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Quick batch extraction method for cotton DNA (Deoxyribonucleic Acid) suitable for PCR (Polymerase Chain Reaction)

A cotton, batch technology, applied in the field of rapid batch extraction of cotton DNA, can solve the problems of increasing the complexity of cotton DNA extraction, affecting molecular biology operations, and unsatisfactory, achieving fast and easy waiting for cotyledons to obtain materials, and fast and easy DNA extraction time , the effect of consistent strip size

Inactive Publication Date: 2013-09-18
HEFEI FENGLE SEED
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This is because a large number of polyphenols and polysaccharides contained in cotton cells are easily oxidized when the cells are broken and irreversibly combined with DNA; in addition, tannins and pigments in cotton can directly affect the activity of Taq enzymes, all of which affect The extraction purity of cotton DNA is affected, which in turn affects a series of molecular biology operations such as subsequent PCR.
Although the improved CTAB method and SDS method can obtain high-purity cotton DNA, most of the samples are taken from cotton leaves. Using EP tubes to extract one by one, not only wastes materials, but also consumes time and labor, which increases the complexity of cotton DNA extraction. , and the operation is still relatively complicated, which cannot meet the demand for cotton DNA in large-scale PCR detection

Method used

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  • Quick batch extraction method for cotton DNA (Deoxyribonucleic Acid) suitable for PCR (Polymerase Chain Reaction)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1 (a method for rapid batch extraction of cotton DNA suitable for PCR in the present invention).

[0019] Take Guofeng Cotton 198 as an example: Take the parental and standard F1 generation of Guofeng Cotton 198, and germinate indoors on sandy soil. After 5-6 days, two cotyledons are unfolded, and the sample can be collected.

[0020] Follow the steps below:

[0021] 1) Take the 0.45-0.55cm of the above two plants of the female parent, male parent and F1 generation at the top of the hypocotyl of healthy cotton in the seedling stage, and place them in each well of a 96-well PCR plate (preferably on ice Operation), and add 40μL of 0.25mol / L NaOH solution to each well;

[0022] 2) Add step 1) the top of hypocotyl and the PCR plate with NaOH solution to each well, and boil it in boiling water in a water bath for 1-1.5min;

[0023] 3) Add 60μL of 0.17mol / L Tris-Hcl solution to each well of the PCR plate after step 2) boiled, and then place it in boiling water in a water bath ...

Embodiment 2

[0026] Example 2 (using the current improved CTAB cotton genomic DNA extraction method, as a comparative example).

[0027] ① The samples were collected by collecting cotyledons from the same plants of the female parent, male parent, and F1 generation of Guofeng Cotton 198 taken in Example 1, put them into 2.0 mL centrifuge tubes, and add 600 μL of freshly prepared extraction buffer , Grinding machine for grinding.

[0028] ②Centrifuge at 10000rpm for 10min (4℃), discard the supernatant.

[0029] ②Add 600 μL of 65 ℃ preheated lysis buffer to the precipitate, and use a copper wire or toothpick to stir and mix well. In a 65 ℃ water bath for 30 minutes, gently shake once every 10 minutes.

[0030] At the end of the water bath, add 600μL of chloroform: isoamyl alcohol (24:1) mixture and turn over 30 times until the lower solution turns black.

[0031] ⑤Centrifuge at 10000rpm for 10min (room temperature), transfer the supernatant to a 1.5mL centrifuge tube, add an equal volume of pre-cooled...

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Abstract

The invention discloses a quick batch extraction method for cotton DNA (Deoxyribonucleic Acid) suitable for PCR (Polymerase Chain Reaction). The method comprises the following steps: filling the top end of the hypocotyledonary axis of a cotton seedling in a hole of a PCR plate; adding NaOH liquor; crushing cells of a sample in a boiling water bath; taking the sample out and adding Tris-Hcl liquor; then putting the sample liquor in a boiling water bath; taking the sample liquor out and cooling; and diluting the sample liquor for 10-30 times by ddH2O to obtain a PCR template of cotton DNA. The banding pattern of the cotton DNA obtained by adopting the quick batch extraction method is highly consistent with that of the DNA extracted by a conventional CTAB (Cetyltrimethyl Ammonium Bromide) method through detection of an SSR (Simple Sequence Repeat) molecular marker. The method disclosed by the invention is simple and quick, fussy procedures of the CTAB method and a SDS (Sodium Dodecyl Sulfate) method are avoided, and time and labor are saved. The method disclosed by the invention is suitable for projects such as DNA fingerprint purity detection of crossbred species of cotton in batches and cotton breeding improvement needing to extract DNA in batches for detection.

Description

Technical field [0001] The invention relates to a method for rapid batch extraction of cotton DNA suitable for PCR. Background technique [0002] Cotton is an important economic crop in my country. In recent years, the phenomenon of cotton varieties being numerous, disordered and mixed is very common. In order to regulate the market and protect the interests of cotton farmers and seed companies, the authenticity and purity of cotton seeds are indispensable. At present, the purity identification of cotton species in my country still relies on the field identification method, because it not only requires a lot of manpower, material resources, high cost, but also time-consuming and poor timeliness, which is of little significance to the market and production. With the development of molecular biology, the use of molecular marker technology to identify the purity of varieties at the genetic level has been widely used in rice, corn, wheat, soybeans, rape and other crops. Among them...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
Inventor 李婧婧周桂林彭家成陈先锋程国旺马惠应黄书伟张庆虎
Owner HEFEI FENGLE SEED
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