Magnetic bead process nucleic acid extraction and transformation kit, and using method thereof

A kit and magnetic bead method technology, which is applied in the field of magnetic bead method nucleic acid extraction and conversion kits, can solve the problems of decreased recovery rate and technical sensitivity, and achieve the effects of short conversion time, saving conversion process time, and improving extraction purity

Inactive Publication Date: 2018-12-18
SUREXAM BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this environment is prone to generate a large number of active oxygen groups at the same time. This active oxygen will fragment and degrade DNA, which will lead to a decrease in the sensitivity of the converted PCR and subsequent analysis techniques, and will also lead to a recovery rate of DNA. decline

Method used

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  • Magnetic bead process nucleic acid extraction and transformation kit, and using method thereof
  • Magnetic bead process nucleic acid extraction and transformation kit, and using method thereof
  • Magnetic bead process nucleic acid extraction and transformation kit, and using method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1, magnetic bead method nucleic acid extraction conversion kit and its use method

[0038] The magnetic bead method nucleic acid extraction conversion kit includes: lysate, proteinase K, magnetic bead solution, conversion reagent, nucleic acid protection agent, binding solution, washing solution I, washing solution II and eluent. The lysate includes Tris-HCl, guanidine isothiocyanate, sodium hydroxide, Nonidet P 40, sodium azide, isopropanol, Triton X-100, catechin and Carrier RNA. The magnetic bead solution includes Tris-HCl, EDTA-2Na and magnetic beads. The content of the magnetic beads in the magnetic bead liquid is 50mg / ml, and the magnetic beads are nano-magnetic particles made of supercis ferric oxide or supercis iron sesquioxide, and the surface is modified with hydroxyl or carboxyl Silica coated. Described transformation reagent comprises bisulfite and / or bisulfite, and urea and hydroquinone; Described bisulfite comprises sodium bisulfite, and descr...

Embodiment 2

[0048] Embodiment 2, conversion step optimization

[0049] Take 6 identical samples and divide them into two groups of A and B respectively, the samples of group A are transformed by the kit described in Example 1, the samples of group B are subjected to nucleic acid extraction according to the steps of the kit of the present invention, and conventional transformation is added in the transformation step The desulfo treatment step conversion in the flow process is to add a strong base and incubate at room temperature for 15 minutes after the primary washing to perform the desulfo treatment, and the other steps are the same as the conversion steps in the kit of the present invention. The two sets of nucleic acid samples after transformation are detected according to the following steps:

[0050] a) Mix the following reagents in a PCR tube:

[0051] 2×PCR Mix

20μl

taq enzyme

0.5μl

transforming DNA solution

10μl

NF water

9.5μl

...

Embodiment 3

[0055] Example 3, Effects of Different Transformation Temperatures and Transformation Times on the Transformation Effect of Nucleic Acid Samples

[0056] By setting the conversion temperature and conversion time of different groups (see Table 1 below for details), the rest of the steps were performed using the method in Example 1 to perform nucleic acid extraction and conversion on the same batch of samples. Twelve groups obtained DNA (referred to as A1-A4, B1-B4, and C1-C4 in sequence) through different transformation temperatures and transformation time methods for methylation detection by real-time fluorescent PCR. The detection results are shown in Table 2.

[0057] Table 1 Transformation condition settings of different experimental groups

[0058]

[0059] Table 2 Methylated DNA detection results after treatment with different experimental groups transformation conditions

[0060]

[0061] It can be seen that in different transformation temperature methods, using r...

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Abstract

The invention provides a magnetic bead process nucleic acid extraction and transformation kit, and a using method thereof. The kit includes a lysate, a protease K, a magnetic bead solution, a transforamtion reagent, a nucleic acid protection agent, a binding solution, a washing solution I, a washing solution II and an eluent. The using method of the kit includes the following steps: (1) lysis; (2)transformation; (3) layering; (4) binding; (5) primary washing; (6) secondary washing; (7) drying; and 8) elution. The kit and the using method thereby can complete the transformation while achievingautomatic extraction of nucleic acid from a sample. The sample is lysed, releases the nucleic acid, and then begins to transform, the nucleic acid is separated from proteins by the binding solution after the transformation is completed, the methylated nucleic acid is purified by a magnetic bead process, and a pre-denaturation step in subsequent qPCR detection of the sample is used to replace a previously omitted sulfo group removal step, so a purification process in the original nucleic acid extraction step is omitted in the entire extraction and transformation process.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a magnetic bead nucleic acid extraction and transformation kit and a method for using the kit. Background technique [0002] Epigenetics is a branch of genetics that studies the heritable changes in gene expression and regulation without changing the nucleotide sequence of the gene. There are many epigenetic phenomena, such as RNA-mediated gene Silencing, histone modification, etc. Methylation is one of the epigenetics of organisms and is closely related to the occurrence of many diseases. Studies have found that abnormal DNA methylation is closely related to the occurrence and development of tumors, and cell carcinogenesis. In recent years, epigenetics and epigenomics represented by methylation analysis have become more active. [0003] DNA methylation refers to the transfer of active methyl groups to specific bases in the DNA chain under the catalysis of DNA methyltransferase (DN...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/1013C12Q2523/308C12Q2527/125C12Q2563/143C12Q2563/149
Inventor 许嘉森吴诗扬刘志明赖炳林严志会
Owner SUREXAM BIO TECH
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