled-up production. The success rate of sequencing is as high as 66% which is more than 5 times of that of the traditional method.

Description

technical field [0001] The invention belongs to the related field of molecular biology, relates to polymerase chain reaction, and more specifically relates to a method for constructing an oligoamino acid plasmid, which has great applications in the fields of genetic engineering, protein engineering, enzyme engineering and medical diagnosis value. Background technique [0002] Point mutation technology is a common technique in molecular biology, widely used to find functional amino acids of proteins, artificially design proteins and so on. To carry out point mutation on the plasmid, two complementary primers can be designed, the primers contain the required mutation, and the polymerase chain reaction is carried out under appropriate reaction system and conditions. The reaction product is digested and transformed into the host cell, cultured on a plate with antibiotics, and the positive single clone is picked and sequenced to determine whether it is the strain carrying the mu...

AI Technical Summary

Problems solved by technology

[0007] However, ligation of blunt ends by ligase has the disadvantages of low efficiency and error-prone

Images