2.3.1. 30
RACE
Total RNA of leaf tissue (1 mg) was used to synthesize the first strand cDNA according to the manufacturer’s instructions of the 30 full RACE Core Set Ver. 2.0 (TaKaRa, Japan). The 30 ends of genes were amplified in two rounds of PCR with the gene-specific primers (Table 1) designed according to the reported NHX1 and VHA-c genes of other plants from GenBank.The first PCR was performed by denaturing the cDNA at 94 C for 3 min followed by 20 cycles of amplification (94 C for 30 s, 53 C for 30 s, 72 C for 120 s (for RrNHX1)) and extension at 72 C for 10 min. The product of the first round
PCR was used as template in the nested PCR amplification and 30 cycles were run under other PCR conditions like in
the case of first PCR.