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Method for extracting genome DNA

An extraction method and genome technology, applied in the fields of molecular genetics and molecular biology, can solve the problems of large reagent environmental pollution, cumbersome operation, eye damage, etc., and achieve the effect of low cost and simple operation

Inactive Publication Date: 2009-04-22
CENT SOUTH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This technology uses chemicals called alkali that attack cells without damaging them while still allowing certain enzymes from their surroundings to work properly on other parts of genome DNA. It allows for quicker analysis than previous methods like extracting nucleus tissue samples beforehand. Additionally it provides precise results at very small sizes (5-20 kilobases) making it ideal for various applications such as genetic testing, medical researchers studying disease symptoms caused by inherited mutations, clinicians working through cancer treatments, environmental scientists looking into pollution levels, and others interested in analyzing these data.

Problems solved by technology

This patented technical problem addressed by the inventors relates how to efficiently collect pure nucleic acid without damaging our environment or causing harmful chemical substances like phenols or other compounds that can be absorbed into living tissues during laboratories' procedures.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1: Extraction of Genomic DNA from Cultured Cells

[0025] 1) Collect 3×10 6 For cultured cells, add 300 μl cell lysate, and mix well by blowing back and forth with a pipette;

[0026] 2) Add 1.0 μl 8mg / ml RNase solution and mix well;

[0027] 3) Digest at 37°C for 30 minutes;

[0028] 4) Place the sample on ice for 1 minute to make it cool rapidly;

[0029] 5) Add 100 μl protein precipitation solution to the cooled sample, vortex and mix thoroughly;

[0030] 6) Centrifuge at 13,000rpm for 2 minutes;

[0031] 7) Prepare a new 1.5ml centrifuge tube, add 400μl 100% isopropanol;

[0032] 8) Carefully transfer the centrifuged supernatant to isopropanol with a pipette and mix thoroughly by inverting back and forth;

[0033] 9) Centrifuge at 13,000 rpm for 2 minutes. At this time, a white mass can be seen at the bottom of the tube;

[0034] 10) Pour off the supernatant, put the centrifuge tube upside down on the filter paper to absorb the residual liquid, then ...

Embodiment 2

[0040] Example 2: Extraction of Human Peripheral Blood Genomic DNA

[0041]1) Add 900 μl of erythrocyte lysate solution to 300 μl of anticoagulated human peripheral blood sample, let it stand at room temperature for 5 minutes, and mix it upside down from time to time until the solution becomes clear;

[0042] 2) Centrifuge the clear solution at 13,000rpm for 1 minute, and a white precipitate can be seen at the bottom of the centrifuge tube; use a 200μl pipette to carefully absorb the supernatant and discard it, and finally keep about 20-30μl of the supernatant, and use it to resuspend the precipitate ;

[0043] 3) Add 300 μl of cell lysate to the resuspended leukocytes, and mix by blowing back and forth with a pipette;

[0044] 4) The remaining steps are the same as step 2) to step 14) in Example 1.

[0045] With this method, 5-15 μg of genomic DNA can be extracted from 300 μl of whole blood.

Embodiment 3

[0046] Example 3: Extraction of Genomic DNA from Human Tissue Specimens

[0047] 1) Take human tissue specimens, first process the samples by grinding, cutting and other methods;

[0048] 2) Add 300 μl of cell lysate to every 10-20 mg of tissue, and mix by blowing back and forth with a pipette;

[0049] 3) After the cell lysate is digested, add 1-5 μl of proteinase K solution, and digest overnight at 55°C until there is no visible tissue mass;

[0050] 4) The remaining steps are the same as step 2) to step 14) in Example 1.

[0051] With this method, 10-30 μg of genomic DNA can be extracted from 10-20 mg of tissue.

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Abstract

The invention discloses a method for extracting genomic DNA. A cell lysate (5-50mmol/L Tris.Cl(pH8.0), 0.2-20mmol/L EDTA(pH8.0),0.1-10 percent SDS) is added into a sample, and a sample cell is lysed; RNase digestion is performed; the sample is cooled, a protein precipitation liquid (1-10mol/L (NH4)2SO4 or 1-10mol/L NH4Ac) is added, and protein is removed through centrifugal precipitation; and a supernatant fluid after the centrifugation is transferred into 100 percent isopropanol, and the DNA is centrifugally precipitated. Compared with the prior art, the method can quickly obtain high-purity high-quality genomic DNA, the whole process only needs 30 minutes, and an obtained DNA fragment is about between 50 and 500kb. And the method has the advantages of simple operation, no need of complicated equipment, low cost, cleanliness, no toxicity, and no harm to users and the environment.

Description

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Claims

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Application Information

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Owner CENT SOUTH UNIV
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