QIAprep 96 Turbo BioRobot Kit (4)

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Screening of genes encoding glycosyl hydrolase from environmental DNA: To amplify partial fragments of genes encoding GHF10, 10–250 ng of each DNA isolated from digestive tracts of horses or termites were added to 50 μl of EX Taq Buffer (TaKaTa‐Bio) containing 0.2 mM each of dNTPs, 1 μM each of primers, XYN‐Fw and XYN‐Rv (Table 3), and 5 U of Ex Taq HotStart version (TaKaRa‐Bio). Polymerase chain reaction was performed with the following cycling programme: 1 cycle of 30 s at 95°C; 30 cycles of 30 s at 95°C, 30 s at 55°C and 15 s at 72°C; and 7 min of final extension at 72°C. The PCR product was purified, ligated to the pCR®4‐TOPO® Vector with a TOPO TA cloning kit (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol, and used to transform ECOSTM‐competent E. coli DH5α cells (Nippongene). Transformants were plated on LB agar containing 50 μg ml−1 ampicillin. Colonies were picked up, suspended in 1.3 ml of LB medium in the wells of a deep‐well 96‐well plate (Qiagen, Hilden, Germany) and grown at 37°C for 20 h. Cells in the cultures were harvested, and plasmid DNA was isolated by using a QIAprep® 96 Turbo BioRobot Kit (Qiagen) and a Qiagen Biorobot 8000 (Qiagen). DNA sequencing was performed by using M13‐reverse and forward primers and a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems), and analysed by using an ABI3130 Genetic Analyser. Sequence data were assembled by using ATGC software (Applied Biosystems).

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