The procedure of sample preparation for UV spectroscopy measurements was the same as that for the CD experiments. Absorbance-versus-temperature profile of 19G4 (at ~3 µM concentration per oligonucleotide strand) was recorded in buffer B in a 600 μL quartz microcuvette (Hellma Analytics, Müllheim, Germany) with an optical path length of 10 mm on a double-beam Hitachi U-2900 UV/visible spectrophotometer (Hitachi, Tokyo, Japan) equipped with a Hitachi thermoelectric controller. Changes in absorbance were monitored between 25 and 85 °C at 295 nm at a heating rate of 0.5 °C/min. Tm was estimated from an extremum value of the first derivative of the fitted curve for data smoothed with the Savitzky–Golay filter.
600 μl quartz microcuvette
Manufactured by Hellma
Sourced in Germany
The 600 μL quartz microcuvette is a laboratory equipment designed for spectrophotometric measurements. It is made of quartz material and has a volume capacity of 600 microliters. The microcuvette is used for holding and analyzing small sample volumes during spectroscopic analysis.
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4 protocols using 600 μl quartz microcuvette
1
Thermal Denaturation Analysis of DNA
2
Characterizing G-Quadruplex DNA Structures
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Oligonucleotides containing the G4 motif (or a d(GT)8 insert) with single-stranded flanks (Figure 4 , Table S4 ) were annealed in 20 mM Tris–HCl buffer (pH 7.3) containing 140 mM NaCl and 5 mM KCl (buffer A) to enable G4 formation. Direct annealing of an equimolar mixture of partially complementary oligonucleotide components (Figure 4 , Table S4 ) in buffer A was used to prepare double-stranded DNA models. Absorbance versus temperature profiles of DNA samples (at ~3 µM concentration per oligonucleotide strand) were recorded in a 600-μL quartz microcuvette (Hellma Analytics, Germany) with an optical path length of 10 mm on a double-beam Hitachi U-2900 UV/visible spectrophotometer (Japan) equipped with a Hitachi thermoelectric controller. Changes in absorbance were monitored between 10 and 85 °C at 295 or 260 nm at a heating rate of 0.5 °C/min. Tm defined as the temperature of the mid-point, was estimated from a maximum/minimum value of the first derivative of the fitted curve for data smoothed with the Savitzky–Golay filter.
3
Characterizing hTERT G4 Motif Stability
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Unlabeled single-stranded 96-nt DNA sample containing the WT hTERT G4 motif with single-stranded flanks (Supplementary Table S1 ) was annealed in 8 mM K-phosphate buffer (pH 7.1) containing 20 mM KCl. Absorbance-versus-temperature profile of DNA oligonucleotide (at ~4 µM concentration per oligonucleotide strand) were recorded in a 600 μL quartz microcuvette (Hellma Analytics, Müllheim, Germany) with an optical path length of 10 mm on a double-beam Hitachi U-2900 UV/visible spectrophotometer (Hitachi, Tokyo, Japan) equipped with a Hitachi thermoelectric controller. Changes in absorbance were monitored between 20 and 85 °C at 295 nm at a heating rate of 0.5 °C/min. Melting temperature (Tm), defined as the temperature of the mid-point, was estimated from a minimum value of the first derivative of the fitted curve for data smoothed with the Savitzky–Golay filter.
4
G4 DNA Melting Temperature Protocol
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DNA oligonucleotides containing the G4 motifs were annealed in 20 mM Tris–HCl buffer, pH 7.3, containing 140 mM NaCl and 5 mM KCl (buffer A), or in 20 mM Tris–HCl buffer, pH 7.3, containing 140 mM NaCl and 0.5 mM KCl (buffer B) to promote G4 formation. One or two equivalents of PSM ligands were added to pre-annealed DNA probes; the mixtures were kept for 30 min at room temperature. The absorbance profiles of DNA samples as a function of temperature (at a concentration of ~3 µM per oligonucleotide strand) were recorded in a 600-μL quartz microcuvette (Hellma Analytics, Müllheim, Germany) with an optical path length of 10 mm on a double-beam Hitachi U-2900 UV/visible spectrophotometer (Tokyo, Japan) equipped with a Hitachi thermoelectric controller. Changes in absorbance were monitored between 20 and 85 °C at 295 nm at a heating rate of 1 °C/min. Tm, defined as the mid-point temperature, was estimated from the extremum value of the first derivative of the fitted curve for data smoothed with the Savitzky–Golay filter and indicated the M ± SD of three independent experiments.
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