Thermal denaturation assays of oligonucleotide duplexes were conducted as described before [15 (link)]. In brief, melting curves were recorded at a heating rate of 0.5 °C/min from 5 to 95 °C in a quartz cell with 0.2 cm light path on a Cary 300-Bio Melt spectrophotometer (Varian, Palo Alto, CA, USA) equipped with a Peltier thermostabilized multicell holder (6 × 6). Prior to melting, the samples in a buffer consisting of 10 mM sodium cacodylate, pH 6.4, 125 mM KCl, and 6 mM MgCl2 (similar to the reaction buffer) were heated to 95 °C, cooled, and held for 5 min at 5 °C for duplex formation. Denaturation melting curves were recorded in a multi-wavelength mode via automatic switching of the monochromator between three wavelengths (260, 270, and 300 nm) with 1 nm bandwidth. The profile of optical density at 300 nm was used for baseline correction of the thermal denaturation curves [35 (link)]. The thermodynamic parameters obtained by the fitting of denaturation curves at 260 and 270 nm were averaged. Thermodynamic parameters were calculated in MS Excel by fitting to various schemes or by a concentration method. Mass balance equations were solved using Newton’s optimization method [15 (link)].
Cary 300 bio melt spectrophotometer
Manufactured by Agilent Technologies
Sourced in Australia
The Cary 300-Bio Melt spectrophotometer is a laboratory instrument designed to measure the absorbance of samples over a range of wavelengths. It is capable of performing melting temperature analysis of nucleic acids.
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Lab products found in correlation
4 protocols using cary 300 bio melt spectrophotometer
1
Thermal Denaturation of Oligonucleotide Duplexes
2
Thermal Denaturation of Oligonucleotide Duplexes
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Oligonucleotide duplexes were obtained by heating equimolar amounts of complementary strands in cacodylate buffer solution at 95 °C for 3 min, followed by incubation at 25 °C until complete cooling. Thermal denaturation experiments were performed using a Cary 300-BioMelt spectrophotometer equipped with a Peltier temperature-controlled cuvette holder (Varian, Sydney, Australia). Equimolar amounts (10 μM, 50 μL of each strand) of complementary oligonucleotides in cacodylate buffer were used. Melting curves were recorded at 260 and 270 nm over a temperature range of 5 °C to 95 °C at a heating rate of 0.5 °C/min.
3
Thermal Denaturation Study of Oligonucleotide Complexes
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Thermal denaturation experiments were carried out in quartz cells (0.2 cm path length) using a Cary 300-Bio Melt spectrophotometer (Varian, Australia) equipped with a Peltier thermostabilized 6 × 6 holder. Melting curves were registered at wavelengths 260, 270, and 300 nm in the 5–95 °C range at a temperature change rate of 0.5 °C min−1. Absorbance at 300 nm (baseline) was subtracted from the values at 260 and 270 nm at each temperature. The maximum of the derivative of a melting curve with respect to temperature was defined as melting temperature, Tm. All oligonucleotide samples were prepared in a buffer composed of 100 mM NaCl, 10 mM sodium cacodylate (CH3)2AsO2, and 10 mM MgCl2, pH 7.2. An equimolar mixture of components M and N at 10 μM in the absence or presence of the S-type oligomer was studied.
4
Thermal Denaturation of Oligonucleotide Complexes
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Thermal denaturation studies of oligonucleotide
complexes were carried out in 0.2 cm path length quartz cells using
a Cary 300-Bio Melt spectrophotometer (Varian, Australia) equipped
with a Peltier thermostabilized multicell holder (6 × 6). Each
melting curve was recorded at a heating rate of 0.5 °C/min in
the range of 5–95 °C. The components of the DNA/DNA duplexes
in equimolar per nucleotide amounts were placed in a buffer solution
at 0.1 mM per nucleotide concentration as in the case analysis with
dA12 and with poly(dA) and at 20 μM (0.2 mM/per nucleotide)
concentration in the case analysis of heteronucleotide oligomers.
The thermal stability for the DNA/RNA duplexes with complementary
oligoribonucleotide 5′-UGUUUGGCGC-3′ was studied at
5 μM (0.05 mM/per nucleotide) concentration and at 10 μM
(0.1 mM/per nucleotide), the concentration for oligonucleotide 14
with the multiple modification. Denaturation melting curves were recorded
in a multi-wavelength mode by automatic switching of the monochromator
between three wavelengths (260, 270, and 330 nm) with 1 nm bandwidth.
The profile of optical density at 330 nm was used for the baseline
correction of thermal denaturation curves. The values of thermodynamic
parameters obtained by fitting denaturation curves at 260 and 270
nm using two-state model were averaged.37 (link) Thermodynamic parameters were calculated in MS Excel by fitting
procedure.
complexes were carried out in 0.2 cm path length quartz cells using
a Cary 300-Bio Melt spectrophotometer (Varian, Australia) equipped
with a Peltier thermostabilized multicell holder (6 × 6). Each
melting curve was recorded at a heating rate of 0.5 °C/min in
the range of 5–95 °C. The components of the DNA/DNA duplexes
in equimolar per nucleotide amounts were placed in a buffer solution
at 0.1 mM per nucleotide concentration as in the case analysis with
dA12 and with poly(dA) and at 20 μM (0.2 mM/per nucleotide)
concentration in the case analysis of heteronucleotide oligomers.
The thermal stability for the DNA/RNA duplexes with complementary
oligoribonucleotide 5′-UGUUUGGCGC-3′ was studied at
5 μM (0.05 mM/per nucleotide) concentration and at 10 μM
(0.1 mM/per nucleotide), the concentration for oligonucleotide 14
with the multiple modification. Denaturation melting curves were recorded
in a multi-wavelength mode by automatic switching of the monochromator
between three wavelengths (260, 270, and 330 nm) with 1 nm bandwidth.
The profile of optical density at 330 nm was used for the baseline
correction of thermal denaturation curves. The values of thermodynamic
parameters obtained by fitting denaturation curves at 260 and 270
nm using two-state model were averaged.37 (link) Thermodynamic parameters were calculated in MS Excel by fitting
procedure.
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