Mj research ptc 200

One hundred microliter amplification reactions were performed in an MJ Research PTC-200 thermal cycler (Bio-Rad Inc, Hercules, CA, USA) containing: 50 mM Tris (pH 8.3), 500 μg/ml bovine serum albumin (BSA), 2.5 mM MgCl2, 250 μM of each deoxynucleotide triphosphate (dNTP), 400 nM of the forward PCR primer, 200 nM of each reverse PCR primer, 4 μl of DNA template, and 2.5 units JumpStart Taq DNA polymerase (Sigma-Aldrich, St. Louis, MO, USA). PCR primers targeted a portion of the SSU and LSU rRNA genes and the hypervariable internal spacer region, with the reverse primers including a 12-bp barcode (Table S2); primer binding sites are the reverse and complement of the commonly used SSU primer 1492R (Frank et al., 2008 (link)) and LSU primer 129F (Hunt et al., 2006 (link)). PCR primers were only frozen and thawed once. Thermal cycling parameters were 94°C for 5 min; 35 cycles of 94°C for 20 sec, 56°C for 20 s, and 72°C for 40 s, and followed by 72°C for 5 min. PCR products were purified using the MinElute 96 UF PCR Purification Kit (Qiagen). DNA sequencing was performed using an Illumina HiSeq 2000 (Illumina, Inc). Clusters were created using template concentrations of 2.5 pM. One hundred base sequencing reads of the 5′ end of the amplicons and seven base barcode reads were obtained using the sequencing primers listed in Table S2.

The ribosomal RNA gene and ITS DNA sequences from P. effusa, P. schachtii, and those of other oomycetes were aligned and examined for differences and similarities using DNasis software (version 2.09; MiraiBio, Inc., Alameda, CA). These alignments included sequences available at the National Center for Biotechnology Information (NCBI). For additional sequence comparisons, DNA from downy-mildew-infected leaves was amplified using primers UF1 (5′-TGAATGCGCATCGTGC-3′) and UR2 (5′-AGATGCCA CACAACCGAAG-3′), designed to amplify the rDNA sequence spanning a portion of the 18S RNA gene, ITS1, 5.8S RNA gene, and a portion of ITS2 from Peronospora spp. DNA was extracted from downy-mildew-infected leaf tissue using a NucleoSpin Plant II kit (Machery-Nagel, Bethlehem, PA). The rDNA sequence was amplified using an MJ Research PTC200 thermal cycler (Bio-Rad, Hercules, CA) using 200 nM each of primers UF1 and UR2 and 1× GoTaq Mix (Promega Corp., Madison, WI). The reaction conditions consisted of an initial denaturation at 94°C for 3 min followed by 35 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 30 s. A final extension at 72°C was for 5 min. The PCR products were cloned into pCR4.0-TOPO (Life Technologies, Carlsbad, CA) and sequenced (Eton Biosciences, San Diego, CA). All of those listed in Supplemental Table 1 were sequenced and submitted to GenBank.