Gelred nucleic acid stain

Genomic DNA (gDNA) was extracted from fresh mycelia (100 mg) of the Alternaria cultures (Table 1) using a ZR Quick-DNA Fungal/Bacterial MicropPrep™Kit (Zymo Research, Tustin, CA, USA). The DNA concentration and purity were determined with a NanoDrop Lite ND-2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The DNA was standardised to a final concentration of 10 ng/µL for polymerase chain reaction.
Polymerase chain reaction (PCR) was performed on the extracted gDNA using Alternaria major allergen (Alt a1) gene primers (Alt-For: 5′-ATG CAG TTC ACC ACC ATC GC-3′ and Alt-Rev: 5′-ACG AGG GTG AYG TAG GCG TC-3′) [37 (link)]. The PCR reaction (50 μL) consisted of 50–100 ng of template DNA, 0.3 μM of each primer, 2.5 mM MgCl₂, 0.3 mM of each dNTP, and 1 U KAPA HiFi HotStart DNA Polymerase (Kapa Biosystems-Roche, Basel, Switzerland).
The T100TM Thermal Cycler conditions (Bio-Rad, Hercules, CA, USA) for PCR amplification entailed an initial denaturation step of 3 min at 95 °C followed by 24 cycles of 20 s at 98 °C, 30 s annealing step at 63.3 °C, 60 s at 72 °C and a final elongation step of 3 min at 72 °C. The PCR products stained with GelRed nucleic acid stain (Thermo Fisher Scientific) were examined using 2% agarose gel electrophoresis, visualised under a UV light “Gel Doc EZ Gel Documentation System” (Bio-Rad).

DNA or nucleosome probes at 35 nM (700 fmol/reaction) were incubated with MBP-tagged CLAMP DBD protein or MBP-tagged FL CLAMP protein in a binding buffer. The binding reaction buffer conditions are similar to conditions previously used to test ZLD nucleosome binding (McDaniel et al., 2019 (link)) in 20 µl total volume: 7.5 µl BSA/HEGK buffer (12.5 mM HEPES, pH 7.0, 0.5 mM EDTA, 0.5 mM EGTA, 5% glycerol, 50 mM KCl, 0.05 mg/ml BSA, 0.2 mM PMSF, 1 mM DTT, 0.25 mM ZnCl₂, and 0.006% NP-40) 10 µl probe mix (5 ng poly[d-(IC)], 5 mM MgCl₂, 700 fmol probe), and 2.5 µl protein dilution (0.5µM, 1 µM, and 2.5 µM) at room temperature for 60 min. Reactions were loaded onto 6% DNA retardation gels (Thermo Fisher Scientific) and run in 0.5× Tris–borate–EDTA buffer for 2 hr. Gels were post stained with GelRed Nucleic Acid Stain (Thermo Fisher Scientific) for 30 min and visualized using the ChemiDoc MP imaging system (Bio-Rad).

Gel electrophoresis was performed in a 1/10 dilution of the PCR amplicon was as a quality control measure to assess successful amplification of a single band. Materials used for electrophoresis were Agarose I™ biotechnology grade (ThermoFisher), 0.5 mL GelRed^® Nucleic Acid Stain (ThermoFisher), 100 bp DNA Molecular Weight Marker (Invitrogen, Waltham, MA, USA), 4X Gel loading buffer (BPB) (ThermoFisher), and distilled water molecular grade. GelRed stain was added to 1% warm agarose prepared in TBE, and 25 mL of agarose was loaded into the gel tray chamber with 1X TBE buffer. Each amplified DNA sample, 2 μL, was mixed with 7 μL loading dye on parafilm and then loaded into gel wells. One well was loaded with 5 μL of DNA molecular weight markers. The run was conducted at a constant 100 V for at least 30 min, and then observed under UV light (254 nm) to visualize the expected band size of approximately 1400 bp.