O generuler 1 kb dna ladder

The total yeast DNA was extracted from the colonies grown on MYGP agar plates using the InstaGene Matrix DNA extraction kit (Bio-Rad Laboratories, Hercules, CA, USA). Rep-PCR was conducted in a 25 μL volume mixture containing 13 μL of Taq DNA Polymerase 2× Master Mix RED (Ampliqon, Odense, Denmark), 5 μL Primer GTG₅ (Integrated DNA Technologies, Denmark), 4 μL sterile Milli-Q water, and 3 μL DNA. The PCR reaction was carried out in a SureCycler 8800 thermocycler (Agilent Technologies, Santa Clara, CA, USA) using the following program: initial denaturation for 7 min at 95 °C, followed by 30 cycles of 95 °C for 1 min, 45 °C for 1 min, and 65 °C for 8 min, and an elongation step of 65 °C for 16 min. The rep-PCR products were separated by 1.5% agarose gel electrophoresis (5 h, 120 V) in Tris-Borate-EDTA buffer (0.5 × TBE), using an O’GeneRuler 1 kb DNA ladder (Thermo Scientific, Roskilde, Denmark) as a reference marker. The rep-PCR profiles were clustered using Bionumerics 7.1 software (Applied Maths, BioMérieux, Schaerbeek, Belgium) based on Dice’s Coefficient of similarity with the Unweighted Pair Group Method and Arithmetic mean clustering algorithm (UPGMA).

Once MDSC differentiation was observed, cells were detached from the Petri dish using 2 mM PBS/EDTA and then centrifuged at 200 g for 5 min to re-suspend them in PBS on ice. Total RNA was extracted using the RNA-Solv kit (OMEGA Biotek) and lysed using a 20G needle and syringe. To degrade genomic DNA, samples were treated with dsDNAseI (ThermoFisher Scientific). cDNA was synthesized using the Maxima H Minus First-Strand cDNA Synthesis Kit (ThermoFisher Scientific) with 0.7–1 µg of total RNA. PCR and overhang PCR (oPCR) reactions were performed according to the instructions of the Platinum SuperFi DNA Polymerase Kit (ThermoFisher Scientific) in a final volume of 20 µL. The PCR products were run on a 1% agarose gel, stained with GelStar (Lonza) 2X, and visualized using a 3UV transilluminator (Epi Chemi Darkroom). The O’Gene Ruler 1 Kb DNA Ladder (ThermoFisher Scientific) was used as a molecular weight marker.

MVs were isolated from Wrinkly Spreader biofilms and planktonic cultures by centrifugation to remove cells and matrix followed by filtration (after [17 (link)] etc.). Two hundred milliliters of 24 h culture and three-day biofilms pooled from four 1 L flasks containing 250 mL KB medium were centrifuged at 8000× g at 4 °C for 20 min and the supernatant recovered. These were then centrifuged at 24,000× g 4 °C for 30 min and the supernatants passed through 0.45 µm pore-size PES filters (MDI, Gurgaon, India). The filtrates were pelleted by centrifugation at 60,000× g at 4 °C for 2 h and finally resuspended in 600 µL PBS. MV-associated proteins were separated by SDS-PAGE and visualized with PageBlue Protein Staining Solution (ThermoFisher Scientific). An aliquot of Pierce™ Unstained Protein MW Marker (ThermoFisher Scientific) was run on the gel for sizing. Samples were also treated with 100 µg/µL DNase I (Sigma-Aldrich) in 20 mM Tris-HCl pH7.4, 10 mM MgCl₂, before the DNA was extracted with phenol-chloroform, precipitated with sodium acetate/ethanol as above, and finally resuspended in TE buffer. Samples were electrophoresed on a 1.3% (w/v) Tris-buffered EDTA (TBE)-agarose gel before being stained with ethidium bromide to visualize the DNA. An aliquot of O’GeneRuler 1 kb DNA ladder (ThermoFisher Scientific) was run on the gel for sizing.