Primer pair, castanopsis hystrix SSR2 (Simple Sequence Repeat 2) marker and preparation method and application thereof
A primer pair, red cone technology, applied in biochemical equipment and methods, DNA preparation, microbial determination/inspection, etc., can solve problems such as poor stability, inability to directly apply molecular marker-assisted breeding and QTL mapping, etc.
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[0028] Extraction and enzyme digestion of total DNA of red cone
[0029] Select the young leaves of Red Cone, and use CTAB method to extract the nuclear genome; digest with EcoRV at 37°C for 3 hours, and inactivate the endonuclease at 65°C for 20 minutes; the reaction system is 20 μL, including 2.0 μL of 10×Buffer, 1.0 μL EcoRV endonuclease, 2 μL of DNA, 15.0 μL of sterilized double distilled water;
[0030] Connection of connector
[0031] Connect the red cone genomic DNA digested by EcoRV in step to the upper and lower linkers (upper linker sequence GTAATACGACTCACTATAGGGCACGCGTGGTCGACGGCCCGGGCTGGT, sequence listing SEQ.ID.No.4; lower linker sequence ACCAGCCC-NH2, sequence listing SEQ.ID.No.5); The 60 μL ligation system contains 9 μL of enzyme-cut red cone genomic DNA, and 100 pmol of the upper and lower adapters; the ligation reaction is carried out under the condition of 1 times T4 ligase buffer, and ligated overnight at 16°C; the ligated mixture is reacted at 65°C for 2...
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