Purified tetranucleosome and dodecamer arrays were diluted to 10 nM and crosslinked with 1% formaldehyde for 1 h at room temperature. Crosslinked sample was dialyzed against 20 mM HEPES-NaOH pH 7.5; 1 mM EDTA; 1 mM DTT, and centrifuged at 20,000 x g to remove aggregates. Crosslinked nucleosome array samples were diluted to 1 nM using 10 mM MOPS pH 7.0 and 5 mM MgCl2, and 3 μL of sample were deposited and incubated for 2 min on freshly cleaved bare mica V1 (Ted Pella Inc.), after which was rinsed with Milli-Q water, and then gently dried under a stream of N2 perpendicular to the mica surface. AFM micrographs were taken with a MultiMode NanoScope 8 atomic force microscope (Bruker Co.) equipped with a vertical engagement scanner E. The samples were excited at their resonance frequency (280-350 kHz) with free amplitudes (Ao) of 2-10 nm and imaged in tapping mode using silicon cantilevers (Nanosensors). The image amplitude (set point As) and A0 ratio (As/A0) was kept at ~0.8 in a repulsive tip-sample interaction regime, and phase oscillations were no greater than ± 5 degrees. The surface was rastered following the fast scan axis (x) at rates of 2 Hz, capturing the retrace line to reconstruct the AFM micrographs. All samples were scanned at room temperature in air, at a relative humidity of 30%.
Bare mica v1
Manufactured by Ted Pella
The Bare Mica V1 is a high-quality mica sheet product offered by Ted Pella. Mica is a naturally occurring mineral known for its excellent electrical insulating properties and thermal stability. This product provides a thin, flexible, and transparent mica sheet that can be used in various industrial and scientific applications where these properties are required.
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Lab products found in correlation
3 protocols using bare mica v1
1
Imaging Crosslinked Nucleosome Arrays
2
AFM Characterization of Chromatin Structures
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Prior to any
dynamic studies, the quality and purity of all the samples—chromatosomes,
nucleosomes, hexasomes, and tetrasomes—were evaluated by AFM
in air. In this case, we imaged formaldehyde cross-linked samples
which were diluted in 10 mM MOPS pH 7.0 and 2 mM MgCl2 to
concentrations ranging between 2 and 4 nM. Three microliters of the
solution was deposited onto freshly cleaved bare mica V1 (Ted Pella
Inc.) and incubated for 2 min, gently rinsed with Milli-Q water, and
dried under a stream of nitrogen. Tetrasomes using the 100W50 template
were visualized by deposition on polylysine mica (0.1% v/v) and rinsed
and dried as above. AFM measurements were performed with a Multimode
AFM Nanoscope 8 (Bruker Co.). The samples were imaged in tapping mode;
the silicon cantilevers (Nanosensors) were excited at their resonance
frequency (280–350 kHz) with free amplitudes of 2–10
nm. The image amplitude (set point As)
and free amplitude (A0) ratio (As/A0) were kept
at 0.8, and the scan rate was kept at 2 Hz (∼0.006 fps). All
samples were imaged at room temperature in air, at a relative humidity
of 30%.
dynamic studies, the quality and purity of all the samples—chromatosomes,
nucleosomes, hexasomes, and tetrasomes—were evaluated by AFM
in air. In this case, we imaged formaldehyde cross-linked samples
which were diluted in 10 mM MOPS pH 7.0 and 2 mM MgCl2 to
concentrations ranging between 2 and 4 nM. Three microliters of the
solution was deposited onto freshly cleaved bare mica V1 (Ted Pella
Inc.) and incubated for 2 min, gently rinsed with Milli-Q water, and
dried under a stream of nitrogen. Tetrasomes using the 100W50 template
were visualized by deposition on polylysine mica (0.1% v/v) and rinsed
and dried as above. AFM measurements were performed with a Multimode
AFM Nanoscope 8 (Bruker Co.). The samples were imaged in tapping mode;
the silicon cantilevers (Nanosensors) were excited at their resonance
frequency (280–350 kHz) with free amplitudes of 2–10
nm. The image amplitude (set point As)
and free amplitude (A0) ratio (As/A0) were kept
at 0.8, and the scan rate was kept at 2 Hz (∼0.006 fps). All
samples were imaged at room temperature in air, at a relative humidity
of 30%.
3
Atomic Force Microscopy of Crosslinked Tetranucleosomes
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Purified tetranucleosomes were diluted to 10 nM and crosslinked with 1% formaldehyde for 1 h at room temperature. Crosslinked sample was dialyzed against 20 mM HEPES-NaOH pH 7.5; 1 mM EDTA; 1 mM DTT, and centrifuged at 20,000 x g to remove aggregates. Crosslinked tetranucleosome sample was diluted to 1 nM using 10 mM MOPS pH 7.0 and 5 mM MgCl2, and 3 µL of sample were deposited and incubated for 2 min on freshly cleaved bare mica V1 (Ted Pella Inc.), after which was rinsed with Milli-Q water, and then gently dried under a stream of N2 perpendicular to the mica surface. AFM micrographs were taken with a MultiMode NanoScope 8 atomic force microscope (Bruker Co.) equipped with a vertical engagement scanner E. The samples were excited at their resonance frequency (280-350 kHz) with free amplitudes (Ao) of 2-10 nm and imaged in tapping mode using silicon cantilevers (Nanosensors). The image amplitude (set point As) and A0 ratio (As/A0) was kept at ~0.8 in a repulsive tip-sample interaction regime, and phase oscillations were no greater than ± 5 degrees. The surface was rastered following the fast scan axis (x) at rates of 2 Hz, capturing the retrace line to reconstruct the AFM micrographs. All samples were scanned at room temperature in air, at a relative humidity of 30%.
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