Sword-like iris vacuolar membrane Na+/H+ antiport protein-2 gene and its application
A technology of antiporter and tonoplast membrane, applied in the field of Na+/H+ antiporter 2 gene
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[0069] 1. Ma Lin Na + / H + Cloning of Partial Fragment of Antiporter 2 Gene
[0070] Trizol method to extract total RNA from 2-week-old Iris seedlings, PowerScript TM The first-strand cDNA was synthesized by reverse transcription with reverse transcriptase, and PCR was carried out with degenerate primers: P1: 5'-GAT TCT GTW TGC ACM YTR CAG GT-'3, P2: 5'-CAT KAG MCC AGC CCA CCA WAT-'3 Amplify, obtain an amplified fragment, clone and sequence.
[0071] Amplify the target gene fragment according to the following conditions:
[0072] Reaction system: 2×GC buffer I 25μl, 10mM dNTP 3μl, 10μM primer 1 and primer 2 1.5μl each, DNA template 250ng, LA GC Taq enzyme 1μl, add water to make up to 50μl.
[0073] The reaction program was: pre-denaturation at 94°C for 3 min, followed by 36 cycles, each cycle including denaturation at 94°C for 40 s, renaturation at 59°C for 1 min, and extension at 72°C for 1 min. Finally, it was kept at 72°C for 10 min.
[0074] 2. Ma Lin Na + / H + Acq...
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