Prism 377

Yeast and microalgae were isolated from winery wastewater sampled from a winery in the Stellenbosch wine region (Stellenbosch, South Africa) in November 2014. The ITS-5.8S rRNA gene was used to identify the yeast, Saccharomyces cerevisiae, while the 18S rRNA gene was used to identify the microalga, Chlorella sorokiniana. All sequencing was performed using an ABI Prism 377 automated DNA sequencer at the Central Analytical Facility at Stellenbosch University and identified using BLAST analysis as described previously [18] (link).

The total volume of the PCR mixture (Takara, Tokyo, Japan) was 25 mL, including 1X PCR buffer, 0.2 mM dNTPs, 0.04 U/mL Taq enzyme, and ~0.6 ng DNA template, with a primer concentration of 0.12 mM. The PCR amplification conditions were: pre-denaturation at 94°C for 2 min; 35 cycles of denaturation at 94°C for 30 s, annealing at 52°C for 30 s, and extension at 72°C for 1 min; followed by extension at 72°C for 10 min and storage at 4°C.
The amplification products were detected by 4% polyacrylamide gel electrophoresis. An ABI PRISM 377 automatic sequencer was used to scan the electropherogram, with the GeneScan 3.1 software used for data extraction. The Matrix file of plastic films was installed, the appropriate internal standard was selected, and analysis parameters were set. The BinThere software was used to set the fragment sizes, and the resulting data were imported into the GeneScan 3.1 software, so that the corresponding peak spectra and related data could be obtained. The sizes and peak areas of the fluorescence signals from the DNA fragments from electrophoresis were analyzed, according to the positions of intra-molecular electrophoresis markers. The alleles were then numbered in ascending order so that the genotype of each sample could be obtained.