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Biotinylated chromatin immunoprecipitation method and kit thereof

A technology of co-immunoprecipitation and biotinylation, applied in the field of molecular biology research, to achieve the effect of convenient use

Inactive Publication Date: 2017-12-19
TONGJI HOSPITAL ATTACHED TO TONGJI MEDICAL COLLEGE HUAZHONG SCI TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is currently no research method for the interaction between DNA and RNA. In molecular biology, the correlation between DNA and RNA is very common. How to verify the interaction between the two has always been a technical problem that needs to be solved in this field.

Method used

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  • Biotinylated chromatin immunoprecipitation method and kit thereof
  • Biotinylated chromatin immunoprecipitation method and kit thereof
  • Biotinylated chromatin immunoprecipitation method and kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: The composition of the kit and the comparison between Chinese and English

[0036] Protein A+G Agarose+Salmon Sperm DNA: Protein A+G Agarose+Salmon Sperm DNA, (manufactured by Millipore, included in both 17-295 / 17-371 kits);

[0037] Glycine Solution (10×): Glycine Solution (10×), (Beiyuntian Company, ST085);

[0038] ChIP Dilution Buffer: chromatin immunoprecipitation dilution buffer, (Millipore, 17-295 / 17-371 included in both kits);

[0039] Low Salt Immune Complex Wash Buffer: Low Salt Immune Complex Wash Buffer;

[0040] High Salt Immune Complex Wash Buffer: High Salt Immune Complex Wash Buffer;

[0041] LiCl Immune Complex Wash Buffer: LiCl immune complex wash buffer;

[0042] TE Buffer: TE buffer;

[0043] 0.5M EDTA (Beiyuntian Company, ST066);

[0044] 5M NaCl (can be prepared by yourself, weigh 292.5g NaCl, dissolve in water, dilute to 1L is 5M NaCl);

[0045] 1M Tris, pH 6.5: 1M Tris, pH 6.5 (Beijing Regen Biotechnology Co., Ltd., NR0070);

[...

Embodiment 2

[0049] Embodiment 2: Other reagents and equipment required for the experiment

[0050] Biotin-labeled small RNA (dsRNA or miRNA): (Synthesized by RiboBio);

[0051] 37% formaldehyde (formaldehyde for industrial use is basically available);

[0052] PBS: Phosphate buffer saline, (Beiyuntian Company, C0221A);

[0053] PMSF: phenylmethylsulfonyl fluoride, (Beiyuntian Company, ST505);

[0054] Tris balanced phenol (Beiyuntian Company, ST792);

[0055] Chloroform (industrial use, basically available);

[0056] 95% ethanol (basically available);

[0057] 70% ethanol (basically available);

[0058] 3M NaAc, pH5.2 (Beiyuntian Company, ST351);

[0059] Cell scraper (Corning, USA, 3008).

Embodiment 3

[0060] Embodiment three: experimental procedure

[0061] 3.1 Transfection of cells after RNA labeling with biotin

[0062] Biotinylate the small RNA (dsRNA or miRNA) of interest.

[0063] 3.2 Cultivate the cells until the time point when the target protein and genomic DNA are expected to bind, add formaldehyde, and incubate the cells

[0064] A. Prepare an appropriate amount of PBS pre-cooled in an ice bath, and 100mM PMSF, warm the SDS Lysis Buffer properly to fully dissolve the SDS in it, and mix well.

[0065]B. Cells were cultured in a 10cm cell culture dish, and the amount of cell culture medium used was 10 ml. The expected time point for the combination of target RNA and genomic DNA is generally 48-72 hours after transfection (see References 1-3 for the selection of this time point). Add an appropriate amount of formaldehyde directly to the cell culture medium, and mix gently until the final concentration of formaldehyde is 1%. This was followed by incubation at 37...

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Abstract

The invention discloses a biotinylated chromatin immunoprecipitation kit. The kit comprises the following components: protein A+G agarose / salmon sperm DNA, a glycine solution, a chromatin immunoprecipitation dilution buffer, a washing buffer, 0.5M EDTA, 5M NaCl, 1M trismetyl aminomethane with a pH value being 6.5, a SDS cracking buffer, a biotin antibody, and a RNA enzyme inhibitor. The kit has the beneficial effects that the traditional immunoprecipitation is improved, a targeting DNA-RNA compound is obtained, interacting between RNA and DNA is researched and verified, the invention provides the corresponding biotinylated chromatin immunoprecipitation kit, which is convenient for usage by scientific researchers.

Description

technical field [0001] The invention relates to the technical field of molecular biology research, in particular to a biotinylated chromatin immunoprecipitation method and a kit. Background technique [0002] The interaction between molecules is the core content of molecular biology. At present, the protein-protein interaction can be clarified by methods such as co-immunoprecipitation (Co-IP) and Pull-down, and the interaction between protein and DNA can be determined by chromatin co-immunoprecipitation ( ChIP) clearly, protein and RNA can be achieved by RNA-co-immunoprecipitation (RIP). There are corresponding kits for the above methods. However, there is currently no research method for the interaction between DNA and RNA. In molecular biology, the correlation between DNA and RNA is very common. How to verify the interaction between the two has always been a technical problem that needs to be solved in this field. [0003] Aiming at the problems in the related technolo...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N33/53
CPCC12Q1/6804G01N33/5308C12Q2563/131
Inventor 胡嘏陈忠李龙承吴焕磊李森茂
Owner TONGJI HOSPITAL ATTACHED TO TONGJI MEDICAL COLLEGE HUAZHONG SCI TECH
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