Figure 4. RT-PCR detection of sodC mRNA in B. neotomae and B. abortus RB51 (A) and identification of their 5′ ends by RACE analysis (B).
A) Total RNA extracted from the bacterial cultures at late log phase was used as template in RT-PCR or direct PCR for amplification of a 5′ portion of sodC mRNA using a pair of specific primers. Post-amplification reaction mixtures of tubes containing RNA extracted from B. neotomae (lanes 1 and 2) or B. abortus RB51 (lanes 3 and 4) were separated on a 2% agarose gel by electrophoresis and stained with ethidium bromide to visualize the DNA fragments. Lanes 1 and 3, RT-PCR reaction mixtures. Lanes 2 and 4, PCR reaction mixtures. Lanes 5 and 6, reaction mixtures from no-template-controls of RT-PCR and PCR reactions, respectively. Lane marked MW contains DNA molecular size marker, and the numbers at the left indicate molecular size in base-pairs. B) Representative nucleotide sequencing chromatograms showing the location of 5′ ends of the cloned sodC cDNAs from B. abortus RB51 (i & ii) and B. neotomae (ii).
