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Method for constructing BAC subclone library

A construction method and fragmentation technology, applied in biochemical equipment and methods, microbial measurement/testing, fermentation, etc., can solve the problems of high cost, affecting the quality of subcloning library, DNA pollution, etc., and achieve the effect of simple operation

Inactive Publication Date: 2006-12-27
CHINA AGRI UNIV
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  • Summary
  • Abstract
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AI Technical Summary

Problems solved by technology

[0006] The technical problem to be solved by the present invention is: the existing methods for constructing BAC subcloning library are complex and cumbersome, expensive, and prone to DNA contamination, which affects the quality of the subcloning library

Method used

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Examples

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Embodiment Construction

[0016] The present invention is explained and illustrated through the construction example of the maize BAC subcloning library below, but the protection scope of the present invention is not limited by the specific examples. It can be foreseen that the construction of BAC subclone libraries from different sources can be realized by referring to this embodiment.

[0017] (1) Extraction of BAC DNA

[0018] 1. Add 200 μl 2×YT (containing 12.5 μg / ml chloramphenicol) medium to a 1.5ml tube, inoculate the corresponding BAC strain, and incubate at 37°C and 150rpm for 6hr;

[0019] 2. Add 50ml of 2×YT (containing 12.51μg / ml of chloramphenicol) medium into a 250ml Erlenmeyer flask, take 50μl of the culture from No. 1, and culture it at 37°C and 250rpm for 14hr;

[0020] 3. Use a 50ml centrifuge tube and centrifuge at 6500rpm for 10min at 4°C to collect the bacteria;

[0021] 4. Suck off the culture medium, centrifuge at 6500rpm at 4°C for 1min to remove the residual liquid, and obtai...

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Abstract

This invention provides a kind of improved construction method of BAC subclone base. The processing steps are: extraction and purification of BAC DNA, the fragememtation and end trimming of BAC DNA; the recollection of target fragment and the attachment of vector; the transformation of attachment. For example, the BAC subclone base built by corn is with more than 1500 clones. The empty clone and coliform DNA pollution rate is lower than 5%. The inserted fragment is 3kb in average. This method is easy, cheap, fast and effective.

Description

1. Technical field: [0001] The invention relates to the field of biotechnology, in particular to the preparation of BAC subclones. 2. Background technology: [0002] Whole-genome sequencing of organisms is of extraordinary significance. A large amount of genetic information can be obtained through the analysis of whole-genome sequences, laying the foundation for functional genomics and comparative genomics research. Determining the genome sequence of an organism, identifying the biological function of the sequence, and studying the evolution law of an organism have become a hot topic in the field of life science research. [0003] Clonal walking is one of the commonly used sequencing methods. The basic steps include: building a BAC library of large genomic DNA fragments; using high-density genetic maps and BAC clone fingerprints to locate BAC clones on chromosomes, and constructing a genome-wide physical map based on BAC clones; subcloning BAC clones Library construction; ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12P19/34
Inventor 徐明良田秀红李建生戴景瑞郑德刚杨琴
Owner CHINA AGRI UNIV
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