Bacteriophage genome DNA (deoxyribonucleic acid) extraction kit and method

An extraction method and phage technology, which is applied in the field of bacteriophage genomic DNA extraction kits, can solve the problems of reducing the operating time of technicians, and achieve the effects of rapid extraction, high extraction yield, and eradication of interference

Active Publication Date: 2016-05-11
SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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Problems solved by technology

[0004] The extraction of phage genomic DNA is the basis for molecular technology-based research on phage; in addition, among the increasing requirements for phage genome sequencing, the most critical step also requires the extraction of high-quality genomic DNA with a certain concentration and free from host nucleic acid contamination. Bacteriophage genomic DNA extraction methods and kits can be widely used, but there is a strong demand for bacteriophage genomic DNA extraction methods and kits in the field of microbiology, and the emergence of general bacteriophage genomic DNA extraction methods and kits will be greatly reduced. Operating time of technicians, speeding up the research process in related fields

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  • Bacteriophage genome DNA (deoxyribonucleic acid) extraction kit and method
  • Bacteriophage genome DNA (deoxyribonucleic acid) extraction kit and method

Examples

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Embodiment 1

[0040] Example 1: Extracting the Genomic DNA of Vibrio alginolyticus Phage ФPE333

[0041] Prepare reagents according to the following formula:

[0042] (1) Reagent A: chloroform;

[0043] (2) Reagent B: 20mg / ml DNaseI prepared with TE buffer, stored at -20°C;

[0044] (3) Reagent C: 20mg / ml RNaseA prepared with TE buffer, stored at -20°C;

[0045] (4) Reagent D: Precipitation buffer containing 400 mg / ml polyethylene glycol-8000 (PEG-8000) and 3M NaCl, stored at room temperature;

[0046] (5) Reagent E: lysis buffer, weigh 5.8gNaCl, 1.23gMgSO 4 ·7H 2 O, prepare 50ml of 1MTris-Cl (1MTris, pH adjustment 8.0), 100ml of 0.5MEDTA, mix the above components and add ddH 2 0 to 1000ml, autoclave for 20min;

[0047] (6) Reagent F: as the main lysate, 200mg / ml sodium dodecylbenzenesulfonate (SDS);

[0048] (7) Reagent G: an auxiliary lysate, 25 mg / ml proteinase K prepared with TE buffer, stored at 4°C;

[0049] (8) Reagent H: impurity removal solution, 6MNaCl;

[0050] (9) Reage...

Embodiment 2

[0064] Example 2: Extraction of coliphage ФP1655 genomic DNA

[0065] Prepare reagents according to the following formula:

[0066] (1) Reagent A: chloroform;

[0067] (2) Reagent B: 10mg / ml DNaseI prepared with TE buffer, stored at -20°C;

[0068] (3) Reagent C: 10mg / ml RNaseA prepared with TE buffer, stored at -20°C;

[0069] (4) Reagent D: Precipitation buffer containing 300 mg / ml polyethylene glycol-8000 (PEG-8000) and 3M NaCl, stored at room temperature;

[0070] (5) Reagent E: lysis buffer, weigh 5.8gNaCl, 1.23gMgSO 4 ·7H 2 O, prepare 50ml of 1MTris-Cl (1MTris, pH adjustment 8.0), 100ml of 0.5MEDTA, mix the above components and add ddH 2 0 to 1000ml, autoclave for 20min;

[0071] (6) Reagent F: as the main lysate, 150mg / ml sodium dodecylbenzenesulfonate (SDS);

[0072] (7) Reagent G: an auxiliary lysate, 15 mg / ml proteinase K prepared with TE buffer, stored at 4°C;

[0073] (8) Reagent H: impurity removal solution, 5M NaCl;

[0074] (9) Reagent I: DNA binding bu...

Embodiment 3

[0088] Example 3: Extracting Genomic DNA of Enterobacter cloacae Phage ФPZJ02

[0089] Prepare reagents according to the following formula:

[0090] (1) Reagent A: chloroform;

[0091] (2) Reagent B: 15mg / ml DNaseI prepared with TE buffer, stored at -20°C;

[0092] (3) Reagent C: 15mg / ml RNaseA prepared with TE buffer, stored at -20°C;

[0093](4) Reagent D: Precipitation buffer containing 330 mg / ml polyethylene glycol-8000 (PEG-8000) and 3M NaCl, stored at room temperature;

[0094] (5) Reagent E: lysis buffer, weigh 5.8gNaCl, 1.23gMgSO 4 ·7H 2 O, prepare 50ml of 1MTris-Cl (1MTris, pH adjustment 8.0), 100ml of 0.5MEDTA, mix the above components and add ddH 2 0 to 1000ml, autoclave for 20min;

[0095] (6) Reagent F: as the main lysate, 180mg / ml sodium dodecylbenzenesulfonate (SDS);

[0096] (7) Reagent G: an auxiliary lysate, 20 mg / ml proteinase K prepared with TE buffer, stored at 4°C;

[0097] (8) Reagent H: impurity removal solution, 5.5M NaCl;

[0098] (9) Reagent...

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Abstract

The invention provides a bacteriophage genome DNA (deoxyribonucleic acid) extraction kit and method. The kit comprises a reagent A (chloroform), a reagent B (DNase I), a reagent C (RNase A), a reagent D (precipitation buffer solution), a reagent E (pyrolysis buffer solution), a reagent F (primary pyrolysis main solution), a reagent G (secondary pyrolysis solution), a reagent H (impurity removal solution), a reagent I (DNA combination buffer solution), a reagent J (rinsing buffer solution) and a reagent K (DNA eluate). The extraction method comprises the following steps: removal of bacterium cells and fragments, degradation of bacterium nucleic acid, bacteriophage particle precipitation, bacteriophage capsid protein structure damage and hydrolysis, impurity removal, DNA liquid separation, DNA adsorption, cleaning of adsorbed DNA and elution of adsorbed DNA. The kit and method can be widely used for extracting the bacteriophage genome DNA, and has the advantages of high extraction speed, high extracted DNA yield and high purity, and the extracted DNA can not be polluted by the host bacterium genome DNA.

Description

technical field [0001] The invention belongs to the technical field of biological nucleic acid extraction, and in particular relates to a bacteriophage genome DNA extraction kit and a method for extracting bacteriophage genome DNA using the kit. Background technique [0002] Phages are viruses that take bacteria as hosts. Like other viruses, bacteriophages are just a clump of genetic material wrapped in a protein coat, and most phages also have a "tail" that is used to inject genetic material into the host. Phages are ubiquitous organisms that often accompany bacteria. Bacteriophages are usually found in places full of bacterial communities, such as river water, soil, and animal viscera. Phages are considered to be the most abundant biological species on earth. [0003] Phages contain only one kind of genetic material, DNA or RNA, and the genetic material of most phages is DNA. In the past two decades, with the advancement of molecular biology techniques, phage research ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/101
Inventor 罗鹏刘秋婷何香燕胡超群田雨顺
Owner SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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