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Method for extracting genome DNA (deoxyribonucleic acid) from peanuts

An extraction method and genome technology, applied in the field of peanut genomic DNA extraction, can solve the problems of harmful operators, multiple samples, complicated steps, etc., and achieve the effects of convenience, quick operability, fewer reagent varieties, and simple process.

Inactive Publication Date: 2012-10-17
CROP RES INST GUANGDONG ACAD OF AGRI SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the widely used technical solutions include CTAB and phenolic method for mass extraction. These methods all have the following defects: 1. Harmful to the operator; 2. More samples are required, the steps are cumbersome and time-consuming, and they also have an impact on the plants.

Method used

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  • Method for extracting genome DNA (deoxyribonucleic acid) from peanuts
  • Method for extracting genome DNA (deoxyribonucleic acid) from peanuts
  • Method for extracting genome DNA (deoxyribonucleic acid) from peanuts

Examples

Experimental program
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Embodiment 1

[0037] A method for extracting peanut genomic DNA, comprising the following steps:

[0038] 1) Put the peanut leaves in a centrifuge tube or mortar, add liquid nitrogen, and grind them into powder;

[0039] 2) Take 0.3-0.5g of the powder obtained in step 1), add 0.75ml of solution A and 4ul of 10mg / ml RNase to make the final concentration 1mg / ml, mix well and let stand at room temperature for 5-10 minutes;

[0040] 3) Take KAc with a volume of 0.25ml, pre-cool it to 4°C before adding it, mix well and then centrifuge at a speed of 8000-15000rpm for 8-15 minutes;

[0041] 4) Take the supernatant obtained in step 3) and add an equal volume of isopropanol, mix well, and at this time, flocculent nucleic acids appear, and then perform centrifugation at a speed of 8000-15000rpm for 8-15 minutes. Then rinse the flocculent material obtained by centrifugation with ethanol with a volume concentration of 75%, dry it after rinsing, and then use 0.05-2ml sterile ddH 2 O dissolved and drie...

Embodiment 2

[0044] A method for extracting peanut genomic DNA, comprising the following steps:

[0045] 1) Put the peanut leaves in a centrifuge tube or mortar, add liquid nitrogen, and grind them into powder;

[0046] 2) Take 5-7g of the powder obtained in step 1), add 9ml of solution A and 45ul of 10mg / ml RNase, mix well and let stand at room temperature for 5-10 minutes;

[0047] 3) Take KAc with a volume of 3ml, pre-cool it to 4°C before adding it, mix well and then centrifuge at a speed of 8000-15000rpm for 8-15 minutes;

[0048] 4) Add an equal volume of isopropanol to the supernatant obtained in step 3), and mix thoroughly. At this time, flocculent nucleic acids appear. Pick out the flocculents and rinse the flocculents with ethanol with a volume concentration of 75%. , rinsed and dried, then 0.05-2ml sterile ddH 2 O dissolved and dried the resulting precipitate to obtain a DNA extract.

[0049] Solution A described in the above steps is 1M Tris-HCl, 0.5M EDTA, 5M NaCl, mass con...

Embodiment 3

[0051] Detect the quality of the peanut gene DNA that embodiment 1 makes with NanoDrop-1000 ultra-micro nucleic acid protein assay instrument, the result is as follows figure 1 shown. Depend on figure 1 It can be seen that the values ​​of D260 / D280 are all between 1.8-2.0, and the measured concentration is above 600ng / ul, indicating that this method is feasible for extracting peanut genomic DNA.

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Abstract

The invention relates to a method for extracting genome DNA (deoxyribonucleic acid) from peanuts, comprising the following steps of: (1) grounding peanut leaves into powder; (2) adding solution A and RNA (ribonucleic acid) enzyme with the final concentration of 1mg / ml; (3) carrying out centrifugal separation after Kac is added; and (4) taking the supernatant obtained in the step 3), adding to isopropanol, carrying out centrifugal separation or picking out flocculent materials, rinsing the flocculent materials by using ethanol with the volume concentration of 75%, drying, and dissolving the obtained precipitate by using sterile ddH2O, wherein in the above steps, the solution A is prepared by evenly mixing 1 Mtris-HCl, 0.5 MEDTA, 5 MnaCl, SDS with the mass concentration of 20%, and H2O according to the volume ratio of 1:1:1:1:6, and adding beta-mercaptoethanol and the RNA enzyme with the final concentration of 40 to 50mg / l according to the ratio of 2% of mixed solution volume. The method provided by the invention is safe, rapid and efficient, and therefore, the method provided by the invention can satisfy different experiment requirements.

Description

[0001] technical field [0002] The invention relates to a method for extracting genome DNA, in particular to a method for extracting genome DNA from peanuts. [0003] Background technique [0004] Peanut, also known as groundnut, is an annual herb with high oil content and is known as "plant meat". In addition to being edible, it can also be used in printing and dyeing, papermaking industries, etc. It is an important oil plant and economic crop in my country. my country's peanut planting area and total production rank among the top in the world, and it has strong international competitiveness. However, my country's current peanut germplasm resources are relatively limited, and some germplasm resources that are resistant to disease and stress are relatively scarce, especially varieties resistant to Aspergillus flavus. Conventional hybrid breeding and mutation breeding are difficult to carry out purposeful genetic improvement of peanut. However, transgenic breeding can ar...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 温世杰刘海燕李洪杰洪彦彬梁炫强
Owner CROP RES INST GUANGDONG ACAD OF AGRI SCI
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