V 650 uv vis spectrophotometer

Ultraviolet absorption spectra were recorded on a Jasco V-650 UV/Vis spectrophotometer. Measurements were performed in PBS at room temperature using 1 cm quartz cells with 3.5 mL capacity.
Fluorescence in solution and within cells (λ_exc = 320 nm) were recorded using a Synergy H1 multi-mode microplate reader in 96-well black plates.

Thermodynamic measurements were performed for nine different concentrations of RNA duplex in the range of 10⁻³–10⁻⁶ M on a JASCO V-650 UV/Vis spectrophotometer in buffer containing 1 M sodium chloride, 20 mM sodium cacodylate, and 0.5 mM Na₂EDTA (pH 7)^{25 (link)}. Oligonucleotide single strand concentrations were calculated from the absorbance above 80 °C, and single strand extinction coefficients were approximated by a nearest-neighbor model. The extinction coefficient of 6-thioguanosine was considered the same as for guanosine^{26 (link)}. Absorbance vs. temperature melting curves were measured at a wavelength of 260 nm with a heating rate of 1 °C/min from 0 to 90 °C on a JASCO V-650 spectrophotometer with a thermoprogrammer. The melting curves were analyzed, and the thermodynamic parameters were calculated from a two-state model with MeltWin 3.5 software^{27 (link)}. For most sequences, ∆H° derived from T_M⁻¹ vs. ln(C_T/4) plots was within 15% of that derived from averaging the fits to individual melting curves, which was expected if the two-state model is reasonable.

Spectral characterization
in the UV/vis range of the PMP films (thickness ≈ 50 μm)
was performed using a JASCO V-650 UV/vis Spectrophotometer (accuracy
0.5 nm, range 190–850 nm, Oklahoma, OK, USA). Both total percentage
transmittance T (%) and total percentage reflectance R (%) were measured using a JASCO ISN-722 integrating sphere
(inside diameter 60 mm, range 200–870 nm).
The PMPs were
further characterized to understand if the metal nanoparticles would
reduce the decay time of the cis isomer of azobenzene.^{51 (link)} The PMPs were irradiated for 10 min using a
405 nm polarized laser at a power density of 1.5 mW/cm². Thereafter, their optical absorbance was measured after 75 min
in the dark. The optical absorbance was calculated as A (%) = 100 – T (%) – R (%).

A sample (the linear plasmid DNA or the artificial chromosome) containing 60 ng (2 μL; 30 ng/μL) of DNA was mixed with 0.1 μL (0.2 units) of DNase I and 1 μL of 10× DNase I reaction buffer. The volume of the digestion mixture was 10 μL. After 20 min of incubation at 37 °C, 0.3 μL of 0.05 M EDTA was added to inactivate the enzyme. Then, the sample was mixed with 1.7 μL of 60% glycerol and electrophoresed for 120 min at 100 V on a 1.1% agarose gel. For visualization, DNA was stained with SYBR Safe DNA Gel Stain (Invitrogen, USA). To evaluate the time-dependent kinetics of DNA degradation, UV absorbance at 260 nm was recorded over time after the addition of DNase I. Each sample (71.2 μL) containing 30 ng/μL of DNA and 10× DNase I reaction buffer (8 μL) was mixed in a cuvette. Then, DNase I (0.8 μL) was added to the cuvette, and the sample was mixed thoroughly by pipetting for ~90−105 s. The measurement of absorbance (A₂₆₀) was performed in a V-650 UV–vis spectrophotometer (JASCO, Japan) using a 10-mm path-length quartz cuvette. The absorbance was recorded at intervals of 15 s.