NdeI (10 U/µL)

Protocol tips

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	PCR-amplified fragments were digested with NdeI and XhoI enzymes (Thermo Fisher Scientific Inc.), ligated into the pET28a plasmid vector by using T4 DNA ligase (Thermo Fisher Scientific Inc.) and used to transform E. coli Top10 competent cells to isolate proper clones which were used for protein expression in E. coli BL21 (DE3) cells.

Protocol tips
PCR-amplified fragments were digested with NdeI and XhoI enzymes (Thermo Fisher Scientific Inc.), ligated into the pET28a plasmid vector by using T4 DNA ligase (Thermo Fisher Scientific Inc.) and used to transform E. coli Top10 competent cells to isolate proper clones which were used for protein expression in E. coli BL21 (DE3) cells.

Publication protocol

Primers were designed using the genome sequence data of T. fusca TM51 [19]. Genomic DNA was isolated from each Thermobifida strains by MoBio UltraClean Microbial DNA Isolation Kit following manufacturer’s instructions. The endomannanase genes man5ATf, man5ATc and man5ATh were PCR-amplified with primers man5ATf forward (5’-GGTGCCATCATATGGCCACCGGGCTCC-3’), man5ATf reverse (5’- GTGCCATCTCGAGTCAGCGAGCGGTG-3’) and the four-fold degenerate primers man5ATfd forward (5’-GGTGCCATCATATGGCCACCGGGCTSS-3’) and man5ATfd reverse 5’-GTGCCATCTCGAGTCAGCGAGCGGWS-3’), with underlined sequences harboring NdeI and XhoI restriction sites. PCR reactions were carried out by using Pfu DNA polymerase (Thermo Fisher Scientific Inc.) for 32 cycles of 30 s at 94°C, 30 s at 60°C, and 3 min at 72°C, preceded by incubation for 5 min at 96°C. PCR-amplified fragments were digested with NdeI and XhoI enzymes (Thermo Fisher Scientific Inc.), ligated into the pET28a plasmid vector by using T4 DNA ligase (Thermo Fisher Scientific Inc.) and used to transform E. coli Top10 competent cells to isolate proper clones which were used for protein expression in E. coli BL21 (DE3) cells.

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