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A method for improving phenol extraction of total RNA

A technology for extracting liquid and solution, applied in the field of genetic engineering, can solve the problems of unstable genomic DNA residual effect, time-consuming and labor-intensive, etc., and achieve the effects of less residual genomic DNA, simple extraction process and high purity

Active Publication Date: 2021-03-23
WANNAN MEDICAL COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The preparation of this "acid phenol" is very time-consuming and laborious, and in actual use, the effect of this "acid phenol" on removing the residual genomic DNA in RNA samples is not stable

Method used

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  • A method for improving phenol extraction of total RNA
  • A method for improving phenol extraction of total RNA
  • A method for improving phenol extraction of total RNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] a) Pick a flat plate and streak culture a single colony of Escherichia coli strain DH5α to 100ml LB liquid medium, culture it overnight at 37°C with shaking. Bacteria were harvested by centrifugation. The bacterial cells obtained by centrifuging 1.5 mL of the bacterial liquid were resuspended in 100 μL of ultrapure water, and the mass of the bacterial cells was 20-50 mg;

[0036] b) Add 100 μL of sodium lauryl sulfate solution with a weight concentration of 2±0.2%, mix thoroughly, and lyse at room temperature for 3 minutes.

[0037] c) Add 200 μL of phenol extract 1, which is a water-saturated phenol: chloroform: isoamyl alcohol mixture with a volume ratio of 25:24:1, repeatedly oscillate and mix; centrifuge at 12000r / min for 10 -15 minutes; draw 200 μL of supernatant;

[0038] d) Add 400 μL of absolute ethanol to the supernatant obtained in step c), and gently invert and mix well. After the precipitation is complete, centrifuge at 12000 r / min for 10 minutes; discard ...

Embodiment 2

[0046] Comparison of this method with the results of phenol extraction of RNA using different pH sodium acetate solutions:

[0047] Repeat steps a) to e) in Example 1; take 50 μL of the total nucleic acid solution in step e) respectively, and add 50 μL of 50 mmol / L sodium acetate solution and 50 μL sodium chloride solution with a pH of 3.13-7.20; repeat g) to i) step, that is, to obtain the RNA sample, and its electrophoresis inspection result is as follows figure 1 shown.

[0048] From figure 1It can be seen that when the buffer pH is less than or equal to 3.51, the purified sample only contains part of the RNA; when the buffer pH is greater than or equal to 4.21, the purified nucleic acid sample contains genomic DNA; when the buffer pH is equal to 3.85 , the method disclosed in Example 1, the purified sample contains intact total RNA and does not contain genomic DNA.

Embodiment 3

[0050] Genomic DNA residue detection:

[0051] Take 1 microgram of the total RNA sample obtained in step i) of Example 1, and the RNA sample obtained by the Trizol method and the phenol extraction method, and use RNase treatment to amplify using genome-specific primers. According to the comparison with the domain cycle number of the standard sample, the standard sample is Escherichia coli genomic DNA with known copy number, it can be seen that the residual genomic DNA of the sample obtained by this method is the least, about 10 per microgram. 4 copy. Residual genomic DNA in samples obtained by Trizol method and phenol extraction method were about 10 per microgram 5 copy and 10 7 copy, such as figure 2 shown.

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Abstract

The invention discloses a method for extracting total RNA by improved phenol. The method comprises the following steps: obtaining total nucleic acid by cracking bacteria and performing extraction by water saturated phenol, chloroform and isoamylol according to the volume ratio of 25:24:1, mixing an acidic sodium acetate solution and the total nucleic acid with the pH value of 3.85+ / -0.10, and performing extraction and purification by water saturated phenol, chloroform and isoamylol according to the volume ratio of 125:24:1 to obtain the total RNA not containing genome DNA. The method is simple in extraction process, and DNA enzyme does not need to be used for digesting and removing the genome DNA in the total nucleic acid; and the total RNA sample obtained by extraction has high purity and high amplification ability.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to an improved method for extracting total RNA with phenol. Background technique [0002] With the continuous development of molecular biology, high-quality RNA samples are the key to many experiments. And some experiments, such as reverse transcription PCR (RT-PCR), gene chips, etc., require high-purity and high-quality RNA samples. A stable and efficient RNA extraction method is crucial for related experimental research. [0003] The most classic method of extracting RNA is the "phenol extraction method". However, the definition of pH value in the process of phenol extraction is not clear in the academic circles at present, and there are even some misunderstandings. "Molecular Cloning Experiment Guide" (Third Edition), written by J. Sambrook and D.W. Russell, Science Press 2002, page 1587 mentions the pH value of the so-called phenol phase, but in fact ph...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/10
CPCC12N15/1003
Inventor 徐蕾孙璐管国宇程星怡张孝普石萌黄钱武吕俊凌烈锋
Owner WANNAN MEDICAL COLLEGE
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