Primer for cloning limit dextrinase gene, kit and cloning method

Abstract

The invention provides a primer for cloning limit dextrinase gene, a kit and a cloning method, and belongs to the technical field of gene cloning. The primer, the kit, and the method can solve the problem that RNA extraction from malt is difficult and complicated at present, and RNA purity and quality are not high, and full-length limit dextrinase gene cannot be obtained. The technical scheme comprises the following steps: designing five gene sequences according to the gene sequence of the limit dextrinase, and aiming at four gene sequences LD-2, LD-3, LD-4, LD-5. Designing upstream and downstream specific primers, using upstream and downstream primers to clone limit dextrinase gene, including: synthesis of limit dextrinase gene fragment, overlap extension PCR, full-length gene acquisition. The invention has the advantages of simple principle and simple operation, and effectively solves the problem that the target gene of the barley long fragment is difficult to be amplified. The purity and yield of malt RNA were low in this method. Gene cloning of limit dextrinase and optimization of its N-terminal sequence lay a foundation for heterologous expression and enzymatic properties.

Description

technical field [0001] The invention belongs to the technical field of gene cloning, in particular to a primer, a kit and a cloning method for cloning a limit dextrinase gene. Background technique [0002] To obtain the target gene of eukaryotes, it is necessary to extract RNA and use reverse transcription PC (RT-PCR) to amplify the target gene product. Target genes with small gene fragments are easy to obtain by RT-PCR, while long-segment genes are difficult to obtain. Large, it is required that the extracted RNA must contain the full-length target gene fragment and the reverse-transcribed cDNA is complete. In addition, the PCR enzyme is required to amplify long fragments and have high enough fidelity. Therefore, obtaining long fragments from eukaryotes Fragmentation of the target gene becomes a difficult point. [0003] Malt endosperm contains a large amount of starch and polyphenols, which makes the extracted RNA easy to degrade and be polluted. At the same time, total R...

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Problems solved by technology

[0005] The present invention aims at the current problem that extraction of malt RNA is relatively difficult, the steps are cumbersome and complicated, resulting in low RNA purity and quality, and the inability to obtain the full-length limit dextrinase gene. The present invention establishes a set of The method of cloning limit dextrinase gene from malt

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