Bigdye terminator kit version 1

A chloroform phenol technique was used to extract DNA, as previously described (Derdak et al., 2021 (link)). Total genomic DNA was isolated from samples and utilized as a template for polymerase chain reaction amplification of a 16S ribosomal DNA fragment (PCR). The PCR reactions have been conducted through a DLAB TC1000-G thermocycler. Two pairs of conserved universal primers were used for amplification: Fd1 5′-AGAGTTTGATCCTGGCTCAG-3 'and RP2 5′-ACGGCTACCTTGTTACGACTT-3′.
The PCR was performed in a total volume of 15 µL composed of approximately 100 ng of genomic DNA, 0.3 μL of each 10 mM dNTP (Promega), 3 μL of 5X buffer (Promega), 0.9 μL of 25 mM MgCl₂ (Promega), 0.12 μL of each 10 mM primer and 0.075 µL of Taq polymerase 5U (Promega). The PCR program used was as follow:
95 °C, 2 '/(95 °C, 40′ '- 55 °C, 40′ '- 72 °C, 1′) x35; 72 °C/5 '/4 °C.
The ExoSAP-IT purification kit (GE Healthcare) was used to purify the PCR products, and the BigDye Terminator Kit version 1.0 was used to sequence them (Applied Biosystems). The sequences obtained were corrected using BioEdit, introduced into Geneious prime and aligned via BLAST to determine the degree of similarity of our sequences with those present in the database.