DNA damage was assessed by using the CometAssay® reagent kit for single cell gel electrophoresis assay (Trevigen, MD USA), following the recommended protocol for neutral conditions, and adapting the gel electrophoresis methods for use in the Sub-Cell GT electrophoresis system (Bio-Rad, CA USA). Briefly, cells were collected from coverslips by treatment with 0.25% trypsin, pelleted and resuspended at 100,000 cells/ml in 1X DPBS (Ca2+ and Mg2+ free; Thermo Fisher Scientific) and verified to be greater than 95% viable by tryptan blue exclusion using an automated cell counter before continuing analysis. Aproximately 5,000 cells were embedded in low melting agarose, plated on slides and lysed overnight. The next day, electrophoresis was run at 30 Volts for 30 minutes in 1X TBE (National Diagnostics). Samples were fixed in 70% ethanol for 5 minutes, and slides were immersed in 1X TE buffer pH 8.0 (Ambion) with 1:10 of 10,000x SYBR green nucleic acid stain (Thermo Fisher Scientific). Fluorescent images were captured using a Leica DMI 400B inverted microscope for scoring.
Sub cell gt electrophoresis system
Manufactured by Bio-Rad
Sourced in United States
The Sub-Cell GT electrophoresis system is a laboratory equipment designed for the separation and analysis of DNA, RNA, and protein samples using gel electrophoresis. It provides a stable and consistent platform for running electrophoresis experiments.
Automatically generated - may contain errors
Lab products found in correlation
Sybr green nucleic acid stain, by Thermo Fisher Scientific (2 mentions)
Dpbs ca2 and mg2 free, by Thermo Fisher Scientific (2 mentions)
Cometassay reagent kit for single cell gel electrophoresis assay, by Bio-Techne (2 mentions)
Dmi 400b inverted microscope, by Leica (2 mentions)
Typhoon fla 7000, by GE Healthcare (1 mentions)
Hybond xl, by GE Healthcare (1 mentions)
Hybond, by Cytiva (1 mentions)
5 protocols using sub cell gt electrophoresis system
1
DNA Damage Quantification by Comet Assay
2
Quantifying Genomic rDNA and ERC Levels
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One-half of an agarose plug, along with 500 ng of lambda HindIII DNA markers, was separated by electrophoresis on a 0.4% agarose gel (15 by 25 cm gel) in 1× Tris-acetate-EDTA (40 mM Tris base, 20 mM acetic acid, and 1 mM EDTA [pH 8.0]) at 1.0 V/cm for ∼48 h at 4°C with buffer circulation in a Sub-cell GT electrophoresis system (Bio-Rad). The buffer was changed every ∼24 h.
DNA was transferred to Hybond-XL (GE Healthcare). Southern blotting was then performed with a probe prepared by PCR amplification of genomic DNA using primers 5′-CATTTCCTATAGTTAACAGGACATGCC and 5′-AATTCGCACTATCCAGCTGCACTC , as described previously (19 (link)). The membrane was exposed to phosphor screens for an appropriate amount of time before any signals were saturated, and the radioactive signal was detected using Typhoon FLA 7000 (GE Healthcare). The membrane was reexposed to the phosphor screen for several days and scanned. The scanned images taken after short and long exposures were used to quantify genomic rDNA and ERC bands, respectively, using FUJIFILM Multi Gauge version 2.0 software (Fujifilm). The ratio of ERCs relative to genomic rDNA was determined.
DNA was transferred to Hybond-XL (GE Healthcare). Southern blotting was then performed with a probe prepared by PCR amplification of genomic DNA using primers 5′-
3
DNA Damage Quantification by Comet Assay
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DNA damage was assessed by using the CometAssay® reagent kit for single cell gel electrophoresis assay (Trevigen, MD USA), following the recommended protocol for neutral conditions, and adapting the gel electrophoresis methods for use in the Sub-Cell GT electrophoresis system (Bio-Rad, CA USA). Briefly, cells were collected from coverslips by treatment with 0.25% trypsin, pelleted and resuspended at 100,000 cells/ml in 1X DPBS (Ca2+ and Mg2+ free; Thermo Fisher Scientific) and verified to be greater than 95% viable by tryptan blue exclusion using an automated cell counter before continuing analysis. Aproximately 5,000 cells were embedded in low melting agarose, plated on slides and lysed overnight. The next day, electrophoresis was run at 30 Volts for 30 minutes in 1X TBE (National Diagnostics). Samples were fixed in 70% ethanol for 5 minutes, and slides were immersed in 1X TE buffer pH 8.0 (Ambion) with 1:10 of 10,000x SYBR green nucleic acid stain (Thermo Fisher Scientific). Fluorescent images were captured using a Leica DMI 400B inverted microscope for scoring.
4
Agarose Gel Electrophoresis of DNA Plugs
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ERC analysis was performed as previously described.51 (link) DNA plugs were cut to 5 mm width and DNA was separated using 0.4% agarose (STAR agarose, RIKAKEN) in 1× TAE on a Sub-cell GT electrophoresis system (Bio-Rad) in 1.5 L of 1× TAE at 1.0 V/cm for 48 h at 4°C with buffer circulation.
5
Genomic DNA Extraction from Yeast Cells
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Cultures grown in 3 ml YPD medium overnight at 28°C were centrifuged at 2,500 g for 3 min, and the cell pellets were washed in 750 μl 50 mM EDTA. Each cell pellet was resuspended in 200 μl lysis buffer (1 % SDS, 2 % triton, 100 mM NaCl, 50 mM EDTA, 50mM Tris, pH=8), 200 μl chloroform/phenol (pH=8) and 300 mg glass beads and mechanically shaken by vortexing for 4 min. Then, 200 μl TE buffer was added and the samples centrifuged for 5 min at 12,000 g. The aqueous phase was transferred to a new tube, and two chloroform extractions were carried out. DNA was precipitated with an equal volume of 100% ethanol and centrifuged for 4 min at 12,000g. The pellet was rinsed with 400 μl of 70 % ethanol, dried at room temperature for 15 min, resuspended in 50 μl TE buffer, and incubated with 2 μl RNase (10 mg ml -1 ) for 30 min at 37°C. The DNA concentration was quantified on a 0.8% agarose gel (wt/vol) with 1X TAE electrophoresis buffer containing 0.2 mg ml -1 ethidium bromide, run at 100 V in a SUB-CELL GT electrophoresis system (Bio-Rad) for 60 min.
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