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Kit and method for extracting microbial DNA

A kit and microbial technology, applied in the field of molecular biology, can solve the problems of unsatisfactory extraction of microbial DNA, cumbersome operation steps, and loss of species, and achieve the effects of good versatility, high purity, and fast extraction speed

Active Publication Date: 2012-07-25
中生方政生物技术股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The direct method adopts the method of cracking soil microorganisms in situ. The genomic DNA obtained by this method is a crude extract, which contains more impurities such as humic acid and yellow acid, which will inhibit downstream experiments and must be purified and recovered before use. ; while the indirect method uses differential centrifugation to recover the bacteria, and then cracks the bacteria to extract genomic DNA, but this extraction method requires special instruments, and the operation steps are cumbersome and time-consuming, and it is easy to cause the loss of species
Aiming at the difficulties and characteristics of microbial DNA extraction in soil, some large biological companies have developed commercial kits for extracting microbial DNA in soil, such as Mo Bio UltraClean Soil DNAKit (MoBio Laboratories, Inc., Solana Beach, Calif.), although this This commercial kit is very convenient and quick to use, but the effect of extracting microbial DNA is not ideal

Method used

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  • Kit and method for extracting microbial DNA
  • Kit and method for extracting microbial DNA
  • Kit and method for extracting microbial DNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Embodiment 1: the kit for extracting microbial DNA according to the present invention

[0069] The kit for extracting microbial DNA of the present invention comprises:

[0070] Extraction buffer: 120mM guanidine isothiocyanate, 181mM Na at pH 7.0 3 PO 4 . Specific preparation method: Weigh 1.42 grams of guanidine isothiocyanate, add enough distilled water to fully dissolve, then add Na with a pH of 7.0 and a concentration of 1M 3 PO 4 18.1ml, mix thoroughly, and dilute to 100ml.

[0071] Lysis solution I: 100 mM Tris-HCl (pH 8.0), 100 mM EDTA (pH 8.0), 1.5 M NaCl, 1% CTAB, 1% PVP. Specific preparation method: Weigh 1 gram of CTAB and 1 gram of PVP10 respectively, add appropriate amount of distilled water to dissolve respectively, then add 10 ml of 1M Tris-HCl (pH 8.0), 20 ml of 0.5 M EDTA (pH 8.0), 30 ml of 5 M NaCl , and then dilute to 100ml.

[0072] Lysis Solution II: SDS at a final concentration of 2%.

[0073] Inhibitor removal solution I: 250 mM ammonium a...

Embodiment 2

[0079] Embodiment 2: extract microbial DNA from soil

[0080] The kit described in Example 1 was used for extraction.

[0081] 1. Extraction reagent

[0082] Extraction buffer: 120mM guanidine isothiocyanate, 181mM Na at pH 7.0 3 PO 4 .

[0083] Lysis solution I: 100 mM Tris-HCl (pH 8.0), 100 mM EDTA (pH 8.0), 1.5 M NaCl, 1% CTAB, 1% PVP.

[0084] Lysis Solution II: SDS at a final concentration of 2%.

[0085] Inhibitor removal solution I: 250 mM ammonium acetate.

[0086] Inhibitor removal solution II: 120 mM ammonium aluminum sulfate.

[0087] Binding solution: 5M guanidine hydrochloride, 20mM Tris-HCl (pH 7.5), 30% isopropanol.

[0088] Rinsing solution: 10 mM Tris-HCl with a pH value of 7.5, 100 mM NaCl, and 80% absolute ethanol.

[0089] Eluent: 10 mM Tris-HCl, pH 8.0.

[0090] Glass cellulose membrane adsorption column: 0.45 μm glass cellulose membrane, Whatman Company, GF / B type, 1.5 mL spin column.

[0091] 2. Extraction method

[0092] (1) Take the soil sam...

Embodiment 3

[0100] Embodiment 3: the microbial DNA concentration and purity detection of extraction

[0101] Same as Example 1, the same amount of soil samples were extracted with Mo Bio UltraClean Soil DNA Kit to extract microbial DNA, and the microbial DNA extracted in Example 1 and the microbial DNA extracted with Mo BioUltraClean Soil DNA Kit were tested for concentration and purity. The results are shown in the table 1.

[0102] Table 1 Detection of microbial DNA concentration and purity

[0103] The method of the invention

[0104] The results show that the concentration of microbial DNA extracted in Example 1 is greater than the concentration of microbial DNA extracted with Mo BioUltraClean Soil DNA Kit; The value is closer to the standard value (1.7-2.0) than the DNA extracted by the MO BIO kit. The above results show that with the same sample size, the DNA extracted by the present invention has a high concentration and less impurities.

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Abstract

The invention relates to the field of molecular biology and discloses a kit and a method for extracting microbial DNA. The kit comprises lysis solution, inhibitor removal solution and binding solution, wherein the lysis solution comprises 50 to 200mM of Tris-HCl, 50 to 150mM of EDTA, 0.5 to 3M of NaCl, 0.5 to 2 percent of CTAB, 0.5 to 2 percent of PVP and 0.5 to 2 percent of SDS; the inhibitor removal solution comprises 100 to 300mM potassium acetate, sodium acetate or ammonium acetate and 50 to 200mM aluminum sulfate, ammonium sulfate or aluminum ammonium sulfate; and the binding solution comprises 3 to 6M guanidine hydrochloride, 10 to 50mM Tris-HCl and 5 to 50 percent isopropanol. The kit and the method for extracting the microbial DNA have the advantages of high purity, high universality, high extraction speed, direct use for a downstream experiment, and application to extracting DNA from a microbe-containing sample.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a kit for extracting microbial DNA, and also to a method for extracting microbial DNA. Background technique [0002] Microorganisms are the general term for all tiny organisms that are invisible or unclear to the naked eye. They are a large group of organisms including bacteria, viruses, fungi, and some small protozoa and microscopic algae. They are small in size and simple in structure, but they are closely related to human life. They cover many beneficial and harmful species, and are widely involved in many fields such as health, food, medicine, industry, agriculture, and environmental protection. [0003] Soil microorganisms are rich in resources and are important life forms in terrestrial ecosystems. They are very sensitive to the external environment, have poor stress resistance, and are easily affected by various adverse external environments. Therefore, soil microorganism...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/10
Inventor 李艳萍
Owner 中生方政生物技术股份有限公司
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