Imagequant 300

Reverse transcription (RT) was carried out with 2.2 μg of total RNA primed with 500 ng of Oligo dT (Life Technologies, USA) using the SuperScript II first-strand synthesis system (Life Technologies, USA) according to the manufacturer’s instructions.
PCR was performed with 1 μl of complementary DNA (cDNA) as template and 0.4 μM of each primer (Table 1). Reactions were performed in a T3 Biometra (Biometra, Germany) thermocycler as follows: 2 min at 94°C, followed by 40 cycles of 94°C for 20 s, 57°C for 20 s, and 72°C for 40 s. The amplified products were resolved on 1.0% agarose gels, stained with Gel Red (Uniscience, Brazil), and visualised using an Image Quant 300 (GE Life Sciences, USA).

The 16S ribosomal RNA gene was amplified by PCR, using a pair of bacterial universal primers: U968F (5’-AACGCGAAGAACCTTAC-3’) and U1391R (5’-ACGGGCGGTGWGTRC-3’), specially designed for the bacterial V6-V8 hypervariable region [16 (link)–17 ]. The total volume was 50 μl, including 4 μL of 2.5 mM dNTP, 1 μL of each primer (10 μM), 0.5 μL of 5U TaKaRa Ex Taq HS (Takara Bio, Otsu, Japan), 5 μL of 10x Ex Taq buffer and 5 μL (10–20 ng) template DNA. The amplification program was conducted using a PxE Thermal Cycler (Thermo Electron Corporation, Milford, MA, USA). The first cycle was initiated at 94°C for 5 min, and followed by 30 cycles; each cycle was 94°C for 30 s, 52°C for 20 s, and 72°C for 45 s. The last cycle was 72°C for 10 min, before cooling at 4°C. Thereafter, PCR products were separated (electrophoresis) and visualized with a UV trans-illuminator (ImageQuant 300, GE Healthcare, Piscataway, NJ, USA).

The integrity of the extracellular extracts was verified by 1-D SDS-PAGE (Laemmli, 1970 (link)). Thirty micrograms of extracellular extracts from each isolate were prepared with sample buffer [0.2 M Tris–HCl, pH 6.8, 40% (w/v) SDS, 2% (w/v) β-mercaptoethanol, and bromophenol blue traces] and heated in the thermoblock at 100°C for 10 min. Subsequently, the samples were submitted to 12% 1-D SDS-PAGE. As reference, the low-molecular-weight marker (GE Healthcare, United Kingdom) was used. Soon after this step, the gels were stained by Coomassie Blue. ImageQuant 300 (GE Healthcare, United Kingdom) was used to obtain the images.