Mighty ta cloning kit

The sequence of Homo sapiens glycogen phosphorylase (Gp, NP_002854.3) was used for a BLAST search of the cDNA database of E. granulosus⁷ with BLAST/N⁸ which identified two genes encoding Gp in E. granulosus. These genes were amplified with Ex Taq DNA polymerase (Takara, Japan) using gene-specific primers. For E. granulosus Gp (EgGp) 1, the primers were 5′-ATGTCCTTAGATGAATAT-3′ (forward) and 5′- CTACTTGCTGGAGGTAGC-3′ (reverse). For EgGp2, the primers were 5′-ATGTCTCTCGATAAGCTT-3′ (forward) and 5′-CTACTTGGCGGCGGCAGG-3′ (reverse). The PCR mixture contained primers (1 μM each), a dNTP mixture (200 μM), 1 × PCR buffer and 0.5 units of ExTaq DNA polymerase. The PCR conditions were as follows: denaturation for 5 min at 95°C, 35 cycles of amplification (40 s at 95°C, 30 s at 60°C, 90 s at 72°C), and extension for 10 min at 72°C. The PCR products were separated on 1.2% agarose gels and purified with a Gel Extraction Kit (Qiagen, Germany). The purified PCR fragments were directly cloned into the pMD19-T vector (Takara, Japan) using a Mighty TA-Cloning Kit (Takara) and transformed into Escherichia coli DH5a cells (Tiangen, China). A single clone for each construct was selected and sequenced (Sunny Biotechnology, Co., Ltd., Shanghai, China). The sequence analyses and alignments were performed using MEGA 6.0⁹ and Clustal Omega¹⁰.

Using an anchor RT primer, total RNAs (500 ng/reaction) were reverse transcribed with ReverTra Ace (TOYOBO, Osaka, Japan) following the manufacturer’s protocol. Using obtained cDNAs, semiquantitative RT-PCR by PrimeStar GXL (Takara Bio Japan) for LTag, STag, and tubulin (as an internal control) mRNA was performed. The thermocycling program was as follows: initial denaturation at 94 °C for 2 min; amplification at 98 °C for 10 s, 60 °C for 15 s, and 68 °C for 3 min; and final extension at 68 °C for 10 min of amplification. The RT-PCR products were separated by 0.6% agarose gel electrophoresis and visualized with a UV transilluminator. Using the Mighty TA cloning kit (Takara Bio), the sequences of the obtained PCR bands were confirmed. The primer sequences used in this experiment are listed in Supplementary File (Table S1).

Target amplicons were separated by electrophoresis and purified using a MiniBEST Agarose Gel DNA Extraction Kit Ver. 4.0 (Takara, Kusatsu, Japan) following the manufacturer’s protocol. The purified amplicons were cloned using both a Mighty TA-cloning Kit (Takara) and chemically competent DH5 α (Enzynomics, Daejeon, South Korea). Six colonies for each amplicon were chosen for seeding in liquid medium for culture. Cloned genes were purified using a HiYield^™ Plasmid Mini Kit (RBC, Banqiao, Taiwan) and sequenced (Macrogen Seoul, South Korea) using a 3730xl DNA analyzer (Thermo Fisher Scientific).

Bisulfite treatment was performed using the CpGenome Turbo Bisulfite Modification Kit (Merck Millipore) according to the manufacturer’s recommendations. The PCR primers are listed in Supplemental Table 1. Amplified products were cloned using Mighty TA-cloning kit (TAKARA BIO INC, Shiga, Japan). Ten randomly selected clones were sequenced with the M13 forward and M13 reverse primers for each gene.