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bac clone dna extraction method

An extraction method and centrifuge tube technology, which are applied in the fields of molecular biology, molecular genetics and cell biology, can solve the problems of DNA being easily hydrolyzed, easily polluted, and high cost, and achieve the effects of simple and stable operation, sufficient quantity, and high quality

Active Publication Date: 2020-05-22
青岛安德贝生命科技有限公司 +1
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  • Claims
  • Application Information

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Problems solved by technology

[0006] The purpose of the present invention is to overcome the problems of easy hydrolysis, small quantity, easy pollution and high cost of DNA existing in the above-mentioned large fragment DNA extraction process, and provide a method of extracting high-quality, sufficient quantity and non-toxic DNA from the large-volume BAC clone bacterial culture solution. , pollution-free, and low-cost DNA extraction technology fundamentally solves the problem of extraction and purification of large fragments of DNA from BAC clones, and at the same time provides the possibility of automatic purification of large fragments of DNA

Method used

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Embodiment

[0033] 1. Main equipment and reagent materials

[0034] Floor type centrifuge: ThermoSorval RC 6+ centrifuge and rotor F12-6x500, F13-14x50cy

[0035] Benchtop 4°C centrifuge: Eppendorf 5424R

[0036] Conventional pocket centrifuge

[0037] 37℃ incubator

[0038] 37°C Bacterial Culture Shaker

[0039] 50mL centrifuge tube: Thermo Scientific, 339653

[0040] 1.5mL Eppendorf centrifuge tube, free of DNase (deoxyribonuclease) and RNase (ribonuclease).

[0041] Ultrafiltration centrifuge tube: Merck Millipore, Amicon Ultra 0.5mL Centrif μge Filters, Ultracel 10K

[0042] LB bacterial culture solution: add the following reagents to 800mL deionized pure water, 10g Bacto-tryptone, 5gyeast extract, 10g NaCl, adjust the pH to 7.5 with NaOH, then add deionized pure water to 1 liter. High temperature disinfection for use.

[0043] Antibiotic working solution: Dilute the antibiotic powder to the required concentration according to the corresponding antibiotic requirements of BAC cl...

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Abstract

The invention relates to the field of molecular biology, molecular genetics and cytobiology, and particularly relates to an extraction and purification method for DNA (deoxyribonucleic acid). The invention adopts a magnetic bead separation and molecular sieve filtration technology, combines a protein enzyme digestion technology and invents a stable extraction method for BAC (bacterial artificial chromosome) cloned DNA. According to the stable extraction method disclosed by the invention, crude DNA precipitates are obtained by utilizing optimized suspension, pyrolysis and neutralization buffer solution; by resuspending the DNA precipitates, under proper pH and ion concentration, DNA polynucleotide macromolecule is nonspecifically and reversely combined to functional groups of a magnetic nano material; by rapid combination and cleaning steps, pollutants, such as ions, proteins, carbohydrate and the like, and impurities, such as micromolecule and the like, in the pyrolysis process are removed; and after further digestion by protease and concentration of a purification and filtering device, a purified DNA product is generated. The extraction method disclosed by the invention is simple and stable to operate, economic, efficient, non-toxic and pollution-free. The extraction method has excellent expansibility, and can implement standardized kit suit series product and full-automatic operation.

Description

technical field [0001] The invention relates to the fields of molecular biology, molecular genetics and cell biology, in particular to a method for extracting and purifying DNA. Background technique [0002] In the past ten years, molecular biology and cytogenetics have developed rapidly, and technologies involving DNA sequencing, cloning, functional analysis and identification have been widely and deeply applied in fields such as medicine, agriculture and forestry, environment, oceanography, and forensic science. BAC (bacterial artificial chromosome) cloning is currently the main method for storing and amplifying large DNA fragments, especially large genomic DNA fragments. The length of BAC clones generally ranges from 100kb to 300kb. Due to the large insert DNA fragment of the BAC clone, the copy number produced after transfection into E. coli is low. Both the existing ion-exchange column method and the manual method of extraction have some defects and difficulties that ...

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/10C12R1/19
CPCC12N15/1013
Inventor 张进平张晓春
Owner 青岛安德贝生命科技有限公司
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