Genomic DNA Extraction and PCR Analysis

C. acetobutylicum ATCC 824 and C. sporogenes ATCC 15579 genomic DNAs for use in cloning and PCR analysis were prepared using QIAGEN DNeasy Blood and Tissue Kit in accordance with the manufacturer’s instructions and recommended pretreatment for Gram-positive bacteria using Lysozyme from chicken egg white (Cat L6876 Sigma-Aldrich) 20 ml/ml phosphate buffer saline (PBS) [25 (link)]. Screening PCRs were performed using DreamTaq™ green PCR master mix (Fisher Scientific UK Ltd) and Failsafe™ PCR system (Epicentre) for downstream cloning in accordance with the manufacturer’s instructions. Primers were designed using web tool available at http://primer3.ut.ee and are listed in Additional file 1: Table S1. All the DNA manipulations including restriction digestion, de-phosphorylation of 5′ end and ligation were carried out according to standard procedures as described in [44 ]. DNA extraction and purification from agarose gel and PCR reaction mixtures were undertaken using QIAquick Gel Extraction and PCR Purification Kit (Qiagen Ltd UK), respectively, and used according to the manufacturer’s instructions.