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Labeled eccDNA is not a product of DNA replication. Charomid DNA (200 ng) was incubated in high speed extracts supplemented with 50 µCi [α-32P]dCTP for 90 min. The purified DNA was cleaved with MluI and PvuI and was then divided into two fractions. One fraction was digested with DpnI (B), whereas the second was not (A). The DNA was separated on a 2D gel, which was blotted and directly exposed to film.
