Clontech smart cdna synthesis kit

Overlapping RT-PCR was carried out using designed pairs of primers (YcCV-F1–YcCV-R1 to YcCV-F6–YcCV-R6) (Table 1) to obtain the sequence of YcCV (31 (link)). A Clontech SMART cDNA synthesis kit (TaKaRa) was used to perform rapid amplification of cDNA ends (RACE) PCR with the aim of identifying the 5′ and 3′ ends of YcCV from diseased yellow catfish. The 5′ region of the cDNA sequence was extended using 5′ RACE with a gene-specific primer (YcCV-rR1–YcCV-rR2) (Table 1). A TaKaRa RNA PCR kit (TaKaRa) was used to carry out 3′ RACE using an oligo(dT) adapter primer and YcCV-rF1–YcCV-rF2 (Table 1). Agarose gel electrophoresis was used to check the PCR product, and then the product was purified using the Wizard SV gel and PCR clean-up system (Promega, Madison, WI, USA) and ligated into vector pMD 19-T (TaKaRa, Japan) at 4°C overnight for cloning and sequencing. The primers listed in Table 1 were used for RT-PCR and RACE to detect the virus.

Overlapping RT-PCR was carried out using designed pairs of primers (YcCV-F1–YcCV-R1 to YcCV-F6–YcCV-R6) (Table 1) to obtain the sequence of YcCV (31 (link)). A Clontech SMART cDNA synthesis kit (TaKaRa) was used to perform rapid amplification of cDNA ends (RACE) PCR with the aim of identifying the 5′ and 3′ ends of YcCV from diseased yellow catfish. The 5′ region of the cDNA sequence was extended using 5′ RACE with a gene-specific primer (YcCV-rR1–YcCV-rR2) (Table 1). A TaKaRa RNA PCR kit (TaKaRa) was used to carry out 3′ RACE using an oligo(dT) adapter primer and YcCV-rF1–YcCV-rF2 (Table 1). Agarose gel electrophoresis was used to check the PCR product, and then the product was purified using the Wizard SV gel and PCR clean-up system (Promega, Madison, WI, USA) and ligated into vector pMD 19-T (TaKaRa, Japan) at 4°C overnight for cloning and sequencing. The primers listed in Table 1 were used for RT-PCR and RACE to detect the virus.