title: San18 :Bacillus nitratireducens San18: soil
design: Chromosomal DNA of the bacterial isolate was extracted by GeneJET Genomic DNA Purification Kit, Catalog number:  K0721 (Thermo Scientific) according to the manufacturers recommended procedure. PCR thermal cycler (Biometra cycler personal) was used for 16S rDNA gene amplification where two universal primers were used including 16S rDNA forward primer:  5-GAG TAA TGT CTG GGA AAC TGC CT-3, and 16S rDNA reverse primer:  5-CCA GTT TCG AAT GCA GTT CCC AG-3. PCR reactions mixtures were prepared as follow: 1 L of each primers were added to 1 L of genomic DNA then 12.5 L of a MyTaq HS Red Mix (2x) (Bioline Reagents Ltd) were added , and finally 9.5 L of nuclease-free water following the manufacturers guidelines were adjusted to a final concentration of 25 L. PCR conditions used in the amplification of 16S rDNA gene were: 95C for 5 min., followed by 35 cycles of 95C for 1.5 min, 59C for 1 min and 72C for 1.5 min, with final 10 min extension at 72C. PCR product was purified using Thermo Scientific Gene JET PCR purification kit ( 2013 Thermo Fisher Scientific, Inc). An automated sequencer AB 5500XL genetic analyzer (applied Biosystems, Foster City, CA, USA) at Macrogen Inc., Korea were used for sequencing the purified PCR product in forward direction. Sequence was analyzed and phylogenetic tree was constructed using Geneious Pro 8.9.0.
library_name: San18