Method for batch extraction of rice endosperm DNA

An endosperm and rice technology, applied in the biological field, can solve the problems of grain damage, cumbersome operation steps, and inability to extract in batches, and achieve the effects of flexible schedule, operator safety, and rapid extraction.

Active Publication Date: 2013-12-11
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, studies on the use of rice grains to extract DNA have been reported, but the existing research procedures are cumbersome and cannot be extracted in batches;

Method used

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  • Method for batch extraction of rice endosperm DNA
  • Method for batch extraction of rice endosperm DNA
  • Method for batch extraction of rice endosperm DNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 This method was used to extract the DNA of rice varieties R173, Nipponbare, Xianxiaozhan, Hanghui 7, and Yuejingsimiao 2.

[0041] The specific method is:

[0042] S1. Take 96 rice seeds with lemma husks, peel off the lemma husks, and cut out 1 / 3 endosperm (about 5-8 mg) with a blade; S2. Put the cut endosperm into the wells of a 96-well PCR plate (T1 plate). The remaining part of the kernel (embryo

[0043] milk and embryo) into the wells of another 96-well PCR plate (T2 plate).

[0044] S3. Use a multi-channel pipette to add 100 μL of 1 mol / L NaOH solution to each well of the T1 plate, put it into a PCR machine, keep it at 99°C for 15 min, and put the T2 plate into a 4°C refrigerator for later use;

[0045] S4. Take out the T1 plate, put 1 tungsten alloy bead into each well, and use a multi-channel pipette to draw 180 μL of 1×DNA extraction solution into each well, and cover the silica gel cover;

[0046] S5. Re-place the T1 plate in the PCR instrument at...

Embodiment 2

[0061] Example 2 The concentration and purity of DNA extracted by the two methods of Example 1 and Comparative Example 1 were determined.

[0062] Specific methods: 1) UV-240 ultraviolet spectrophotometer is turned on and preheated for 10 minutes; 2) Wash the cuvette with double distilled water, blot it dry with absorbent paper, add TE buffer, put it on the S cell rack in the sample room, and close it. Cover plate; 3) Calibrate to zero after setting the slit; 4) After properly diluting the standard sample and the sample to be tested (DNA 5 μl or RNA 4 μl diluted to 1000 μl with TE buffer), record the number and dilution; 5) Put the standard sample Put the sample or the cuvette of the sample to be tested on the S shelf of the sample room, and close the cover; 6) Set the wavelength of ultraviolet light, and measure the OD value at 230nm, 260nm, and 280nm wavelength respectively; 7) Calculate the OD value of the sample to be tested Concentration and purity, DNA sample concentrati...

Embodiment 3

[0063] Example 3 Using gel electrophoresis to detect the extracted DNA in Example 1 and Comparative Example 1

[0064] Specific method: Prepare 1% agarose gel for electrophoresis detection, observe the results and save them by taking pictures.

[0065] The concentration and purity of DNA extracted by the two methods were compared. The DNA extracted by this method can reach about half of the amount of DNA extracted by the CTAB method. The concentration of DNA extracted by the CTAB method is between 740-1100ng / μL, and the concentration of DNA extracted by this method is 300-480ng / μL. The OD260 / OD280 of the DNA extracted by the CTAB method is between 1.8 and 2.0, indicating that the quality is intact; the OD260 / OD280 of the DNA extracted by this method is between 1.8 and 2.0. The results of electrophoresis showed that the DNA extracted by the two methods could be used in the next research. In terms of time consumption, taking the extraction of 96 samples of rice as an example, ...

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Abstract

The invention relates to the technical field of biology, and discloses a method for batch extraction of rice endosperm DNA. The method comprises: S1, cutting endosperm from rice seeds; S2, pouring the endosperm into holes of a PCR plate (T1 plate); S3, adding an alkali solution to every hole of the T1 plate, and placing the T1 plate into a PCR instrument to be heated; S4, taking the T1 plate, and pouring tungsten alloy beads and a 1*DNA extraction solution to every hole; S5, re-placing the T1 plate into the PCR instrument to be heated; S6, taking the T1 plate, and carrying out vibration; S7, placing the T1 plate into a centrifuge to be centrifuged; S8, taking the T1 plate, and transferring the supernatant to another PCR plate (T3 plate), wherein every hole of the T3 plate is added with double distilled water in advance; and S9, placing the T3 plate in a refrigerator to be stored. The method has characteristics of rapidness and simple operation, wherein batch extraction of rice endosperm DNA can be achieved with the method.

Description

[0001] technical field [0002] The invention relates to the field of biotechnology, more specifically, to a method for extracting rice endosperm DNA in batches. Background technique [0003] Rice is an important food crop in my country, and the cultivation of new high-yield, high-quality, and multi-resistant rice varieties is an important part of improving my country's food security. With the emergence of molecular markers, the use of molecular marker-assisted selection to improve the efficiency of breeding new varieties has been widely used. [0004] The preparation of rice DNA is the prerequisite for molecular marker-assisted selection. During extraction, the relative integrity of its structure should be ensured according to different research needs; contamination by other macromolecular components such as proteins and polysaccharides should be excluded as much as possible. Using rice leaves as materials, using CTAB to extract DNA is a widely used method at present. T...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 郭涛罗文龙陈志强王慧陈海英刘永柱张建国
Owner SOUTH CHINA AGRI UNIV
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