Pol III* replicates a primed template to the last nucleotide before it transfers to an acceptor. In the cycling assay, the donor template was M13mp18 single-stranded DNA and the acceptor was phiX174 single-stranded DNA. After 40 seconds of replication using [32P]TTP, the reaction was quenched with 1% SDS, 40 mM EDTA, the DNA was purified and digested with Bgl II, and the reaction was analyzed in a 0.6% sequencing gel (lane labeled "cycle"). The only fragment small enough to enter the gel is the fragment from the Bgl II site to the point at which Pol III* dissociates from its beta clamp. The reference sizing ladder was produced using a synthetic DNA 20-mer annealed to M13mp18 at the Bgl II site.