Zizania latifolia complementary deoxyribonucleic acid (cDNA) library construction method
A construction method and library technology, applied in the field of molecular biology, can solve problems such as complex operation steps, hindering the research and application of rice breeding excellent gene resource bank, and easy degradation of RNA, so as to reduce redundancy rate, improve homogenization efficiency, The effect of large storage capacity
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[0081] The method for constructing a homogenized cDNA library of wild rice wild rice of the present invention mainly includes the following steps: extraction of total RNA, synthesis of cDNA double strands by using the SMART method, re-hybridization of cDNA after denaturation, and completion of cDNA degrading of double strand cDNA by double strand specific nuclease Homogenization process, the homogenized single-stranded cDNA components are amplified by PCR and inserted into vectors, etc.
[0082] Specific steps are as follows:
[0083] (1) Total RNA obtained from wild rice:
[0084] a. Take 1g of young leaves of wild rice, grind them into powder with liquid nitrogen in a pre-cooled mortar, and then transfer them to a 15mL RNasefree centrifuge tube;
[0085] b. Add 10 mL of Trizol reagent, vortex and mix, and let stand at room temperature for 10 minutes to fully lyse the cells;
[0086] c. Add 2 mL of chloroform, shake vigorously for 15 sec, and let stand at room temperature f...
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