To study the impact of test compounds on DNA secondary structure, 300 ng of the pUC19 plasmid (2686 bp) were incubated with the test compounds (50 μm ) at 37 °C for different periods of time (15 min to 6 h) with continuous shaking. 4 μL of a 6x DNA loading dye were added to the 20 μL reaction solution, and the DNA samples were loaded on an agarose gel (1 % w/v in 1 x TBE) and subjected to electrophoresis initially at 60 V for 5 min and subsequently at 120 V for 90 min, in 1×TBE buffer. DNA visualization was achieved by ethidium bromide (EtBr) staining of the agarose gel in 1 x TBE (0.75 μg mL−1) for 20 min. Images were captured with the GelDoc‐It Imaging System Fusion Fx7 (Vilber Lourmat, Germany).
Geldoc it imaging system fusion fx7
Manufactured by Vilber
Sourced in Germany
The GelDoc-It Imaging System Fusion Fx7 is a versatile imaging system designed for a range of laboratory applications. It features a high-resolution camera and advanced optics to capture images of various samples, such as gels, blots, and other specimens. The system provides a user-friendly interface and offers various imaging modes to accommodate different experimental requirements.
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5 protocols using geldoc it imaging system fusion fx7
1
Investigating DNA Structural Changes
2
Probing DNA Structural Changes
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For detecting changes in DNA secondary structure upon drug treatment, 400 ng of the pUC19 plasmid (2686 bp, New England Biolabs, Ipswich, MA, USA) was incubated with the test compounds at a final concentration of 50 µM (dissolved and diluted in MilliQ water). The incubation was performed at 37 °C for different time intervals (15 min to 6 h) with continuous shaking. Additionally, 4 µL aliquots of a 6× DNA loading dye (Thermo Fisher) were added to the 20 µL reaction solution, and the DNA samples were separated in a 1% agarose gel in 1 × Tris-Borate-EDTA (TBE) buffer. Electrophoresis was initiated at 60 V for 5 min and continued at 120 V for 90 min in 1 × TBE buffer. For visualization of the DNA modification degree, the agarose gel was stained with ethidium bromide (EtBr) in 1 × TBE (0.75 µg/mL) for 20 min. Images were captured with the GelDoc-It Imaging System Fusion Fx7 (Vilber Lourmat, Eberhardzell, Germany), and data were quantified with ImageJ/Fiji 1.46 software.
3
Plasmid DNA Damage Kinetics
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Stock solutions of 3a and 3d (5 mM) were prepared in MilliQ water and respective volumes were added to reaction solutions to obtain 50 µM of the test compounds. 0.1 µg µL -1 pUC19 dsDNA (2686 bp) plasmid (New England BioLabs) was exposed to the compounds for different time intervals (15 min to 6 h) at 37 °C under continuous shaking. 20 µL of the samples was added to 4 µl of 6x DNA loading dye (ThermoFisher Scientific) and loaded onto a 1% agarose gel in 1× TBE buffer. Electrophoresis was performed at 60 V for 5 min, followed by 120 V for 90 min. Ethidium bromide (Serva) staining was carried out in 1× TBE (0.75 µg ml -1 ) for 20 min. Images were taken using the GelDoc-It Imaging System Fusion Fx7 (Vilber Lourmat, Germany).
4
DNA Plasmid Damage Assay
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Stock solutions of
the test compounds
were prepared in DMSO (Fisher Scientific) and diluted in Milli-Q water.
A 400 ng portion of pUC19 dsDNA (2686 bp) plasmid (New England BioLabs)
was incubated with 50 μM of the test compounds or cisplatin
for different time intervals (15 min to 6 h) at 37 °C under continuous
shaking. In addition to the untreated control, a linear pUC19L vector
(ThermoFisher Scientific) was used. A 20 μL portion of the samples
was added to 4 μL of 6× DNA loading dye (ThermoFisher Scientific)
and loaded into the pockets of 1% agarose gel in 1× TBE buffer.
Electrophoresis was carried out at 60 V for 5 min, followed by 120
V for 90 min. Ethidium bromide (SERVA) staining was performed in 1×
TBE (0.75 μg/mL) for 20 min. Images were taken by the GelDoc-It
Imaging System Fusion Fx7 (Vilber Lourmat, Germany). For quantification
of the spots, ImageJ/Fiji1.46 was used.
the test compounds
were prepared in DMSO (Fisher Scientific) and diluted in Milli-Q water.
A 400 ng portion of pUC19 dsDNA (2686 bp) plasmid (New England BioLabs)
was incubated with 50 μM of the test compounds or cisplatin
for different time intervals (15 min to 6 h) at 37 °C under continuous
shaking. In addition to the untreated control, a linear pUC19L vector
(ThermoFisher Scientific) was used. A 20 μL portion of the samples
was added to 4 μL of 6× DNA loading dye (ThermoFisher Scientific)
and loaded into the pockets of 1% agarose gel in 1× TBE buffer.
Electrophoresis was carried out at 60 V for 5 min, followed by 120
V for 90 min. Ethidium bromide (SERVA) staining was performed in 1×
TBE (0.75 μg/mL) for 20 min. Images were taken by the GelDoc-It
Imaging System Fusion Fx7 (Vilber Lourmat, Germany). For quantification
of the spots, ImageJ/Fiji1.46 was used.
5
Evaluating DNA Interaction with Compounds
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