Platinum hot start pcr master mix 2x

All the sample DNAs from the L. vannamei, C. hongkongensis, organisms in pond water were amplified in duplicate PCRs at a 25 μL reaction volume, containing 10 μL Platinum^TM Hot Start PCR Master Mix (2X) (13000014, Thermo Fisher Scientific, Lithuania), 1 μL (each) primer 1391f and primer EukBr (10 μM), 1 μL template DNA, 8–12 μL nuclease-free water, and 0–4 μL blocking primer (10 μM). The PCR thermal cycling conditions were as follows: 3 min at 94°C; 35 cycles of 30 s at 94°C, 30 s at 55°C, 1 min at 72°C; and finally 10 min at 72°C. The PCR products were checked by electrophoresis on a 1.5% agarose gel stained with DNA_GREEN (UV) (TIANDZ). In the electrophoresis results, the decrease in intensity of the band compares to the no-blocking, reflects the inhibitory effect of the blocking primer on the host DNA. The less intense the band, the better the blocking effect.

To fill the gaps that remained after mapping Ion reads, we designed 11 primer sets (Table 4) against the NCBI MERS-CoV reference genome assembly (NC_019843.3). Primers were designed and checked using Primer Express software (v3.0) (Applied Biosystems, Foster City, CA, USA) and NetPrimer (Premier Biosoft, Palo Alto, CA, USA), with gap flanking regions incorporated into the primers. Sequences for all the major gaps were amplified by PCR using Platinum^TM Hot Start PCR Master Mix 2X (Thermo Fisher Scientific, Waltham, MA, USA). PCR products were sequenced using the BigDye^® Terminator Sequencing Kit (Applied Biosystems, Foster City, CA, USA) on an Applied Biosystems 3730xl DNA Analyzer. Most minor gaps were sample-specific; to ensure complete genome coverage for phylogenetic analysis, these gaps were closed manually after performing multiple sequence alignment (MSA) on the fully-sequenced genomes. MSA was carried out using Geneious software [34 (link)].

To extract DNA, coral tissue (~0.2 cm²) was first placed in a Power Bead Tube (Qiagen) and homogenized horizontally with a vortex adapter for 10 minutes. The samples were then processed using the DNeasy PowerSoil kit (Qiagen, Germantown, MD) following the manufacturer’s protocol. Extracted DNA was PCR amplified with 16S rRNA gene V4 primers^{25 (link)} using the Earth Microbiome Project (EMP) protocols²⁶ . Briefly, samples were processed using the Platinum Hot Start PCR Master Mix (2X) (ThermoFisher Scientific, Waltham, MA) in a 50-µl reaction with 2 µl of DNA template or 2 µl of PCR grade water for the negative control. DNA was amplified with the following parameters: 94 °C for 3 minutes (1X), 94 °C for 45 seconds (35X), 50 °C for 60 seconds (35X), 72 °C for 90 seconds (35X), and 72 °C for 10 minutes (1X). PCR products were cleaned using AMPure XP beads (Beckman Coulter, Brea, CA). Each cleaned, amplified, and barcoded DNA sample was normalized to 4 nM (except for the negative control due to its low DNA quantity). After, 5 µl of each sample and the PCR negative control were all pooled, and sent to the Hussman Institute for Human Genomics University of Miami Miller School of Medicine for sequencing on the MiSeq with PE-300v3 kits.