2.3. Isolation of Genomic DNA and Amplification of 16S rRNA
Gene
Genomic DNA of halophilic archaeal strains from the rhizospheric and
non-rhizospheric soils was isolated by CTAB method [31]. The quality
and quantity of extracted DNA was observed by agarose gel
electrophoresis in TAE buffer (Tris base, 242 g/L; glacial acetic acid,
57 ml/L; 0.5 M EDTA, 100 ml/L) and the Nanodrop, respectively.
For PCR amplification of 16S rRNA gene, universal forward primer HA1
(5’-ATTCCGGTTGA
TCCTGCCGGAGGTC-3’), and reverse primer HA1465
(5’-GATCCAGCCGCAGATTCCCC-3’), for prokaryotes were used [32].
Denaturation temperature was 95 ℃ for 5 min followed by 35 rounds of 94
℃ for 60 sec, 55 ℃ for 50 sec and 72 ℃ for 90 sec and final extension at
72 ℃ for 10 min. A reaction mixture of 25 μL was prepared by using Taq
buffer 2.5 μL (10X), MgCl2 3 μL (25 mM), Taq polymerase
1 μL, dNTPs 2 μL (2.5 mM), 2 μL of forward and reverse primers (10 pmol)
and the template DNA 2 μL (>50ng/ μL). PCR products were
purified by using gel extraction kit (Fermantas USA). Agarose gel (1%)
method and NanoDrop equipment were used to determine the quality and
quantity of the sample, respectively. Purified PCR products were
sequenced by using forward and reverse primers (Eurofins, Germany).