Ezna microelute genomic dna kit

Sampled cod eggs were extracted for DNA using the E.Z.N.A MicroElute Genomic DNA Kit (Omega Bio‐tek, Norcross, GA), following the manufacturer's instructions for tissue samples with only one minor modification: the last elution buffer step being done twice through the same filter (25 µl was eluted). Genomic DNA from juvenile and spawning cod was extracted from a small piece of the dorsal fin, using E.Z.N.A Tissue DNA kit (Omega Biotek) following the protocol. DNA from all individual cod samples was quality‐verified and quantified with a NanoDrop instrument (NanoVue Plus, GE healthcare).

The genomic DNA was extracted by using the E.Z.N.A.^® MicroElute Genomic DNA Kit (Omega Bio-tek, Norcross, GA, USA), according to the manufacturer’s protocol, and quantified by Nanodrop spectrophotometry (Thermo Scientific, Waltham, MA, USA). The purified DNA was sequenced by using PacBio Sequel Single Molecule Real-Time (SMRT) sequencing technology. After genome assembly, the coding sequences (CDSs) were predicted using prodigal software (v2.6.3). Common function annotation was performed by BLAST against the NCBI nonredundant protein (NR), cluster of orthologous groups of proteins (COG), gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), Swiss-Prot, and CAZyme databases.

To quantify how food treatment affected sporozoite production for each infected mosquito, we performed genomic DNA extraction and qPCR analysis for Plasmodium genomes in midguts saved from oocyst dissection. Plasmodium genomic DNA was extracted from midguts using the E.Z.N.A. MicroElute Genomic DNA kit (Omega Bio-Tek, as per the manufacturer’s protocol). DNA was eluted in 20 μL of molecular grade water, and the number of parasite genomes present in midguts was quantified using a previously developed qPCR assay [47 (link)]. Briefly, reactions were run on an ABI Prism 7500 Sequence Detection System (TaqMan). Initial denaturation was 20 seconds at 95°C followed by 40 cycles of a three-second 95°C denaturation period and a 30-second 60°C period of annealing and extension. Primers and probes were designed to amplify DNA from several Plasmodium species. We constructed standard curves for P. yoelii genome detection by extracting DNA from a known number of infected mouse red blood cells using the BloodPrep kit (Applied Biosystems) on the ABI Prism 6100 Nucleic Acid Prep Station (as per the manufacturer’s protocol). Parasite production per oocyst was evaluated by dividing the total number of parasite genomes by the number of oocysts quantified for each midgut. We used both sporozoite production per midgut and per oocyst as measures of the efficiency of parasite replication.

DNA was extracted from specimens obtained from various sources (Table 1) using the Omega Bio-tek EZNA MicroElute Genomic DNA Kit (Omega Bio-tek, Norcross, GA) or with a MO-BIO Powermax Soil DNA Isolation Kit. As most of the newly sequenced taxa were small-bodied, in most cases entire specimens were placed directly into lysis buffer, and if size permitted, were ground with a sterile pestle prior to digestion to break open shells. DNA concentration was measured using a Qubit 4 Fluorometer (Thermo Fisher Scientific, Waltham, MA) with the ds DNA HS kit. Samples that yielded too little DNA for library preparation (Rissoella morrocayensis and Omalogyra atomus) were amplified with multiple strand displacement amplification using the Illustra Single Cell GenomiPhi DNA Amplification Kit (GE Healthcare, Chicago, IL). Dual-indexed sequencing libraries were prepared with the Illumina Nextera Kit (Illumina, San Diego, CA). Library size was assessed via agarose gel following a test PCR (run with provided Illumina primers 1.1 and 2.1, run 95 °C for 10 min followed by 40 cycles of [95° for 10 s, 60° for 30 s]). Pooled libraries were sequenced with a 2 × 100 bp paired-end TruSeq 3000/4000 SBS kit on an Illumina HiSeq4000 (Macrogen, South Korea) using 1/24 lane each.