Pyrolysis solution, extracting solution, pyrolysis and extraction method, kit and application and PCR (Polymerase Chain Reaction) system

A technology of lysate and extract, which is applied in the field of genetic engineering, can solve problems such as unfavorable fish research, and achieve the effects of easy mastery, simple and convenient operation, and expanded material sources

Active Publication Date: 2017-01-04
CHINA JILIANG UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The existing animal tissue DNA extraction method is to remove the protein and fat in the tissue
Obviously, existing methods for extracting DNA from animal tissues are not conducive to fish research

Method used

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  • Pyrolysis solution, extracting solution, pyrolysis and extraction method, kit and application and PCR (Polymerase Chain Reaction) system
  • Pyrolysis solution, extracting solution, pyrolysis and extraction method, kit and application and PCR (Polymerase Chain Reaction) system
  • Pyrolysis solution, extracting solution, pyrolysis and extraction method, kit and application and PCR (Polymerase Chain Reaction) system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment A1～A4

[0096] Examples A1 to A4, Examples B1 to B4 and Examples C1 to C4

[0097] DNA extraction from small yellow croaker tissue. The small yellow croaker tissues used are scales, bones, muscles, and liver.

[0098] Examples A1~A4

[0099] Wash the small yellow croaker tissue with sterilized distilled water, take 25mg of the cleaned small yellow croaker tissue, cut it and put it into a 1.5mL centrifuge tube, add 400μL of lysate, shake for 20 seconds, keep in a water bath at 55℃ for 10min, then add 120μL The neutralization solution was shaken for 20 seconds, and then centrifuged at 10,000 g for 2 minutes, and the supernatant was taken to obtain a solution containing DNA.

[0100] The difference between the reaction conditions of Examples A1 to A4 and Examples B1 to B4 and Examples C1 to C4 is only that the respective reagent ratios in the lysis solution and the neutralization solution are different, as shown in Table 1:

[0101] Table 1 Composition of reagents in lysis solution and neutraliz...

Embodiment D～ Embodiment S

[0119] The fish tissue DNA extracts used in Examples D to S are the same as those used in Examples C1 to C4, except for the type of small yellow croaker tissue, the amount of small yellow croaker tissue, the amount of lysis solution and neutralization solution, and the water bath conditions.

[0120] Example Organization Type Tissue (mg) Lysis solution (μL) Neutralizing solution (μL) Water bath temperature (℃) Bath time (min) D muscle254001205510 E muscle10200605020 F muscle252001007530 H liver254001205510 I liver10200605020 G liver252001007530 K skeleton254001205510 L skeleton10200605020 M skeleton252001007530 N fin254001205510 O fin10200605020 P fin252001007530 Q Scales 254001205510 R Scales 10200605020 S Scales 252001007530

[0121] The DNA concentration of the DNA-containing solution prepared in Example D to Example S was determined. The inventors found that the DNA concentration in the DNA-containing solution obtained by processing muscle and liver was 50-200 ng / μL, and the...

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Abstract

The invention discloses a pyrolysis solution, a DNA extracting solution, a pyrolysis and extraction method, a NDA extraction kit and application and a PCR (Polymerase Chain Reaction) system. The pyrolysis solution is used for splitting fish tissue to release DNA, and comprises ethylenediamine tetraacetic acid and/or ethylenediamine tetracetic acid disodium salt, sodium chloride, lauryl sodium sulfate, strong base and water, wherein the mole concentration of the ethylenediamine tetraacetic acid and/or the ethylenediamine tetracetic acid disodium salt is 2-10mM relative to the pyrolysis solution; the mole concentration of the sodium chloride is 1-10mM relative to the pyrolysis solution; the mass percentage of the lauryl sodium sulfate is 0.1-10% relative to the pyrolysis solution; the mole concentration of hydroxyl ions of the strong base is 100-300mM. Compared with the prior art, the pyrolysis solution disclosed by the invention is capable of providing a DNA template for fish tissue PCR amplification within relatively short time, reduction or loss of sample DNA can be avoided in the whole process, and moreover, operation is simple and convenient and easy to master.

Description

Technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a lysis solution, fish tissue DNA extraction solution, a lysis method, a method for extracting fish tissue DNA, a fish tissue DNA extraction kit and its application, and amplifying fish tissue DNA 的PCR system. Background technique [0002] The main methods of DNA extraction from animal tissues are phenol-chloroform extraction after protease cleavage, isopropanol or isoamyl alcohol extraction and SDS method, as well as cesium chloride precipitation method, chelex-100 or Tween (Tween) for mitochondrial DNA extraction. ) Method and urea extraction method. At present, relatively simple and convenient commercial animal tissue DNA extraction kits are widely used. The genomic DNA obtained by these methods can generally remove proteins and fats in the original tissue samples, and can also remove phenols and other reagents through alcohol precipitation. The DNA obtained is of high ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/1003C12Q1/686
Inventor 管峰薛超波赵进王萍亚
Owner CHINA JILIANG UNIV
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