Sophora japonica L. transcriptome sequence-based and developed EST-SSR (expressed sequence tags-simple sequence repeats) marker primer and application thereof
A technology of transcriptome sequence and labeled primers, which is applied in the direction of recombinant DNA technology, microbial measurement/inspection, biochemical equipment and methods, etc., can solve problems affecting the innovative research and utilization of Chinese Sophora japonica germplasm resources, and achieve electrophoretic bands Clear, polymorphic rich, stable amplification results
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Embodiment 1
[0043] 1 Test materials and reagents
[0044] 1.1 Sophora japonica material
[0045] The experimental materials of this study are 233 germplasms of ancient Chinese pagoda tree (Table 1), mainly from Shandong Weifang, Tai'an, Dongying, Linyi, Heze, Yantai, Binzhou, Dezhou, Zibo, Jining, Rizhao, Liaocheng, Zaozhuang. old tree. All the germplasm of Chinese pagoda japonica ancient trees were collected from the original site in 2012 and then grafted and preserved in Yinmaquan Nursery and Zhangqiu Experimental Base of Shandong Academy of Forestry Sciences. The germplasm materials of Chinese pagoda tree ancient trees used in this study basically involve the ancient Chinese pagoda tree of various cities, counties and townships in Shandong Province.
[0046] Table 1 233 germplasms of Chinese pagoda japonica ancient trees tested
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[0052] 1.2 Primer acquisition
[0053] The high-throughput sequencing platform Illumina HiSeq ...
Embodiment 2
[0057] 2.1 Extraction of Genomic DNA from Sophora japonica
[0058] The genomic DNA of Sophora japonica was extracted using the improved CTAB method, and the young leaves of Sophora japonica were extracted as materials. And improve according to the characteristics of Chinese pagoda tree containing more phenols and protein.
[0059] 1. Preheat DNA extraction buffer 2×CTAB (100mM Tris-HCl (PH8.0), 1.4mM NaCl, 50MmEDTA (PH8.0), 2% CTAB) 700μL at 65℃, 20μL of β-mercaptoethanol (add now use), L, mix thoroughly.
[0060] 2. Weigh 0.5g of young Sophora japonica leaves, put them into a 2mL centrifuge tube, add two steel balls with a diameter of about 2mm, put them in a box filled with liquid nitrogen, freeze for about 15 seconds, and grind them with a retsch mixing grinder About 1.5min, until the leaves of Sophora japonica are in the form of fine powder, quickly add preheated L, and then put it into a 65°C water bath for about 30 minutes. During the water bath, pay attention to ...
Embodiment 3
[0098] 3.1 Detection of the concentration and purity of Genomic DNA of Sophora japonica
[0099] High-quality DNA is an important prerequisite to ensure the success of PCR amplification. The leaves of Sophora japonica contain secondary substances such as phenols and polysaccharides. These substances can inhibit the activity of Ex Taq enzyme and should be removed as much as possible during DNA extraction. In this study, PVP and β-mercaptoethanol were added to the DNA extraction liquid, and the modified CTAB method was used to extract the extracted DNA. After the UV spectrophotometer detected the extracted DNA, the results showed that the OD260 / OD280 value of the DNA was between 1.7 and 2.0 . The DNA was detected by 0.8% agarose gel electrophoresis, and the results were as follows: figure 1 As shown, it can be seen from the figure that the extracted DNA showed clear and complete bands after agarose gel electrophoresis without obvious tailing. This shows that the concentration ...
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