Method for constructing cotton bacterial artificial chromosome (BAC) library by non-dark cultured leaves

A technology of dark culture and leaves, which is applied in the field of molecular biology, can solve the problems of time-consuming, narrow range of material collection, hard seed shells, etc., and achieve the effects of simple and easy acquisition, avoiding a large number of germination, and saving experimental costs

Inactive Publication Date: 2012-10-24
INST OF COTTON RES CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the traditional cotton BAC library construction method has a narrow range of materials and requires dark culture treatment of the materials, which is complex and time-consuming; the construction of the BAC library requires a large amount of materials, generally more

Method used

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  • Method for constructing cotton bacterial artificial chromosome (BAC) library by non-dark cultured leaves
  • Method for constructing cotton bacterial artificial chromosome (BAC) library by non-dark cultured leaves
  • Method for constructing cotton bacterial artificial chromosome (BAC) library by non-dark cultured leaves

Examples

Experimental program
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Effect test

Embodiment 1

[0036] Example 1 Construction of BAC library using cotton wool non-dark culture adult leaves

[0037] 1. Selection of wool cotton adult plant materials:

[0038] The non-dark cultured young leaves with normal growth, no disease and no pests were selected as experimental materials.

[0039] 2. Extraction of high molecular weight nuclear DNA:

[0040] Take 40g of leaves, quickly transfer to liquid nitrogen and grind for about 1h, then transfer the powder to 400mL pre-cooled Washbuffer (0.08M KCl, 0.01M Trizma base, 0.01M EDTANa 2 , 1mM Spermidine, 1mMSpermine, 0.5M Sucrose, 2%PVP40, 0.13%DIECA, 0.15%β-mercaptoethanol and 0.5%TritonX-100), stirred gently on ice for 15-20min, covered with two layers of gauze and two layers of microfine Filter the filter cloth into a pre-cooled 50mL centrifuge tube, centrifuge at 4°C, 3200rpm for 20min, and remove the supernatant. If there are still some green precipitates during this centrifugation, use a brush to gently brush out the green p...

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Abstract

The invention relates to the field of molecular biology and especially relates to a method for constructing a cotton bacterial artificial chromosome (BAC) library by non-dark cultured leaves. The method comprises the following steps of 1, selecting cotton leaves, 2, extracting a high-molecular weight nucleus DNA, 3, partly digesting the high-molecular weight nucleus DNA and carrying out selection twice, 4, carrying out recovery, connection and transformation of a large-fragment DNA, and 5, carrying out detection of an insert fragment. The method is suitable for construction of BAC libraries of all cotton materials which comprise cotton materials having low seed amounts and persistent cotton materials having no seed.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a method for constructing a cotton BAC library using non-dark cultured leaves. Background technique [0002] At present, most of the constructed cotton Bacterial artificial chromosome (BAC) libraries are cultivars, and the selected materials are the cotyledons of etiolated seedlings obtained from the dark culture of cotton seeds after accelerated germination. The dark culture treatment can greatly reduce the production of chloroplast and phenolic substances, reduce its influence in the DNA extraction process and make the cells easy to rupture, and it is easier to obtain purer DNA from seedlings cultured in dark. However, the traditional cotton BAC library construction method has a narrow range of materials and requires dark culture treatment of the materials, which is complex and time-consuming; the construction of the BAC library requires a large amount of materials, generally m...

Claims

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Application Information

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IPC IPC(8): C40B50/06C12N15/10C12N15/63
Inventor 高海燕王坤波马峙英刘方王省芬王春英张香娣王玉红彭仁海
Owner INST OF COTTON RES CHINESE ACAD OF AGRI SCI
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