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Chicken whole blood simple, convenient and quick DNA extraction method

An extraction method and a simple technology, applied in the field of design biological detection, can solve the problems of affecting PCR results, DNA integrity damage, etc., and achieve the effect of reducing DNA damage, avoiding damage and degradation, and simplifying steps and time

Inactive Publication Date: 2018-03-27
GUANGXI AGRI VOCATIONAL COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method extracts DNA, and there are still some proteins in the supernatant; the boiling process causes certain damage to the integrity of the DNA, and sometimes it will affect the PCR results.
This method is selective for samples, and there are no reports on the use of samples such as chicken and coagulated blood.

Method used

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  • Chicken whole blood simple, convenient and quick DNA extraction method
  • Chicken whole blood simple, convenient and quick DNA extraction method
  • Chicken whole blood simple, convenient and quick DNA extraction method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Embodiment 1 The method of the present invention extracts and compares the DNA that the kit carries out

[0047] 1.1 Materials and reagents

[0048] Guangxi bantam three-yellow chicken hen and high-legged cock hybrid F1 male; proteinase K, absolute ethanol, NaCl, etc. were purchased from Nanning Tiandiyang Biotechnology Co., Ltd.; TIANamp Blood DNA Kit kit was purchased from Tiangen Biochemical Technology (Beijing) Ltd.

[0049] 1.2 Genomic DNA extraction

[0050] Select 3.8% sodium citrate, with anticoagulant:blood ratio of 1:9, collect anticoagulant blood samples from 4 F1 generation roosters, and use the following two methods to extract DNA respectively.

[0051] 1.2.1 The kit method is carried out according to the method of the TIANamp Blood DNA Kit kit. Take 20 μL of anticoagulated blood, add 20 μL of Proteinase K solution, and mix well; add 200 μL of buffer GB, fully invert and mix well, and place at 56°C for 10 minutes. Mix by inverting several times during th...

Embodiment 2

[0068] Example 2 DNA Extraction and GHR Gene Detection of Guangxi Ma Rooster

[0069] 1. Test materials and reagents

[0070] Materials: 12 Guangxi hemp roosters were sampled in this experiment.

[0071]Reagent: 3.8% sodium citrate (weigh 3.8g sodium citrate, add 90mL deionized water, after dissolving, set the volume to 100mL, autoclave, refrigerate at 2-8°C, and reserve), lysate (50mM Na 2 EDTA·2H 2 O, 2% SDS, 0.19M NaCl; before use, add proteinase K to a final concentration of 0.4 μg / μL), separation liquid, washing liquid, eluent, self-prepared, and reagents of analytical grade.

[0072] 2. Test method

[0073] 1. Template preparation

[0074] a) About 900 μL of whole blood was collected from chicken wing veins, added to a centrifuge tube (with 100 μL of 3.8% sodium citrate anticoagulant added), covered with the cap of the centrifuge tube, mixed well, and refrigerated at 2-8°C for later use.

[0075] b) Open the cap of the centrifuge tube, take 100 μL of collected antic...

Embodiment 3

[0098] Example 3 Extraction of genomic DNA from whole blood of green-shell layer hens and detection of SLCO1B3 gene

[0099] 1. Test materials and reagents

[0100] Material: 12 roosters with green shell eggs.

[0101] Reagent: Na 2 EDTA·2H 2 O (weighing 1.5g, adding 90mL deionized water, after dissolving, set the volume to 100ml, autoclave, refrigerate at 2-8°C, and set aside), lysate (same as Example 2)

[0102] 2. Test method

[0103] 1. Template preparation

[0104] a) About 900 μL of whole blood was collected from the chicken wing vein, and added to a centrifuge tube (1.5% Na 2 EDTA·2H 2 O sodium anticoagulant (100 μL), cover the centrifuge tube, mix well, and refrigerate at 2-8°C for later use.

[0105] b) The following steps for preparing the DNA template are the same as in Example 2.

[0106] 2. Identification of DNA samples

[0107] Ultraviolet spectrophotometry: the steps are the same as in Example 2.

[0108] 3. PCR test

[0109] PCR system: 10 μL of 2×ta...

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Abstract

The invention discloses a chicken whole blood simple, convenient and quick DNA extraction method which comprises the following steps of after adding chicken whole blood in an anticoagulant, adding a mixture in lysate for mixing, putting a mixture in a water solution boiler with the temperature being 56-60 DEG C for 20-30min, adding a separating medium in a product, putting a mixture in the water solution boiler with the temperature being 70-80 DEG C for 20-30min for centrifugation, taking a supernate, transferring the supernate into another EP pipe, adding a precipitated solution, performing centrifugation, reserving precipitates, adding a washing solution, and performing uniform mixing and centrifugation; and carefully discarding the supernate, collecting the precipitates, drying the precipitates at the room temperature, and adding a TB buffer liquid to obtain a chicken DNA extracting solution. By adopting the chicken whole blood simple, convenient and quick DNA extraction method provided by the invention, the purity and the integrity of extracted DNA can both reach test requirements, and the concentration and the purity are superior to that of DNA extracted by an existing kit. Bycombining with application on a chicken molecular breeding aspect, proved that the method is an environment-friendly DNA extraction method with low cost and simple operation.

Description

technical field [0001] The invention relates to the technical field of biological detection, and in particular relates to a simple and rapid DNA extraction method from chicken whole blood. Background technique [0002] Since Watson and Crick proposed the DNA double helix model in the 1950s, molecular biology has achieved unprecedented development, and animal molecular breeding has been applied more and more in production. The extraction of genomic DNA is a basic and important technology in animal molecular breeding, which is directly related to the effect of genetic testing. At present, there are many extraction methods for poultry genomic DNA, such as kit method, salting out method, protease digestion method, organic solvent extraction method, etc., but each method has certain advantages and disadvantages. [0003] The phenol-chloroform method is the most classic method for extracting DNA. Its principle is to use the organic solvent phenol to denature proteins and inhibit ...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12Q1/6806
CPCC12N15/1003C12Q1/6806
Inventor 王树艳沈前程李军成凌丁
Owner GUANGXI AGRI VOCATIONAL COLLEGE
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