Newly extracted plasmids and old plasmids (500 ng each) were run together on a gel at 160V for 30 min (Figure 1). Lane 1: old plasmid. Lanes 2-4: new plasmid. The results show that the new plasmid also exhibits four bands.

When the run time was extended to 50 min at 160V (Figure 2), an additional band (a1) appeared in all plasmid samples, while an extra band (g) appeared specifically in the old plasmid.

When using the new plasmid as a template to test E. coli Topo I activity, an additional band (f1) appeared in both the experimental groups (with varying Topo I concentrations) and the negative control group (plasmid in reaction buffer). Furthermore, the experimental and negative control groups produced similar results, suggesting that E. coli Topo I activity appears minimal or undetectable under these conditions.

However, this observation seems inconsistent with the initial activity data I obtained (Figure 4), which was generated using the old plasmid.

I'm uncertain whether my interpretation of the plasmid conformation results is correct. I have noted the running conditions and my proposed band conformations below each image.