Improvement of Astragaloside content by endogenous gene overexpression technology
An overexpression and endogenous gene technology, applied in genetic engineering, plant gene improvement, recombinant DNA technology, etc., can solve the problems of long growth cycle of transgenic plants, more genes and less
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[0024] 1) Total RNA extraction (one-step method)
[0025] Preparation of reaction reagents: denaturing solution (26mM sodium citrate pH 4.0, 0.5% sodium lauryl sarcosinate, 0.125M 2-mercaptoethanol, 4M guanidine isothiocyanate); NaAc (2M, pH 4.7); phenol : chloroform: isoamyl alcohol (25:24:1); 70% ethanol. All reagents are prepared with RNase-free water treated with 0.1% DEPC water, and all glass containers are RNase-free.
[0026] Operating procedures: Take an appropriate amount of fresh plant tissue and place it in a ceramic mortar, add liquid nitrogen and quickly grind it to a fine powder, transfer it to a 1.5ml centrifuge tube containing 650 μl denaturing solution and mix well; add 65 μl 2M NaAc (pH 4.0), and invert repeatedly Mix well; then add 650 μl phenol: chloroform: isoamyl alcohol, mix well, and ice-bath for 15 min; 4°C, 12000 rpm, centrifuge for 20 min; transfer the supernatant to a new 1.5 ml centrifuge tube, add an equal volume of isopropanol, mix After homoge...
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