Method for high-throughput amplifying plasmid for sequencing

Abstract

The invention discloses a method for high-throughput amplifying plasmids for sequencing, belonging to the technical field of plasmid amplification. The method comprises the following steps: uniformly mixing a 10*phi29 DNA polymerase buffer liquid, an Hexamer primer, a sample to be tested and deionized water, heating for 3 minutes at 95 DEG C, and putting on ice for cooling, thereby obtaining a reaction liquid (I); adding dNTP, 100*BSA and phi29 DNA polymerase to the reaction liquid (I), uniformly mixing, culturing for 8 hours at 30 DEG C, and heating for 10 minutes at 65 DEG C, thereby obtaining a reaction liquid (II); and diluting the reaction liquid (II) for 3-6 times with sterilized deionized water, thereby obtaining the DNA for sequencing. As a mixed plasmid library or single plasmid transformed escherichia coli bacterial colony can be used as a template, the method has the advantages that the time for large-scale plasmid extraction is shortened, and the labor consumption is reduced.

Description

technical field [0001] The invention belongs to the technical field of plasmid amplification, and in particular relates to a high-throughput amplification plasmid method for sequencing. Background technique [0002] In the process of conventional plasmid sanger sequencing, the general method is to prepare the sequencing template. The plasmid is extracted by overnight culture enrichment and traditional alkaline lysis method. The process of shaking the bacteria and extracting the plasmid takes at least 16 hours. If the plasmid is extracted with low copy The yield and quality are relatively low, which directly affects the sequencing results. If a large number of plasmids are extracted (more than 100 samples), it will take longer and consume more manpower and cost. Contents of the invention [0003] In order to quickly obtain a large number of plasmids and avoid large-scale extraction of plasmids, rolling circle amplification (Rolling circle amplification, RCA) technology can...

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Problems solved by technology

[0002] In the process of conventional plasmid sanger sequencing, the general method is to prepare the sequencing template. The plasmid is extracted by overnight culture enrichment and traditional alkaline lysis method. The process of shaking the bacteria and extracting the plas

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