Dot Blot Assay for 5-hmC Detection

Dot blot assays were performed as previously described19 (link). Briefly, genomic DNA was sonicated, 5-hmC was conjugated to UDP-6-N3-Glu18 (link), and biotinylated by addition of 4.5 nm Click-IT Biotin DIBO Alkyne (Life Technologies). The biotin labeled DNA was purified and spotted on HyBond N + membrane (GE Healthcare Life Sciences). Membranes were probed with Avidin-HRP and dots were visualized with Western Lightning ECL reagent (PerkinElmer).

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Chapman C.G., Mariani C.J., Wu F., Meckel K., Butun F., Chuang A., Madzo J., Bissonnette M.B., Kwon J.H, & Godley L.A. (2015). TET-catalyzed 5-hydroxymethylcytosine regulates gene expression in differentiating colonocytes and colon cancer. Scientific Reports, 5, 17568.

Publication 2015

Click-IT Biotin DIBO Alkyne HyBond N + membrane Western Lightning ECL reagent

Alkyne Assays Avidin hrp Biotin Dot blot Genomic Membranes

Corresponding Organization :

Other organizations : University of Chicago

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Variable analysis

independent variables

Sonication of genomic DNA
Conjugation of 5-hmC to UDP-6-N3-Glu
Biotinylation of DNA by addition of 4.5 nm Click-IT Biotin DIBO Alkyne

dependent variables

Presence and abundance of biotinylated DNA on the membrane

control variables

Purification of biotin labeled DNA

controls

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

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