18S amplicon sequencing of estuarine water: Haulover Canal March 2019 replicate a
The EMP primers Euk1391F (5 GTACACACCGCCCGTC 3) and EukBR (5 TGATCCTTCTGCAGGTTCACCTAC 3) were used to amplify the eukaryotic hypervariable V9 region of 18S rRNA. Primers were synthesized by Integrated DNA Technologies (Coralville, IA, USA). Each 20 ?L PCR reaction contained: 1X Phusion Hit-Start II High-Fidelity Master Mix, 200 ?M bovine serum albumin, 250 nM of each primer, nuclease-free water, and 1 ?L template DNA Reactions. These reactions were run on the SimpliAmp Thermal Cycler (Applied Biosystems, Foster City, CA). Thermocycler conditions consisted of an initial denaturation at 98 C for 1:30 minutes, followed by 30 cycles of 98 C for 10 seconds, 56 C for 20 seconds, and 72 C for 30 seconds, and then a final extension at 72 C for 5 minutes. Purified PCR amplicons were sent to GeneWiz (South Plainfield, NJ, USA) for 18S sequencing on the HiSeq platform.