Genelute genomic dna extraction kit

MicroLYSIS^®-PLUS (Microzone Ltd., Haywards Heath, West Sussex, United Kingdom) was used to release DNA from single colonies of each of the ACC positive and 18 ACC negative isolates following the manufacturer’s protocol and utilized as the template DNA in polymerase chain reaction (PCR) reactions for amplifying the and acdS gene fragments. These pseudomonad isolates were selected for full genome sequencing, in which high quality genomic DNA was extracted from 10 mL LB cultures of each isolate using the GenElute genomic DNA extraction kit (Sigma-Aldrich), as per the manufacturer’s protocol. DNA quantity was determined using the Qubit Fluorometric Quantification (Life Technologies) using the manufacturer’s instructions. DNA quality was determined using the NanoDrop Microvolume UV spectrophotometer (Thermo Fisher Scientific Inc.) by OD at 260 nm, the 260/280 ratio was used to determine DNA quality in addition to running the samples on a 1.5% (w/v) agarose gel in 1 × Tris-borate-EDTA (TBE) stained with EtBr (0.2 μg mL^–1) with TBE as the running buffer. Bands of DNA were viewed under UV light to visually detect smears arising from any degraded DNA.

Twenty-one isolates were sampled with a plankton net from 15 natural populations (NP) in the eastern United States, including ponds in Georgia, Illinois, Indiana, Missouri, Oklahoma, Pennsylvania, South Carolina and Texas (Fig. 1, S1 Table in S1 File). Daphnia were sampled from ponds accessed via public roadsides or on private land with the permission of the land owner. No specific permissions are required to sample Daphnia as they are not endangered or protected species.
Cultures were initiated in the laboratory from single females that were allowed to propagate clonally to increase the tissue available for DNA extraction [27] . DNA was extracted using the GenElute genomic DNA extraction kit (Sigma Chemical, St. Louis, MO, USA). A NanoDrop ND-8000 spectrophotometer was used to determine the concentration (ng/µL) of each sample. Isolates were coded based on their state, population and individual number. For example, IL1.2 is individual 2 from population 1 in Illinois. All 21 isolates, hereafter referred to as the NP isolates, were analyzed using qPCR but only 19 of them were analyzed using TED due to lack of sufficient DNA from the other two samples.