Quant it picogreen assay kit

The Click-iT EdU Alexa Fluor 488 Imaging Kit (ThermoFisher Scientific) was used with a 17 h pulse of 5-ethynyl-2′-deoxyuridine (EdU) at 10 μM. Samples were then immunostained for DAPI as previously described. The Quant-iT PicoGreen Assay kit (ThermoFisher) was used to quantify the dsDNA content according to the manufacturer's instructions for the Quant-iT PicoGreen Assay kit (ThermoFisher).

Libraries were generated using barcoded PCR primers 27F 5′-AGAGTTTGATCCTGGCTCAG-3′ and 338R 5′-TGCTGCCTCCCGTAGGAGT-3′ annealing to the V1–V2 region of the 16S rRNA gene^{36 (link)}. PCR reactions were carried out in quadruplicate using Q5 High-Fidelity DNA Polymerase (New England Biolabs, Ipswich, MA). After amplification, quadruplicate PCR reactions were pooled and then purified using a 1:1 volume of SPRI beads (GE HealthCare, Chicago, IL). DNA in each sample was then quantified using Quant-iT PicoGreen Assay Kit (Thermo Fisher) and pooled in equal molar amounts. The resulting library was sequenced on the MiSeq instrument (Illumina, San Diego, CA) using 2 × 250 bp chemistry. Extraction blanks and DNA-free water were subjected to the same amplification and purification procedure to allow for empirical assessment of environmental and reagent contamination. Positive controls, consisting of eight artificial 16S gene fragments synthesized in gene blocks and combined in known abundances, were also included^{37 (link)}.

Extraction method for extracellular matrix of S. davawensis was based on a previous study.⁴⁷ The colonies were grown on a cellophane membrane placed onto MS medium. At each of the designated incubation time, the cellophane was peeled off and then soaked in 3 mL of 1.5 M NaCl solution for 5 min. The solution was transferred to a tube for centrifugation at 3000 × g for 3 min to remove cells. The resultant supernatant was used for further analyses. The treated cellophane was not used for any experiment. DNA concentration in the extract was quantified using Quant-iT PicoGreen assay kit (Thermo Fisher, MA, USA). For agarose gel electrophoresis, 400 μL of the extract was subjected to ethanol precipitation and then dissolved in 40 μL of 10 mM HEPES-NaOH (pH7.5). Saccharide content was quantified by phenol-sulphate method. One aliquot of 5% phenol was added to the extract, and then 5 aliquots of sulphate was added. After cooling, absorbance at 490 nm was measured. Standard curve of glucose was used for calculation of the saccharide content in the extract. Protein concentration was quantified using Pierce BCA protein assay kit (Thermo Fischer). Standard curve of bovine serum albumin was used for calculation of the protein content in the extract.