A kind of enzyme-free whole nucleic acid extraction kit

A nucleic acid extraction reagent and kit technology, applied in the field of molecular biology, can solve problems such as unfavorable purification process, influence on product purity, extraction difficulty, etc., and achieve the effects of simple and convenient operation, good integrity, and guaranteed safety.

Active Publication Date: 2021-08-27
INTEGRATED BIOSYSTEMS CO LTD +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The cell wall of Gram-positive bacteria is mainly composed of a network structure formed by peptidoglycan. The content of peptidoglycan in the cell wall of Gram-negative bacteria is low, there is no teichoic acid, and the peptide bridge, peptide tail and disaccharide in the middle reaches of the cell wall The formed network structure is relatively loose, and the lipid content is high. Therefore, in the process of nucleic acid extraction, Gram-positive bacteria are not easy to be lysed, which makes the extraction difficult. Often scientists use lysozyme to break up the bacterial cells. It is not conducive to the subsequent purification process and affects the purity of the product

Method used

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  • A kind of enzyme-free whole nucleic acid extraction kit

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Embodiment 1

[0034] Extraction of whole nucleic acid of Escherichia coli.

[0035] 1) Take 1ml of Escherichia coli cultured overnight and centrifuge at 10,000rpm for 2 minutes.

[0036] 2) Discard the supernatant described in step 1.

[0037] 3) Add 400ul of lysate to the Escherichia coli cell precipitate obtained according to step 2, shake and mix well and let it stand for 5 minutes to completely lyse the E. coli cell;

[0038] The lysate consists of 4M guanidine isothiocyanate, 20mM Tris-HCl, 12mM EDTA, 0.8M NaAc, and 0.15% (volume ratio) Triton-X100.

[0039] 4) Add 10ul of magnetic bead solution and 200ul of binding solution to the solution obtained in step 3, vortex and mix well, and let stand at room temperature for 3 minutes;

[0040] The magnetic bead solution contains 20mg / ml magnetic microspheres, 0.4M NaCl, and 20mM Tris-HCl. The magnetic microspheres are characterized by surface modification with silanol and superparamagnetism, with a diameter of 300nm. % ethanol (volume rat...

Embodiment 2

[0053] Extraction of whole nucleic acid of Proteus.

[0054] 1) Take 1ml of Proteus cultured overnight and centrifuge at 10,000rpm for 2 minutes.

[0055] 2) Discard the supernatant described in step 1.

[0056] 3) Add 500ul of lysate to the Proteus cell precipitate obtained according to step 1 and step 2, shake and mix well and let stand for 10 minutes to completely lyse the Proteus cells;

[0057] The components of the lysate are 4.5M guanidine hydrochloride, 30mM Tris-HCl, 10mM EDTA, 1.2M NaAc, 0.5% (volume ratio) Triton-X100.

[0058] 4) Add 20ul of magnetic bead solution and 250ul of binding solution to the solution obtained in step 3, shake and mix well, and let stand at room temperature for 3 minutes;

[0059] The components of the magnetic bead liquid are 20mg / ml magnetic microspheres, 0.42M NaCl, 20mM Tris-HCl. The magnetic microspheres are surface-modified with silanol and have superparamagnetism, with a diameter of 300nm. The binding solution consists of 75% Etha...

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Abstract

The invention belongs to the field of molecular biology, and in particular relates to the extraction and purification of bacterial whole nucleic acid, which is an enzyme-free whole nucleic acid extraction kit, which is characterized in that the kit includes: a lysate, a binding liquid, a magnetic bead liquid, and a washing liquid A. Washing solution B. Dissolving solution. The bacterial cells are lysed by the lysate, and the released nucleic acid is captured by the magnetic beads. The entire extraction process does not require enzyme reagents, and the complete nucleic acid molecule can be extracted with high purity. It can be directly applied to downstream biological experiments. The extraction process is fast, non-toxic, and easy to operate. Can be used with automated instruments.

Description

technical field [0001] The invention belongs to the field of molecular biology, in particular to the extraction and purification of bacterial nucleic acid. Background technique [0002] Nucleic acid is a biological macromolecular compound polymerized by many nucleotides. According to different chemical compositions, nucleic acid can be divided into ribonucleic acid (RNA for short) and deoxyribonucleic acid (DNA for short). DNA is the main material basis for storing, replicating and transmitting genetic information. RNA plays an important role in the process of protein synthesis, so nucleic acid plays a decisive role in a series of major life phenomena such as growth, inheritance, and variation. The research on nucleic acid can essentially reveal the structure, composition, and even the mystery of the pathogenic mechanism of microorganisms, and can more effectively study how to prevent and treat the resulting diseases. Therefore, the research on nucleic acid is of vital sign...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/10
CPCC12N15/1013
Inventor 张瑜蔡亦梅高静范东雨代有来李洁昆任鲁风
Owner INTEGRATED BIOSYSTEMS CO LTD
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