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Rapid separation and detection kit and method for fragmented crosslinked DNA

A technology for detection kits and detection methods, which is applied in the direction of biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of cumbersome separation and purification, cannot faithfully and accurately reflect the actual size of the DNA fragment of the sample to be tested, and achieve saving Sample resources, the effect of improving the success rate

Inactive Publication Date: 2016-08-10
BEIJING JIAOTONG UNIV +1
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Problems solved by technology

[0005] Because the process of ultrasonic fragmentation of chromatin DNA is irreversible, people tend to reduce the output power and prolong the ultrasonic time for optimization. Phenol chloroform) or expensive (kit) DNA fragment separation and purification, time-consuming more than 1 day, and because of the problem of recovery effect, it cannot faithfully and accurately reflect the actual size of the DNA fragment of the sample to be tested ( figure 1 )

Method used

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  • Rapid separation and detection kit and method for fragmented crosslinked DNA
  • Rapid separation and detection kit and method for fragmented crosslinked DNA

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Experimental program
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Effect test

Embodiment 1

[0027] Embodiment 1: experimental method establishment

[0028] 1. Ultrasonic Fragmentation of Chromatin DNA

[0029] step:

[0030] 1) Prepare cell lysate, transfer 10 μL of cell lysate to a 1.7 μL microcentrifuge tube (EP tube), labeled as S0;

[0031] 2) DNA fragmentation conditions: use a 1 / 16-inch probe of a Sonicator 3000 (MISONIX, Part#S3000) sonicator to enter a depth of 2-3 mm below the liquid surface of the cell lysate sample. Ultrasonic lysis of chromatin DNA in the sample, set the power of the ultrasonic instrument: 0.5W, time: 2sec on / 15sec off, a total of 8 times as a round.

[0032] Sonicate according to the above conditions, transfer 10 μL of cell lysate to 1.7mL EP tubes after each round of sonication, and mark them as S1, S2, S3, etc. respectively.

[0033] If foam appears in the sample, stop the ultrasound immediately, centrifuge the sample at 6000g for 1 min at 4°C to eliminate the foam, and continue to sonicate after the foam is eliminated.

[0034] If...

Embodiment 2

[0043] Embodiment 2: traditional method and the method contrast of the present invention

[0044] The comparison of the traditional method and the method of the present invention in aspects such as time and effect, the comparison results are shown in Table 1.

[0045] Table 1: Contrast of traditional method and method of the present invention contrast

[0046]

[0047] It can be seen from the comparison that the method of the present invention saves time and effort, and can greatly improve the success rate of ChIP.

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Abstract

The invention discloses a rapid separation and detection kit and method for fragmented crosslinked DNA. The kit includes a reagent for decrosslinking and separation extraction of fragmented DNA, and the reagent is Chelex 100. The kit can further include a protein degradation reagent, a DNA ladder marker, a sample loading buffer solution and / or an RNA degradation reagent. Chelex 100 can integrate multivalent metal ions, has high metal ion selectivity and binding force, can separate DNA and prevent DNA degradation. The rapid separation and detection method for fragmented crosslinked DNA can determine whether a to-be-detected sample needs further ultrasound or is used for a next step precipitation experiment according to the effect of rapid quality control ultrasound fragmentation, or can be used for ultrasound optimization of a plurality of to-be-detected samples so as to select optimum conditions. The kit and the method provided by the invention can be used for rapid detection of chromatin ultrasound fragmented sample quality in chromatin immunoprecipitation assay (ChIP) and other technologies, and can greatly improve the success rate of ChIP.

Description

technical field [0001] The invention relates to quality control of fragmented DNA. More specifically, it relates to a rapid separation and detection of fragmented cross-linked DNA. Background technique [0002] Chromatin immunoprecipitation assay (ChIP) is currently the only method for studying the interaction between proteins and DNA in vivo, and plays an irreplaceable role in the study of gene expression regulation. Eukaryotic genomic DNA interacts with histones to form chromatin, which is the carrier and form of genetic material. Studying the interaction between protein and DNA in chromatin is the basic way to elucidate the regulation mechanism of gene expression. [0003] The basic principle of ChIP is to immobilize DNA-protein complexes with formalin in the state of living cells, ultrasonically shear randomly to form small chromatin fragments, and then precipitate the target protein-specific antibody to specifically enrich the DNA bound to the target protein Fragment...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6806C12Q2521/537C12Q2521/327C12Q2563/173C12Q2563/149
Inventor 黄家强景坤玉林舒晔林博楠
Owner BEIJING JIAOTONG UNIV
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