阅读说明：本技术 磁珠法血液基因组dna提取试剂盒的使用方法 (Use method of magnetic bead method blood genome DNA extraction kit ) 是由 包文静 金安娜 高伙妮 龚伟 于 2018-08-20 设计创作，主要内容包括：本发明公开了磁珠法血液基因组DNA提取试剂盒的使用方法,包括以下步骤：取100μL血液样品,加入300μL细胞裂解液,混匀后室温孵育5min,12000rpm离心1min,去除上清液；加200μL裂解液和10μL蛋白酶K,混匀后在56℃、800rpm的条件下孵育15min；放置5min后离心,转移上清并在其中加入100μL磁珠混匀孵育5min,离心,放置磁力架上至溶液澄清；移除上清后加入乙醇溶液混匀,放在磁力架上离心去除乙醇溶液后静置至磁珠不反光；加入30μL的无核酸酶水低盐缓冲液,静置后置于磁力架上2min,将上清转移至新的样品管中备用。本方法提取过程不涉及有毒有害试剂,操作安全。(The invention discloses a use method of a kit for extracting blood genome DNA by a paramagnetic particle method, which comprises the following steps: adding 300 mul of cell lysate into 100 mul of blood sample, uniformly mixing, incubating at room temperature for 5min, centrifuging at 12000rpm for 1min, and removing supernatant; adding 200 μ L lysate and 10 μ L proteinase K, mixing, and incubating at 56 deg.C and 800rpm for 15 min; standing for 5min, centrifuging, transferring supernatant, adding 100 μ L magnetic beads, mixing, incubating for 5min, centrifuging, and standing on magnetic frame until the solution is clear; removing the supernatant, adding an ethanol solution, mixing uniformly, placing on a magnetic frame, centrifuging to remove the ethanol solution, and standing until the magnetic beads do not reflect light; adding 30 μ L of nuclease-free water low-salt buffer solution, standing, placing on a magnetic frame for 2min, and transferring the supernatant to a new sample tube for later use. The method has the advantages of no toxic and harmful reagent involved in the extraction process, and safe operation.)

1. A use method of a kit for extracting blood genome DNA by a magnetic bead method is characterized by comprising the following steps:

(1) taking 100 mu L of blood sample into a sample tube, then adding 300 mu L of cell lysate, uniformly mixing, incubating at room temperature for 4-6min, centrifuging at room temperature for 1-2min under the conditions of 10000-12000rpm, and removing supernatant;

(2) sequentially adding 200 μ L lysis solution and 10 μ L proteinase K into the obtained precipitate, mixing well, and incubating for 12-15min at 55-57 deg.C and rotation speed of 800-;

(3) standing at room temperature for 5-8min, centrifuging at room temperature under 10000-12000rpm for 1-2min, transferring the supernatant to a new sample tube, adding 100 μ L of magnetic beads into the sample tube, vortex mixing by using a vortex mixer, standing at room temperature for incubation for 5-7min, centrifuging, and putting the sample tube on a magnetic frame until the solution is clear;

(4) removing the supernatant, adding 500 mu L of 75% ethanol solution, continuing to uniformly mix by vortex, then placing on a magnetic frame for 30-40S, repeating the steps once, centrifuging, then removing the residual ethanol solution at the bottom by using a pipettor, and standing at room temperature for 2-5min until the magnetic beads do not reflect light;

(5) adding 30 mu L of nuclease-free water/low-salt buffer solution, taking the sample tube from the magnetic frame to remove the heavy suspension magnetic beads, standing at room temperature for 4-6min, placing the sample tube on the magnetic frame for 2-3min, sucking the supernatant by a pipette, transferring the supernatant to a new sample tube, and carrying out quantitative quality inspection for later use.

Technical Field

The invention belongs to the field of molecular biology, and particularly relates to a use method of a magnetic bead method blood genome DNA extraction kit.

Background

In recent years, molecular biology technology has been rapidly developed, and researches on genome level have become hot spots for researches of extensive researchers, and many routine experiments of molecular biology are premised on extraction of genomic DNA. Whole genome DNA is a vector of genetic information, including all genetic information including coding and non-coding sequences. The extraction of high quality DNA from human or animal blood is an important prerequisite for gene detection, precision medicine and other molecular biological studies.

The method for extracting the blood genome DNA commercialized in the market at present is mainly a centrifugal column method, and a centrifugal column kit is simpler to operate than the traditional DNA extraction method, and has the defects that the operation in a centrifugal tube is needed, a DNA product can be obtained after repeated centrifugal cleaning is needed, the operation process needs to frequently transfer a centrifugal column out of the centrifugal tube and transfer the centrifugal column into the centrifugal tube and cooperate with a large amount of centrifugal work, so that the operation time is increased, a large amount of labor is needed, the operation efficiency can be accepted for one or more samples, but the operation is still not ideal for large-scale high-throughput DNA extraction.

In recent years, the magnetic bead method is beginning to be applied to the purification work of different systems of DNA. The magnetic affinity adsorbent is prepared by utilizing groups introduced by the way of copolymerization, surface modification and the like in the surface of a proper magnetic bead and a buffer solution. When a biological sample is incubated with the affinity adsorbents with the magnetic effect, target molecules can be rapidly captured by the magnetic beads, adsorbed from a liquid phase under the action of a magnetic field, and separated through simple washing to obtain a magnetic bead compound with the surface bound with the target molecules. Although the method can be used for quickly extracting the blood sample, the extraction time is obviously increased along with the increase of the samples due to the pretreatment step of the red blood cells, and the workload of experimenters is correspondingly increased.

Disclosure of Invention

The invention aims to solve the problems and provides a use method of a kit for extracting blood genome DNA by a magnetic bead method.

In order to achieve the purpose, the invention is realized by the following technical scheme:

a use method of a kit for extracting blood genome DNA by a magnetic bead method is characterized by comprising the following steps:

(1) taking 100 mu L of blood sample into a sample tube, then adding 300 mu L of cell lysate, uniformly mixing, incubating at room temperature for 4-6min, centrifuging at room temperature for 1-2min under the conditions of 10000-12000rpm, and removing supernatant;

(2) sequentially adding 200 μ L lysis solution and 10 μ L proteinase K into the obtained precipitate, mixing well, and incubating for 12-15min at 55-57 deg.C and rotation speed of 800-;

(3) standing at room temperature for 5-8min, centrifuging at room temperature under 10000-12000rpm for 1-2min, transferring the supernatant to a new sample tube, adding 100 μ L of magnetic beads into the sample tube, vortex mixing by using a vortex mixer, standing at room temperature for incubation for 5-7min, centrifuging, and putting the sample tube on a magnetic frame until the solution is clear;

(4) removing the supernatant, adding 500 mu L of 75% ethanol solution, continuing to uniformly mix by vortex, then placing on a magnetic frame for 30-40S, repeating the steps once, centrifuging, then removing the residual ethanol solution at the bottom by using a pipettor, and standing at room temperature for 2-5min until the magnetic beads do not reflect light;

(5) adding 30 mu L of nuclease-free water/low-salt buffer solution, taking the sample tube from the magnetic frame to remove the heavy suspension magnetic beads, standing at room temperature for 4-6min, placing the sample tube on the magnetic frame for 2-3min, sucking the supernatant by a pipette, transferring the supernatant to a new sample tube, and carrying out quantitative quality inspection for later use.

The invention has the beneficial effects that: the use method of the kit for extracting the high-quality genomic DNA from the blood by the paramagnetic particle method is suitable for separating and purifying the high-quality genomic DNA from 100 mu L of whole blood, the A260/280 of the kit is more than 1.8, the total amount can reach 1.5-2 mu g, the whole extraction process does not involve toxic and harmful reagents, the operation process is safe, the extraction of the high-quality DNA can be completed in about 45 minutes in the whole process, and the kit is convenient, rapid and stable. Through matching of an efficient lysate system and unique magnetic beads, a sample (whole blood) is subjected to cracking digestion under the action of the efficient lysate and proteinase K, DNA is released into the lysate, the magnetic beads are added to enable the sample to be specifically combined with the DNA, in ethanol solution with a certain proportion, protein and other impurities in the sample system are removed, only the DNA combined on the magnetic beads is left, and finally the DNA is eluted in water solution or low-salt buffer solution, so that the obtained DNA can be used for experiments such as high-throughput sequencing experiments, enzyme digestion, PCR amplification, fluorescence quantitative PCR and the like.

Detailed Description

The technical solution of the present invention will be further described with reference to the following examples.

The use method of the kit for extracting the blood genome DNA by the paramagnetic particle method is characterized by comprising the following steps of:

(1) adding 100 mu L of blood sample into a sample tube, adding 300 mu L of cell lysate, uniformly mixing, incubating at room temperature for 5min, centrifuging at the room temperature of 12000rpm for 1min, and removing supernatant;

(2) sequentially adding 200 μ L of lysate and 10 μ L of proteinase K into the obtained precipitate, mixing well, and incubating for 15min at 56 deg.C and rotation speed of 800 rpm;

(3) standing at room temperature for 5-8min, centrifuging at room temperature under 10000-12000rpm for 1-2min, transferring the supernatant into a new sample tube, adding 100 μ L of magnetic beads into the sample tube, vortex mixing by using a vortex mixer, standing at room temperature for incubation for 5min, centrifuging, and putting the sample tube on a magnetic frame until the solution is clear;

(4) removing the supernatant, adding 500 mu L of 75% ethanol solution, continuing to uniformly mix by vortex, then placing on a magnetic frame for 30S, repeating the step once, centrifuging, then removing the residual ethanol solution at the bottom by using a pipettor, and standing at room temperature for 4min until the magnetic beads do not reflect light;

(5) adding 30 μ L of nuclease-free water/low-salt buffer solution, taking the sample tube from the magnetic frame, standing at room temperature for 5min, placing the sample tube on the magnetic frame for 2min, sucking the supernatant with a pipette, transferring the supernatant to a new sample tube, and performing quantitative quality inspection for later use.

The use method of the kit for extracting the high-quality genomic DNA from the blood by the paramagnetic particle method is suitable for separating and purifying the high-quality genomic DNA from 100 mu L of whole blood, the A260/280 of the kit is more than 1.8, the total amount can reach 1.5-2 mu g, the whole extraction process does not involve toxic and harmful reagents, the operation process is safe, the extraction of the high-quality DNA can be completed in about 45 minutes in the whole process, and the kit is convenient, rapid and stable. Through matching of an efficient lysate system and unique magnetic beads, a sample (whole blood) is subjected to cracking digestion under the action of the efficient lysate and proteinase K, DNA is released into the lysate, the magnetic beads are added to enable the sample to be specifically combined with the DNA, in ethanol solution with a certain proportion, protein and other impurities in the sample system are removed, only the DNA combined on the magnetic beads is left, and finally the DNA is eluted in water solution or low-salt buffer solution, so that the obtained DNA can be used for experiments such as high-throughput sequencing experiments, enzyme digestion, PCR amplification, fluorescence quantitative PCR and the like.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.