Zymo quick dna fungal bacterial miniprep kit

Microbial Plaque DNA was extracted from plaque samples using the Zymo Quick-DNA Fungal Bacterial Miniprep KIT (Zymo Research, Irvine, CA, USA), and extraction was performed according to the manufacturer’s protocol. DNA quality and quantity were determined using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The Qubit 4.0 Fluorometer was used to quantify the metagenomic DNA (mgDNA) using the Qubit dsDNA HS assay kit according to the manufacturer’s protocol (MAN0017455 Rev. A.0). The mgDNA purity was established using the NanoDrop ND-1000 spectrophotometer, while the LapChip GXII was used to determine the genomic quality score following use of the DNA Reagent Kit (PerkinElmer, Waltham, MA, USA) and Genomic DNA (gDNA) Quality Control kit according to the manufacturer’s protocol (CLS140166 Rev. C; Supplementary Report). A genomic score between 0 and 5 was used, with 5 indicating high-quality gDNA.

A modified protocol for qPCR was performed based on previously published methods (43 (link)). Spn strains were streaked and grown through secondary culture as above, grown in triplicate. One culture remained untreated, one was treated with 1/10th MIC, and the final culture was treated with 192 μg/mL erythromycin. Cultures were grown for 1 h before the bacteria were pelleted at 4K rpm for 5 min and frozen at −80°C. Genomic DNA was isolated from the pellets using the Zymo Quick-DNA Fungal/Bacterial Miniprep kit (Zymo Research) according to the manufacturer’s protocol. Genomic DNA concentration was determined using a NanoDrop 8000 spectrophotometer. The samples were diluted to 15 ng/μL, and qPCR was performed with standard curves using 10-fold serial dilutions of untreated genomic DNA. Gene copy numbers were determined relative to the fab(K) control using the ΔCt method.