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. Author manuscript; available in PMC: 2018 Apr 13.
Published in final edited form as: J Vis Exp. 2018 Mar 5;(133):10.3791/57413. doi: 10.3791/57413

Figure 1. Generation of a DPC-containing plasmid.

Figure 1

(A) An oligonucleotide containing an 8-oxo-guanine residue (red) is annealed to single-stranded DNA (bold black circle) and extended to create double-stranded plasmid (primer extension product indicated by dashed line). Following electrophoresis in ethidium bromide, the band corresponding to supercoiled DNA (red box) is excised from a low-melt agarose gel and digested with β-agarase. Lanes: (1) Molecular weight marker, (2) single-stranded DNA, (3) double-stranded DNA, (4) primer extended sample. (B) Oxoguanine glycosylase (abbreviated hOGG1, depicted as an orange circle) is crosslinked to the 8-oxo-guanine residue via sodium cyanoborohydride and the resulting DPC substrate digested to generate a 2800 base pair free DNA fragment and a 4400 base pair fragment attached to the protein. SDS is added to the sample which is then divided into two portions, one of which is treated with KCl and centrifuged to sediment DPC-containing DNA (depicted as an orange pellet). The supernatant from this latter sample (depicted as blue fluid in centrifuge tube) and the sample not exposed to KCl are subjected to gel electrophoresis. Lanes: (1) Molecular weight marker, (2) –KCl, (3) +KCl.