Phusion high fidelity dna polymerase f530 s

Cloning Dockerin-containing glycoside hydrolases were cloned from R. champanellensis genomic DNA using appropriate primers (Table S1) and Phusion High Fidelity DNA polymerase F530-S (New England Biolabs). The genes were restricted using Fastdigest enzymes (Thermo Scientific, USA) and ligated either into pET21a or pET28a using T4 DNA ligase (Fermentas UAB, Vilnius, Lithuania). The constructs were designed to contain a His-tag for subsequent purification.
The CBM-Coh gene cassette (Barak et al., 2005) consists of a family 3a CBM from the C. thermocellum CipA scaffoldin cloned into plasmid pET28a (Novagen, Madison, WI, USA), into which any cohesin gene can be introduced between BamHI and XhoI restriction sites of the plasmid. The Coh-CBM gene cassette is the same as the CBM-Coh cassette, only in reverse order of the modules. Any cohesin gene can be introduced between NcoI and BamHI restriction sites of the plasmid. The full list of fused cohesins used in this article is given in Table 3.
The PCR products were purified using a HiYield TM Gel/PCR Fragments Extraction Kit (Real Biotech Corporation, RBC, Taiwan), and plasmids were extracted using Qiagen Miniprep Kit (Netherlands). The cloning of each gene was confirmed by DNA sequencing. Competent E. coli XL1 competent cells were used for plasmid transformation.