Primer pair, castanopsis hystrix SSR4 (Simple Sequence Repeat 4) marker and preparation method and application thereof
A primer pair, red cone technology, applied in biochemical equipment and methods, DNA preparation, microbial assay/inspection, etc., can solve problems such as inability to directly apply molecular marker-assisted breeding and QTL positioning, poor stability, etc.
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[0028] Extraction and Restriction Digestion of Total DNA from Rhododendron
[0029] Select the young leaves of C. erythraea and extract the nuclear genome by CTAB method; digest with EcoRV for 3 hours at 37°C and inactivate endonuclease at 65°C for 20 minutes; reaction system 20μL, including 2.0μL of 10×Buffer, 1.0μL of EcoRV endonuclease, 2μL DNA, 15.0μL sterilized double distilled water;
[0030] Connector connection
[0031] Steps EcoRV digested red cone genomic DNA is connected to the upper and lower linkers (upper linker sequence GTAATACGACTCACTATAGGGCACGCGTGGTCGACGGCCCGGGCTGGT, sequence listing SEQ.ID.No.4; lower linker sequence ACCAGCCC-NH 2 , Sequence Listing SEQ.ID.No.5); 60μL ligation system contains 9μL of digested red cone genomic DNA, and the upper and lower adapters are 100pmol each; the ligation reaction is carried out under 1x T4 ligase buffer conditions and ligated overnight at 16°C; ligation The latter mixture was reacted at 65°C for 20 minutes to inactivate the...
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