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Method for simply, conveniently and quickly extracting trace total deoxyribonucleic acid (DNA) of single roes and fries

A technology for fish eggs and larvae, which is applied in the field of DNA extraction, can solve the problems of large influence on elution efficiency, large final volume of extraction, and low DNA concentration, and achieves the effect of avoiding freezing equipment, ensuring efficiency and wide application range.

Inactive Publication Date: 2011-08-24
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method also has the disadvantages of large final extraction volume and low DNA concentration.
The commercial trace DNA kit mainly completes the collection and purification of DNA through membrane adsorption and elution. Although a higher concentration of DNA can be extracted, the operation is relatively complicated, the cost is high, and the elution efficiency is limited. greater impact

Method used

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  • Method for simply, conveniently and quickly extracting trace total deoxyribonucleic acid (DNA) of single roes and fries

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] 1) Sample fixation:

[0026] Collect 0.5mL of fertilized eggs of turbot, observe with a dissecting microscope that the fertilized eggs have developed to the 2-cell stage, filter out the seawater and add 1mL of absolute ethanol. After 24 hours, the anhydrous ethanol was replaced once. Store at room temperature;

[0027] 2) Removal of fixative in the sample:

[0028] Take 1 fixed fish roe obtained in step 1), add 0.9% NaCl solution in normal saline to wash, centrifuge at 1500g for 30 seconds, discard excess normal saline, dry the water with filter paper, repeat the above steps Operate 2 times;

[0029] 3) Sample digestion:

[0030] Put the washed fish eggs into a 1.5 mL centrifuge tube, add 100 μL DNA extraction buffer and 10 μL cell lysis buffer. Then use clean dissecting scissors to cut the egg membrane, add 3 μL 10 mg / mL proteinase K and 3 μL 10 mg / mL RNase. Shake and mix and incubate at 55°C, invert and mix once every 10 minutes, digest for 20 minutes until the ...

Embodiment 2

[0042] 1) Sample fixation:

[0043] Collect 1 mL of fertilized eggs of flounder, observe with a dissecting microscope that the fertilized eggs have developed to the embryo body stage, filter off the seawater and add 5 mL of absolute ethanol. After 24 hours, the anhydrous ethanol was replaced once. Store at -20°C;

[0044] 2) Removal of fixative in the sample:

[0045] Take 1 fixed fish roe obtained in step 1), add a mass-volume ratio of 0.9% NaCl solution and wash with normal saline; centrifuge at a speed of 1000g for 60 seconds, absorb and discard excess normal saline, dry the water with filter paper, repeat 2 Second-rate.

[0046] 3) Sample digestion:

[0047]Put the washed fish eggs into a 1.5 mL centrifuge tube, add 200 μL DNA extraction buffer and 20 μL cell lysis buffer. Then use clean dissecting scissors to cut the egg membrane. Add 2 µL of 20 mg / mL proteinase K and 2 µL of 20 mg / mL RNase. Shake and mix and incubate at 60°C, invert and mix once every 15 minutes, ...

Embodiment 3

[0059] 1) Sample fixation:

[0060] Collect 10 newly hatched flounder larvae, filter the seawater and add 2 mL of absolute ethanol. After 24 hours, the anhydrous ethanol was replaced once. Store at room temperature;

[0061] 2) Removal of fixative in the sample:

[0062] Take 1 fixed newly hatched larvae obtained in step 1), add a mass-volume ratio of 0.9% NaCl solution, and wash with saline; centrifuge at 2000g for 20 seconds, dry the water with filter paper, and repeat the above operation 3 times.

[0063] 3) Sample digestion:

[0064] Put the washed larvae into a 1.5 mL centrifuge tube, add 300 μL DNA extraction buffer and 30 μL cell lysis buffer. Then cut the larvae into pieces with clean dissecting scissors. Add 5 µL of 20 mg / mL proteinase K and 5 µL of 20 mg / mL RNase. Shake and mix and incubate at 55°C, invert and mix once every 20 minutes, digest for 1 hour until the solution is clear; the DNA extraction buffer is pH=8.0 with a final concentration of 0.01mol / L, Tr...

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Abstract

The invention relates to a deoxyribonucleic acid (DNA) extraction technology, in particular to a method for simply, conveniently and quickly extracting trace total DNA of single roes and fries. The method comprises the following steps of: fixing and preserving collected roes or fries; washing to remove stationary liquid, adding DNA extraction buffer solution and lysis buffer solution, shearing the roes or the fries, adding proteinase K and ribonucleic acid (RNA) enzyme, incubating and digesting; and precipitating protein by using high-concentration NaCl solution, adding isopropanol to precipitate DNA, washing a precipitate by using 70 percent ethanol to remove salt ions, and adding ultrapure water or tris-ethylenediaminetetraacetic acid (TE) solution and dissolving after the ethanol is volatilized. The method solves the problem of the limitation of the application the conventional total DNA extraction method to samples with the low content of genomic DNA, such as roes or fries and thelike.

Description

technical field [0001] The invention relates to a DNA extraction technology, in particular to a simple and quick method for extracting a trace amount of total DNA from a single fish roe and larvae. Background technique [0002] In order to study the role of DNA molecules in life metabolism, it is often necessary to extract DNA from different biological materials. The preparation of high-quality total DNA is one of the foundations for molecular biology research and applications. Common extraction methods for total DNA mainly include high-salt method, organic solvent extraction, Chelex-100 extraction, enzymatic cleavage, and commercial kits. The traditional high-salt method is suitable for a large number of tissue samples, and it is difficult to extract enough DNA from samples with a small total DNA content such as single fish eggs and larvae. Although the organic solvent extraction method can obtain relatively pure DNA, it takes a long time, causes a large loss of DNA, and ...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 尤锋吴志昊王伟徐冬冬张毅张培军徐永立
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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