Clontech advantage 2 pcr kit

The data analysis of the hepatopancreas transcriptome of H. cumingii (unpublished data) revealed a sequence of C1qDC gene (HcC1qDC6). Then, the 5′ and 3′ fragments of HcC1qDC6 were amplified using gene-specific primers (HcC1qDC6-F: 5′-GCGTGCGAATGCCAAATAGCGGAG-3′ and HcC1qDC6-R: 5′-CCACTCCGCTATTTGGCATTCGCAC-3′) and a Universal Primer A Mix (UPM). The PCR was performed in accordance with the following procedures: 1 cycle at 94°C for 2 min; 30 cycles at 94°C for 30 s, 68°C for 30 s, and 72°C for 3 min; and 1 cycle at 72°C for 2 min using the Clontech Advantage 2 PCR Kit (Takara, Dalian, China). The full-length HcC1qDC6 cDNA was obtained by overlapping the 5′ and 3′ fragments.

The partial cDNA sequences of HcToll6 and HcToll7 were retrieved from our previous hepatopancreas transcriptome database (unpublished). In this experiment, 3′ and 5′ RACE were performed by using a Clontech Advantage^® 2 PCR kit from Takara (Japan) with two pairs of specific primers (HcToll6-F:5′-TGGATAAACGATGTGCTAAGCGACCCC-3′, HcToll6-R: 5′-CTGTGAAGACCACGCAAATAGAGACGGAAC-3′; HcToll7-F: 5′-GAGAGAACTTGGACAGGAAAGGGGGCT-3′, HcToll7-R: 5′-CGTATCGGCAGGTCGCCAAGGGTAAC-3′) to obtain the full-length cDNAs of HcToll6 and HcToll7. The amplification products were purified using a DNA gel extraction kit (Shanghai Generay Biotech Co., Ltd., Shanghai, China), inserted into the pEasy-T3 vector, and transformed into Escherichia coli Trans1-T1 cells (TransGen Biotech, Beijing, China). The putative clones were identified by PCR with M13F and M13R primers. The selected clones were sequenced by a commercial company (Springen, China).