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A method for extract genomic DNA of Lonicera japonica Maxim

An extraction method and genome technology, applied in the field of molecular biology, can solve problems such as DNA browning

Inactive Publication Date: 2019-01-11
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most reports add water-soluble PVP to the extract, but it is found in experiments that this cannot completely solve the problem of DNA browning

Method used

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  • A method for extract genomic DNA of Lonicera japonica Maxim

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] (1) Take 0.1 g of the leaves in a pre-cooled mortar, add a small amount of PVP powder, and immediately add 0.7-1.2 mL of SET nuclei separation buffer after liquid nitrogen grinding, quickly grind to a homogenate, and carefully transfer to a 2 mL centrifuge tube. Let stand at 2-8°C for 10min, centrifuge at 4000r / min for 10min at 4°C, and discard the supernatant.

[0027] (2) Add 700-1000μl 2×CTAB extraction buffer preheated at 60-65℃ for precipitation, then add 1-10μL of β-mercaptoethanol, mix well, put in water bath at 60-65℃ for 1 hour, shake gently several times during the period .

[0028] (3) After cooling to room temperature in a water bath, add 700-1000 μL of chloroform / isoamyl alcohol (22:3), gently invert to mix, and centrifuge at 12000 r / min for 10 min at room temperature.

[0029] (4) Take the supernatant to another tube, add 1 / 8-1 / 10 volume of preheated 10-15% CTAB solution and mix well, then add an equal volume of chloroform / isoamyl alcohol (22:3), lightly ...

example 2

[0035] (1) Take 0.1-0.35g leaves in a pre-cooled mortar, add a small amount of PVP powder, immediately add 0.7-1.2mL SET nuclei separation buffer after grinding with liquid nitrogen, quickly grind into a homogenate, and carefully transfer to a 2mL centrifuge tube . Let stand at 2-8°C for 10min, centrifuge at 4000r / min for 10min at 4°C, and discard the supernatant.

[0036] (2) Add 700-1000 μL of 2×CTAB extraction buffer preheated at 60-65°C to the precipitate, then add 1-10 μL of β-mercaptoethanol, mix well, and bathe in water at 60-65°C for 1 hour. During this period, shake gently for several times. Second-rate.

[0037] (3) After cooling to room temperature in a water bath, add 700-1000 μL of chloroform / isoamyl alcohol (23:2), mix gently by inversion, and centrifuge at room temperature at 12000 r / min for 10 min.

[0038] (4) Take the supernatant to another tube, add 1 / 8-1 / 10 volume of preheated 10-15% CTAB solution and mix well, then add an equal volume of chloroform / isoam...

example 3

[0044] (1) Take 0.1-0.35g of leaves in a pre-cooled mortar, add a small amount of PVP powder, and immediately add 0.7-1.2mL of SET nucleus separation buffer after liquid nitrogen grinding, quickly grind into a homogenate, and carefully transfer to a 2mL centrifuge tube middle. Let stand at 2-8°C for 10min, centrifuge at 4000r / min for 10min at 4°C, and discard the supernatant.

[0045] (2) Add 700-1000 μL of 2×CTAB extraction buffer preheated at 60-65°C to the precipitate, then add 1-10 μL of β-mercaptoethanol, mix well, and bathe in water at 60-65°C for 1 hour. During this period, shake gently for several times. Second-rate.

[0046] (3) After cooling to room temperature in a water bath, add 700-1000 μL of chloroform / isoamyl alcohol (24:1), mix gently by inversion, and centrifuge at room temperature at 12000 r / min for 10 min.

[0047] (4) Take the supernatant to another tube, add 1 / 8-1 / 10 volume of preheated 10-15% CTAB solution and mix well, then add an equal volume of chlo...

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Abstract

A method for extractING genomic DNA of Lonicera japonica Maxim belongs to field of molecular biology. The method mainly comprises the following steps: firstly, cell walls and cell membrane are brokenso that DNA is fully released into buff; secondly, the interference of protein, polysaccharide, pigment and other impurities was eliminated; finally, DNA degradation is prevented. The sample was treated with SET nucleus separation buffer, which could separate the nucleus from the secondary substances such as polysaccharides and polyphenols in the supernatant, PVP added in the grinding process andbeta-Mercaptoethanol, can effectively prevent the oxidation of polyphenols, chloroform / isoamyl alcohol extraction, the addition of diluted CTAB to remove polysaccharides. The leaves of Caprifoliaceaeplants are rich in carbohydrates and polyphenols, These substances not only affect the extraction of DNA, but also affect the activity of Tag enzyme in PCR. By this method, DNA with high yield and good quality can be obtained. DNA electrophoretic bands are clear and bright, and there is no tail-trailing phenomenon, which can meet the further research at molecular level.

Description

Technical field: [0001] The invention belongs to the field of molecular biology, and mainly relates to a method for extracting genome DNA of Lonicera japonica. Background technique: [0002] Plant DNA extraction is a routine experimental technique, but for plants and tissues with high polysaccharide and phenolic substances, the extraction work is not easy, and it may take a lot of time and energy to obtain ideal DNA. The most fundamental requirement for DNA extraction is to maintain the integrity of nucleic acid. The molecular weight of genomic DNA products should be above 50kb. During the DNA extraction process, there are many factors that can cause DNA to degrade into small fragments, such as mechanical tension or high temperature. Molecules are broken, so the operation should be as gentle and gentle as possible, and excessive solution transfer and violent oscillation should be avoided as much as possible to reduce the damage of mechanical tension to DNA. DNase can degrad...

Claims

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Application Information

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IPC IPC(8): C12N15/10
CPCC12N15/1003
Inventor 霍俊伟张妍刘化禹孙丰秦栋
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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