2.3 Genomic DNA isolation and PCR analysis
The colonies on the selection media were inoculated into 5 mL LB medium, and incubated at 30 °C for 12 h to extract genomic DNA. Then, the cells were harvested and suspended in 100 μL extraction buffer (200 mM lithium acetate, 1% SDS), after which they were incubated at 70 °C for 10 min (Looke et al., 2011). Afterward, 300 μL of 96% ethanol was added to the tubes and the samples were vigorously mixed in a vortex mixer. Genomic DNA and cell debris were precipitated by centrifugation at 15,000×g for 10 min, and the pellets were dissolved in 100 μL distilled water. The cell debris was separated by centrifugation at 15,000×g for 10 min, and 1 μL of the supernatant was used for PCR analysis of the transformants and gene cloning of the LdhAp and LdhAt homologous arms from C. glutamicum , using the primers in additional file 2: Table S1. Transformants were analyzed with the LdhAp (-1042) sense, Knr anti-sense, and Knr sense/LdhAt (+1029) primer pairs (PCR 1 and PCR 2, respectively). The over-expression strains appearing on streptomycin solid media were selected by PCR analysis with LdhAp (-1042) sense and sucE anti-sense primer.