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Method for extracting mammalian blood DNA without anticoagulant

A technology for mammals and extraction methods, applied in the direction of DNA preparation, recombinant DNA technology, etc., can solve the problems of poor DNA purity and quality, achieve easy operation and realization, solve the difficult problem of blood coagulation DNA extraction, and facilitate storage and transportation condition control Effect

Inactive Publication Date: 2019-02-19
天津市农业科学院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these measures do not have a unified operating standard, and secondly, the purity and quality of the extracted DNA are not good

Method used

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  • Method for extracting mammalian blood DNA without anticoagulant
  • Method for extracting mammalian blood DNA without anticoagulant
  • Method for extracting mammalian blood DNA without anticoagulant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] 1. Taking sheep as an example, without adding any anticoagulant, draw 2 ml of blood from the jugular vein of the sheep through a veterinary syringe, transport the veterinary syringe together with blood and ice packs at low temperature, and store at -20°C after 24 hours of low temperature. Prior to cryopreservation, natural separation of serum and blood clots is generally seen. The coagulated blood stored at -20°C can guarantee that the quality of DNA extraction will not decrease for at least one year.

[0059] 2. The pretreatment method for DNA extraction of coagulated sheep blood without adding anticoagulant, comprising the following steps:

[0060] (1) The coagulated blood of sheep frozen at -20°C should be fully thawed at a low temperature of 4°C.

[0061] (2) When the serum in the liquid part is completely separated from the blood clot, cut about 0.2g of the blood clot and set aside.

[0062] (3) Put the 0.2g cut blood clot into a 2ml centrifuge tube, first add 20...

Embodiment 2

[0092] Comparative Test:

[0093] Conventional method:

[0094] For blood clots formed by mammalian blood without anticoagulants, the conventional treatment method generally adopts the homogenizer crushing method, that is, adding double distilled water to wash the blood clots, breaking the blood clots through a homogenizer, and after centrifugation, Discard the supernatant and add lysate and proteinase K, and then use kit method or conventional phenol imitation method to extract DNA. If the kit method is adopted, it is easy to cause blockage when passing through the column, and it is not easy to extract DNA; if the conventional phenol imitation method is adopted, the concentration and purity of the proposed DNA are low (such as Figure 4 , Figure 5 ), which is not conducive to subsequent PCR amplification tests (such as Image 6 ). Taking sheep coagulated blood as an example, the Nanodrop detection peak pattern of the extracted DNA is as follows Figure 4 As shown, its 2...

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Abstract

The invention discloses a method for extracting mammalian blood DNA without an anticoagulant. The method includes the following steps: (1) extracting mammalian jugular vein blood through a veterinarysyringe without adding any anticoagulant, performing low-temperature transportation on the veterinary syringe together with blood and an ice bag, and freezing at -20 DEG C after 24 hours of low temperature; (2) fully unfreezing at a low temperature of 4 DEG C within one year of freezing, and cutting about a blood clot of 0.2 g for subsequent DNA extraction when the serum of the liquid part is completely separated from the blood clot; (3) adding the cut blood clot into 500 microliters of DPBS, and using a scissor to fully cut until a yellow gun head can be blown and sucked smoothly; (4) addinglysate and protease K to solid precipitate left after centrifugation for digestion overnight, and then completing DNA extraction by extraction of saturated phenol, chloroform: isoamyl alcohol and alcohol. As DNA with good quality and high concentration is extracted at low cost for solidified mammalian blood without the anticoagulant under the condition of not using red blood cell lysis buffer, themethod can be used for subsequent gene amplification and high-throughput sequencing.

Description

technical field [0001] The invention relates to a method for extracting mammalian blood DNA without adding an anticoagulant, and belongs to the field of molecular biology technology. Background technique [0002] Mammalian blood contains three types of blood cells, namely red blood cells, white blood cells and platelets, and DNA mainly exists in the nucleated white blood cells in the blood. Usually, when extracting mammalian blood, it is necessary to add anticoagulant to prevent blood clotting, so as to ensure that nucleated leukocytes can be lysed to release the DNA in the nucleus. However, in sampling practice, there are often situations where the conditions are not allowed or the shaking is not sufficient after adding anticoagulant, and finally only coagulated blood clots can be obtained. For mammalian blood clots, the blood DNA extraction kits on the market and the DNA extraction methods after routine blood clot processing are no longer effective, which brings research ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/1003
Inventor 郭晓飞张效生张金龙李义海刘海军杨晶
Owner 天津市农业科学院
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