DNA extraction kit applied to transgenic mouse gene identification

A technology of transgenic mice and kits, applied in the field of molecular biology, to achieve the effect of improving efficiency, less operation steps, and low price

Pending Publication Date: 2021-03-16
SHUNDE HOSPITAL SOUTHERN MEDICAL UNIV (THE FIRST PEOPLES HOSPITAL OF SHUNDE FOSHAN)
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The invention discloses a DNA extraction kit applied to gene identification of transgenic mice, in order to ...

Method used

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  • DNA extraction kit applied to transgenic mouse gene identification
  • DNA extraction kit applied to transgenic mouse gene identification
  • DNA extraction kit applied to transgenic mouse gene identification

Examples

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Effect test

Embodiment 1

[0024] Example 1: Kit 1 was prepared and applied to DNA extraction and gene identification from toes of T-bet transgenic mice.

[0025] Kit formula 1: The lysate contains: 0.5% SDS, 0.1M NaCl, 0.05M Tris-HCl, 0.05M EDTA, adjust the pH to 8.0. Proteinase K concentration: 15mg / ml, added to the lysate before use. Composition of the binding solution: 1M NaCl, 70% isopropanol, 5% PEG 8000, adjust the pH to 7.0. The silica microspheres have a concentration of 0.4 g / ml and a pH of 7.0, and are added to the binding solution before use to form a microsphere suspension. Composition of washing solution: 10mM Tris, 70% ethanol, adjust pH to 8.0. The eluent was ultrapure water at pH 8.0.

[0026] Gene identification of T-bet flox series mice in C57BL / 6J background using kit 1:

[0027] Paired T-bet fl / + The mice were purchased from Jackson Laboratory in the United States, and the toes of offspring mice were cut off after weaning for genotype identification.

[0028] The kit prepared ...

Embodiment 2

[0042] Example 2: Kit 2 preparation and application to T-bet transgenic mouse toe DNA extraction and gene identification.

[0043] Kit formula 2: The lysate contains: 1% SDS, 0.5M NaCl, 0.07M Tris HCl, 0.08M EDTA. Adjust the pH to 8.0. Proteinase K concentration: 17 mg / ml, added to lysate before use; composition of binding solution: 1.2M NaCl, 75% isopropanol, 6% PEG 8000, adjust pH to 7.0. The concentration of silica microspheres is 0.5g / ml, and the pH is 7.0. Add it to the binding solution before use to form a suspension of microspheres; the composition of the washing solution: 12mM Tris, 72% ethanol, adjust the pH to 8.0; the eluent Ultrapure water with a pH of 8.0.

[0044] The steps of using Kit 2 to identify genes of T-bet flox series mice with C57BL / 6J background are the same as in Example 1.

[0045] figure 2 It is the mouse gene identification result of this embodiment, and the single band of 650bp is T-bet fl / fl Mouse, 650bp and 470bp double bands are T-bet fl...

Embodiment 3

[0046] Example 3: Kit 3 preparation and application to T-bet transgenic mouse toe DNA extraction and gene identification

[0047] Kit formula 3: The lysate contains: 2% SDS, 1M NaCl, 0.1M Tris-HCl, 0.1M EDTA. Adjust the pH to 8.0. Proteinase K concentration: 20mg / ml, added to the lysate before use; composition of the binding solution: 1.5M NaCl, 80% isopropanol, 8% PEG 8000, adjust the pH to 7.0. The silica microspheres are 0.6g / ml, and the pH is 7.0, and they are added to the binding solution before use to form a microsphere suspension; the composition of the washing solution is: 15mM Tris, 75% ethanol, and the pH is adjusted to 8.0; the eluent is Ultrapure water at pH 8.0.

[0048] The steps of using kit 3 to identify the genes of T-bet flox series mice with C57BL / 6J background are the same as those in Example 1.

[0049] image 3 It is the mouse gene identification result of this embodiment, and the single band of 650bp is T-bet fl / fl Mouse, 650bp and 470bp double bands...

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Abstract

The invention discloses a DNA extraction kit applied to transgenic mouse gene identification. The DNA extraction kit comprises a tissue lysis solution, protease K, a binding solution, adsorption microspheres, a washing solution and an eluent, wherein the adsorption microspheres are silicon dioxide microspheres with the diameter of 50 to 100 [mu]m. Compared with a traditional kit, the DNA extraction kit disclosed by the invention has the advantages of low price, few operation steps and high extraction purity; used reagents are non-toxic, safe and stable; and the DNA extraction kit is suitable for extracting DNA of tail tips and toes of mice on a large scale. The invention also discloses a method for performing high-throughput extraction on mouse tissue genome DNA by using the kit. The provided extraction method has the advantages that the operation process is simplified, the experiment time is shortened, and the quality of the extracted DNA completely meets the requirements of subsequent PCR. The transgenic mouse gene identification efficiency is obviously improved.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a DNA extraction kit for gene identification of transgenic mice. Background technique [0002] With the development of gene editing technology, transgenic mice came into being. At present, many scholars at home and abroad have used transgenic mice to carry out research work. With the rapid development of transgenic technology, transgenic mice are no longer expensive. Therefore, the strains and numbers of transgenic mice in many domestic laboratories have gradually increased. However, the acquisition of homozygous transgenic mice requires the crossing of heterozygous parents, so the genetic identification of transgenic mice—including DNA extraction and PCR amplification—becomes a tedious and repetitive work. [0003] Traditional DNA extraction methods include phenol / chloroform extraction and magnetic bead adsorption. Although these methods can obtain relatively high-p...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12Q1/6806
CPCC12N15/1006C12Q1/6806C12Q2521/537C12Q2523/308C12Q2527/125
Inventor 曾小康肖强姚婕
Owner SHUNDE HOSPITAL SOUTHERN MEDICAL UNIV (THE FIRST PEOPLES HOSPITAL OF SHUNDE FOSHAN)
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