Smarter cdna cloning kit

The bodies of A. japonica were flash frozen in liquid nitrogen and ground to powder form. Total RNA was extracted with Isogen solution (Nippon Gene, Tokyo, Japan). Poly(A) RNA was collected using an Oligotex-dT30 mRNA purification kit (Takara, Otsu, Japan), and cDNA was synthesized using a SMARTer cDNA Cloning Kit (Clontech). The desired DNA fragment was amplified by polymerase chain reaction (PCR) using two degenerate primers: DF3, 5′-TAYTAYYTIGAYCCITAYGTIATIAA-3′, and DR1, 5′-AARTAICCRAAIACRTCIGCRCA-3′. This DNA fragment was cloned into a pTAC-2 vector using E. coli JM109 cells (Clontech) and sequenced with an ABI PRISM 3130 Genetic Analyzer (Applied Biosystems). The amino acid sequence deduced from this DNA fragment agreed with the peptide sequences determined from the purified protein. Therefore, 3′- and 5′-rapid amplification of cDNA ends (3′-RACE and 5′-RACE) was preformed using primers F2, F3, R2, and R4, with F3 and R4 designed from this region using a SMARTer cDNA Cloning Kit and used for a nested PCR.