3500 sequencer

PCR was performed on individual bacterial colonies using a ScreenMix-HS reaction mixture (Evrogen, Moscow, Russia) in an Eppendorf MasterCycler Personal cycler. Direct and reverse primers for bacterial DNA amplification were 785F (5′-GGATTAGATACCCTGGTA) and 1492R (5′-TACGGYTACCTTGTTACGACTT) [17 (link)], respectively. PCR was performed in the following regime: Denaturation at 94 °C for 4 min, 35 cycles of 94 °C for 30 s, 52 °C for 30 s, and 72 °C for 45 s, and final elongation at 72 °C for 10 min. Amplified fragments were isolated from the gel and purified with a Cleanup Standard kit (Evrogen, Moscow, Russia). Purified PCR products were sequenced with an Applied Biosystems 3500 sequencer using a BigDye Terminator v3.1 Cycle Sequencing Kit, and obtained nucleotide sequences were analyzed using the GenBank database. Organisms were assigned to certain taxa if their DNA sequences coincided by no less than 99% with the corresponding sequences already deposited in the databases by at least five different authors.

GoTaq Green Master Mix was used to mix a single colony. Reaction mixes for 25 µL reaction volume were prepared in accordance with the manufacturer’s instructions. PCR was operated on individual bacterial colonies using Applied Biosystems^® Veriti^® 96-Well Thermal Cycler (ThermoFisher SCIENTIFIC, Waltham, Massachusetts, United States of America). Polymerase chain reaction (PCR) was performed for exponentially amplifying target sequences from a DNA template. For bacterial DNA amplification, 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-TACGGYTACCTTGTTACGACTT-3′) primers were used. Denaturation at 94 °C for 10 min, 35 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min 50 s, and final elongation at 72 °C for 7 min were used in the PCR. The amplified fragments were isolated from the gel and purified with a QIAquick PCR Purification Kit (QIAGEN). Purified PCR products were sequenced on an Applied Biosystems 3500 sequencer with a BigDye Terminator v3.1 Cycle Sequencing Kit and the obtained nucleotide sequences were finally aligned against the NCBI database for bacterial identification. The BlastN program was used to approximate taxonomic identification of sequences obtained from 16S rDNA of endophytic bacterial isolates [14 ]. This method was used for a total of six culturable isolates from PM_2.5 samples.