Method for preparing tobacco-leaf-tissue PCR template

A tobacco leaf and template technology, which is applied in the preparation of tobacco leaf tissue PCR templates and in the field of transgenic tobacco leaf tissue, can solve the problems of time-consuming and increased sample cross-contamination, and achieve low sample consumption, rapid extraction and identification, and repeatability Good results

Inactive Publication Date: 2017-02-22
TOBACCO RES INST CHIN AGRI SCI ACAD
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Problems solved by technology

These additional steps will obviously increase the cross-contamination betwee

Method used

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  • Method for preparing tobacco-leaf-tissue PCR template
  • Method for preparing tobacco-leaf-tissue PCR template
  • Method for preparing tobacco-leaf-tissue PCR template

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Screening test for suitable concentration of NaOH extract: such as figure 1 As shown, the DNA template prepared by NaOH solution between 0.01mol / L and 0.1mol / L can all amplify bands, indicating that the crude extract of NaOH solution can be directly used as a template for PCR reaction. Although the template extracted by 0.001mol / L NaOH solution can also amplify the band, the brightness of the band is weak, while the DNA template prepared by the NaOH solution higher than 0.25mol / L (including 0.25mol / L) cannot be amplified. Extra strips. From the perspective of amplification effect and saving reagents, 0.01mol / L NaOH solution was finally selected as the extraction solution for the experiment.

Embodiment 2

[0026] A preparation method for tobacco leaf tissue PCR template, adding steel balls with a diameter of 0.25mm and 250μL of NaOH solution with a molar concentration of 0.01mol / L into a 2mL centrifuge tube, and then taking 10mm 2 The left and right plant leaf tissues were put in, and then the sample was ground on a grinder at a frequency of 23 Hz for 2 minutes, and 1 μL of the supernatant was taken as a template for PCR reaction. According to this method, be applied in the embodiment 3~6.

Embodiment 3

[0028] Use different brands of PCRMix to amplify the results: such as figure 2As shown, the commonly used 4 different brands of PCR Mix amplify the template prepared with 0.01M NaOH concentration using primers Ntab0067020-SF / Ntab0067020-S and Ntab0104030-SF / Ntab0104030-SR, all 4 kinds of PCR Mix can amplify bright single There was no significant difference between the amplification results.

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Abstract

The invention relates to a method for preparing a PCR template, in particular to a method for preparing a tobacco-leaf-tissue PCR template. The method includes the steps that a NaOH solution is an extracting liquid, the extracting liquid and plant leaf tissue are put into a centrifuge tube, the mixture is directly subjected to sample grinding on a sample grinding machine, and the obtained supernatant liquid directly serves as the template to carry out a PCR reaction. The method has the advantages that the heating step, the boiling step, the neutralizing step and the like are not required, the supernatant liquid can be directly used for the PCR reaction template; the prepared DNA template PCR amplification result is stable, accurate and good in repeatability, and compared with the DNA obtained with the traditional SDS extracting method and the kit extracting method, the PCR result is free of obvious difference; the method is also applied to plants such as wheat, sorghum, rice and corn; devices used for the method are small, sample consumption is small, the method is rapid, simple and convenient, low in cost and free of cross pollution, a large quantity of samples can be treated in the short time, and therefore a large quantity of transgenic tobacco samples can be subjected to DNA rapid extracting and DNA identification.

Description

technical field [0001] The invention relates to a method for preparing a PCR template, in particular to a method for preparing a PCR template for tobacco leaf tissue, especially for transgenic tobacco leaf tissue. Background technique [0002] Tobacco (Nicotiana tabacum) is currently an important economic crop in China. Cultivating high-quality, low-harm, disease-resistant and stress-resistant tobacco varieties suitable for production is the main goal of tobacco breeding at present. With the rapid development of molecular biology and the gradual maturity of transgenic technology, transgenic technology has become an important method of tobacco breeding. In recent years, with the development of Crispr-Cas9 technology, it has become possible to site-directed mutation of one or more genes or even the entire gene family in the tobacco genome. This technology can accurately improve the target traits and obtain stable expression transgenic lines through screening, which can not o...

Claims

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Application Information

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IPC IPC(8): C12N15/10
CPCC12N15/1003
Inventor 孔英珍徐宗昌王萌石大川
Owner TOBACCO RES INST CHIN AGRI SCI ACAD
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