Pmd 19 cloning vector

We designed a pair of specific primers (Forward: 5′-GAG CCG ATT GTT GAA GCG GAA AAA G-3′; Reverse: 5′-TGG CCT TGA TAG CGT TGT TCA AAA T-3′) of Garfield_BM (Zhang et al. 2014 (link)) using its internal sequence to determine its distribution in some insects. PCR was performed with an initial denaturation step of 4 min at 95 °C followed by 30 cycles of 40 s at 95 °C, 40 s at 58 °C, and 2 m at 72 °C. Then, purified PCR products were cloned into PMD-19 cloning vector (TaKaRa). One or two random clones of each species were selected and sequenced.

To validate the presence of Merrow, Garfield, and Conan identified computationally, their polymerase chain reaction (PCR) primers were designed using their flanking or internal sequences (supplementary table S2, Supplementary Material online). PCR was carried out with an initial denaturation step of 4 min at 95 °C followed by 32–35 cycles of 40 s at 95 °C, 40 s at 55–58 °C, and 2 m at 72 °C. Then, PCR products were run in 1% agarose gels in 1× Tris acetate–ethylenediaminetetraacetic acid buffer and visualized under UV light. Purified PCR products were cloned into PMD-19 cloning vector (TaKaRa). One random clone of each species was selected and sequenced.