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Novel method for extracting phage genome DNA

An extraction method and phage technology, applied in the field of genetic engineering, can solve the problems of phage reduction, large differences in biological characteristics, degradation, etc., and achieve the effects of reducing experimental costs, shortening extraction time, and wide application range

Inactive Publication Date: 2019-02-22
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most phages can be collected by PEG sedimentation, but there are many kinds of phages with great differences in biological characteristics, and the extraction method of genomic DNA lacks wide applicability
For example, chloroform-sensitive phages may cause a large loss during chloroform extraction, which reduces the number of phages and cannot extract a sufficient amount of genomic DNA; for temperature-unstable phages, proteinase K 56 ° C may also be added. Loss, inactivation of phage, genome degradation, so that the extraction quality cannot fully meet the requirements of use

Method used

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  • Novel method for extracting phage genome DNA
  • Novel method for extracting phage genome DNA
  • Novel method for extracting phage genome DNA

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Embodiment 1

[0046] According to the above embodiment, the genomic DNA of the chloroform-sensitive phage is extracted, wherein the name of the phage is ΦH2, and the classification name is Pseudomonas aeruginosa PA14Phage. The host bacterium lysed by phage ΦH2 is named: Pseudomonas aeruginosa PA14, and the taxonomic name is Pseudomonas aeruginosa.

[0047] ①Take the same preparation method of phage lysate

[0048] ②The effect of chloroform extraction on the titer of phage ΦH2

[0049] During the extraction of the phage genome, after resuspension in TM buffer, chloroform was added to extract to remove residual PEG6000. The titer of phage after extraction with chloroform was determined by double-layer plate method.

[0050] Chloroform pumped ahead of time, ΦH2 titer was 6×10 11 pfu / ml, after chloroform extraction, the titer is 3.6×10 10 pfu / ml, the loss reached 94%, seriously affecting the concentration of the extracted phage genomic DNA.

[0051] ③The effect of chloroform extraction on ...

Embodiment 2

[0055] Extraction of Genomic DNA of Staphylococcus aureus Phage Z-1

[0056] (1) Cultivate the host bacteria overnight at 37°C, inoculate 2% (V / V) in 100mL fresh LB medium, add phage according to the optimal MOI, culture at 30°C, 220r / m shaking for 8h

[0057] (2) Add NaCl to a final concentration of 0.1M, mix and dissolve, then ice-bath for 1h, centrifuge at 10000r / / m for 20min

[0058] (3) Add DNase I and RNase A to the lysate to a final concentration of 5 μg / mL, mix well, and let stand at 37°C for 1 hour

[0059] (4) After centrifugation, transfer the supernatant to another centrifuge tube, add PEG6000 to a final concentration of 10%, fully shake and dissolve, and place at 4°C overnight

[0060] (5) Centrifuge the overnight treatment solution at 10000r / m for 20min, discard the supernatant

[0061] (6) Resuspend the pellet with 1 / 50 volume of TM buffer, add DNase I and RNase A to a final concentration of 10 μg / mL, and let stand at 37°C for 1 hour

[0062] (7) Add EDTA (pH...

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Abstract

The invention provides a novel method for extracting phage genome DNA. The novel method comprises the following steps: after amplifying phage lysate, removing cell debris and nucleic acid substances of host bacteria, adding PEG6000 to settle phage, after resuspending by TM buffer liquid, avoiding chloroform extraction, directly adding nuclease, removing the genome of the remaining host bacteria, then replacing protease K by using urea, after degeneration of coat protein, separating the protein from the genome DNA by agarose gel electrophoresis, and then recycling the genome DNA of the phage byusing a freeze thawing and recycling method. The method is simple and convenient to operate, is particularly suitable for phage sensitive to chloroform, and is wide in application range, experiment costs are greatly reduced, and extracting time is shortened.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a new method for rapidly and economically extracting bacteriophage genome DNA. technical background [0002] Bacteriophage, known as the "natural killer of bacteria", is a virus that specifically infects bacteria. Phages can be found everywhere in nature. Wherever various microorganisms exist, there are corresponding types of phages, and their number is proportional to the number of host bacteria. Phages are considered to be the most abundant and diverse biological entities in nature, and their number is 10-100 times that of bacteria. Phages have a simple structure consisting of genetic material wrapped in a protein coat. The phage genome can be composed of DNA or RNA, the genetic material of most phages is DNA, and the structure of nucleic acid can be single-stranded or double-stranded, closed circular or linear molecules. [0003] With the advent of c...

Claims

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Application Information

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IPC IPC(8): C12N15/10
CPCC12N15/101
Inventor 杨洪江张茜茜黄志伟张志强李东航
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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