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Preparation method of PCR template for kelp sporophore seedlings and amplifying method

A sporophyte and template technology, applied in the field of molecular biology, can solve the problem of insufficient sample sampling amount, and achieve the effects of saving labor, simple method and clear results.

Inactive Publication Date: 2018-09-18
SHANDONG ORIENTAL OCEAN SCI TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] The present invention aims to provide a PCR template preparation method and amplification method for kelp sporophyte seedlings, which are used to overcome the deficiencies of the existing methods, without using any toxic reagents, and quickly obtain templates for subsequent PCR reactions through simple steps; at the same time, only Relevant experiments can be completed with a very small amount of samples to solve the problem of insufficient sample volume

Method used

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  • Preparation method of PCR template for kelp sporophore seedlings and amplifying method
  • Preparation method of PCR template for kelp sporophore seedlings and amplifying method
  • Preparation method of PCR template for kelp sporophore seedlings and amplifying method

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Embodiment 1

[0038] The present invention provides a method for preparing PCR templates for kelp sporophyte seedlings. In the present embodiment, the amplification verification of primers with different annealing temperatures is carried out, and the method comprises the following steps:

[0039] 1) Take the kelp sporophyte seedlings and absorb the water with filter paper and wipe them clean. Cut the leaves with a size of about 1mm×2mm and put them into a 1.5mL centrifuge tube.

[0040] 2), add 50μL ddH 2 O Soak the leaves of the seedlings and grind them repeatedly with a special grinding rod for a 1.5mL centrifuge tube until ddH 2 There were no sporophytic tissue pieces in O.

[0041] 3) Centrifuge at 6000 rpm for 5 minutes at room temperature.

[0042] 4) Aspirate the supernatant and transfer it to a new centrifuge tube, which can be used as a PCR reaction template.

[0043] The PCR template obtained by the preparation method of the present invention is used for amplification, and the ...

Embodiment 2

[0045] The present invention provides a method for preparing PCR templates for kelp sporophyte seedlings. In this embodiment, the comparison of sampling tissues of different sizes is carried out, and the method comprises the following steps:

[0046] 1) Take kelp sporophyte seedlings and use filter paper to absorb water and wipe them clean. Cut out a total of 4 leaves with a size of (a) 1mm×2mm (b) 2mm×4mm (c) 3mm×8mm (d) 4mm×20mm were placed in 1.5mL centrifuge tubes.

[0047] 2), add 50μL ddH 2 O Soak the leaves of the seedlings and grind them repeatedly with a special grinding rod for a 1.5mL centrifuge tube until ddH 2 There were no sporophytic tissue pieces in O.

[0048] 3) Centrifuge at 6000 rpm for 5 minutes at room temperature.

[0049] 4) Aspirate the supernatant and transfer it to a new centrifuge tube, which can be used as a PCR reaction template.

[0050] Apply the PCR template obtained by the preparation method of the present invention to amplify, the method ...

Embodiment 3

[0052] The present invention provides a method for preparing PCR templates for kelp sporophyte seedlings. In this embodiment, the comparison of different storage times for preparing templates is carried out. The method includes the following steps:

[0053] 1) Take the kelp sporophyte seedlings and absorb the water with filter paper and wipe them clean. Cut the leaves with a size of about 1mm×2mm and put them into a 1.5mL centrifuge tube.

[0054] 2), add 50μL ddH 2 O Soak the leaves of the seedlings and grind them repeatedly with a special grinding rod for a 1.5mL centrifuge tube until ddH 2 There were no sporophytic tissue pieces in O.

[0055] 3) Centrifuge at 6000 rpm for 5 minutes at room temperature.

[0056] 4) Aspirate the supernatant and transfer it to a new centrifuge tube for direct amplification, storage at 4°C for 24 hours, amplification after storage at 4°C, and amplification after storage at 4°C for 48 hours, then it can be used as a PCR reaction template.

...

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Abstract

The invention discloses a method for rapidly preparing a PCR template for kelp sporophore seedlings and an amplifying method of the PCR template. The method comprises the following steps: fetching thekelp sporophore seedlings, cutting leaves, and putting the leaves into a centrifuge tube; adding distilled water for soaking the leaves of the seedlings, and adequately grinding the leaves of the seedlings, carrying out centrifugation; and transferring supernate into a new centrifuge tube as a PCR temperature for amplification. According to the method, the steps are obviously simplified on the basis that genomic DNA is conventionally extracted for PCR detection, various reagents and kits do not need to be used, the genomic DNA does not need to be extracted, the kelp sporophore seedlings are directly ground and centrifuged, supernate is directly extracted for PCR amplification, so that the method is simple and convenient, the result is definite, a large number of manpower, material resources and financial resources are saved, and the pollution and injury which are caused to the environment and experimenters are greatly reduced.

Description

technical field [0001] The present invention belongs to the field of molecular biology. In particular, it relates to a method for quickly preparing a PCR template for kelp sporophyte seedlings, and also relates to an amplification method for the PCR template. Background technique [0002] Laminaria is a typical economically important seaweed with an alternate life history of heterosexual generations, and its gametophyte and sporophyte morphology are significantly different. Cultivating high-yield and stress-resistant new varieties and providing farmers with high-quality seedlings are important prerequisites for ensuring the sustainable development of the industrial chain. In the actual production process of kelp, the sporophyte seedlings are usually cultivated in the seedling cultivation workshop. After the seedlings grow to 2-3cm, and the sea conditions are suitable, they are temporarily raised in the sea; after the seedlings grow to 30-40cm, the seedlings are separated. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6806C12Q1/686
CPCC12Q1/6806C12Q1/686C12Q2565/125
Inventor 彭捷姜黎明刘延岭李言李晓捷田萍萍张壮志潘金华孙娟史良
Owner SHANDONG ORIENTAL OCEAN SCI TECH