Two-Dimensional DNA Electrophoresis Analysis

Two micrograms of DNA were loaded on a 25 cm 0.4% SeaKem Gold (Lonza) agarose gel and run in 0.5X Tris-borate-EDTA buffer for 16 h at 40 V. Next, each sample lane was excised and soaked in ethidium bromide (Thermo Fisher Scientific) for 20 min. Samples were then placed perpendicular to the original running direction and a 0.4% agarose gel containing ethidium bromide (Thermo Fisher Scientific) was cast around the gel blocks. The gel was resolved for another 16 h at 40 V and then nicked, denatured, and processed per the Southern blot protocol described above^{25 (link)–27 (link)}.

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Nath S., Caron N.S., May L., Gluscencova O.B., Kolesar J., Brady L., Kaufman B.A., Boulianne G.L., Rodriguez A.R., Tarnopolsky M.A, & Truant R. (2022). Functional characterization of variants of unknown significance in a spinocerebellar ataxia patient using an unsupervised machine learning pipeline. Human Genome Variation, 9, 10.

Publication 2022

SeaKem Gold ethidium bromide

Agarose Cast Ethidium bromide Gold Southern blot Tris borate edta buffer

Corresponding Organization : Hamilton Health Sciences

Other organizations : McMaster University, British Columbia Children's Hospital, University of British Columbia, University of Toronto, University of Pittsburgh

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Variable analysis

independent variables

Agarose gel concentration (0.4%)
Running voltage (40 V)
Running time (16 h)

dependent variables

DNA separation and visualization

control variables

Tris-borate-EDTA buffer (0.5X)
Ethidium bromide staining

controls

Positive control: Not explicitly mentioned.
Negative control: Not explicitly mentioned.

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