genome sequencing of Chlorophyta sp.: Agricultural_soil
Genomic DNA was extracted using a FastDNA Spin Kit for Soil (MP Bio, USA) following the Amplification was carried out in a CFX-96 thermal cycler (Bio-Rad, USA) with the following conditions: 95 C for 3 min; 39 cycles of 95 C for 15 s, 55 C for 30 s, and 72 C for 30 s. manufacturers protocol. Reactions (25 L) contained 1 L DNA template, 0.5 L each of forward and reverse primers (10 M), 2.5 L dNTPs (10 M), 2.5 L 10buffer, 2.5 L MgCl (25 M), 0.2 L SynTaq polymerase (5 U L), and 15.3 L ddHO. Gene-specific primers SS5 (5'-GGTGATCCTGCCAGTAGTCATATGCTTG-3') and SS3 (5'-GATCCTTCCGCAGGTTCACCTACGGAAACC-3') were used. PCR products were sequenced by Sanger sequencing using SS5/SS3 primers and the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, USA). Reactions (10 L) contained 1 L primer (3.2 pmol), 0.8 L Ready Reaction Mix, 1.6 L 5 Sequencing Buffer, 25 L purified PCR product (1050 ng, depending on fragment size), and nuclease-free water to volume.