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.