The following two images show E. coli Topoisomerase I activity assay samples run under different electrophoresis conditions, with running parameters annotated. Figure 1: 2 V/cm for 12 hours; Figure 2: 160 V for 32 minutes. Each lane is labeled with its sample type. I am uncertain why my DNA template displays 5-7 bands during electrophoresis. I have annotated the presumed conformational states of bands in the images. However, under both experimental conditions, the results consistently show disappearance of the supercoiled band, increased prominence of the linear band, and an additional bright band above the relaxed circular plasmid. Does the presence of multiple bands in my template indicate degradation, rendering it unsuitable for activity assays? Should I re-prepare negatively supercoiled plasmid as substrate and repeat the tests?
The bands above band b may be concatenated plasmid (plasmids linked together like the links in a chain). This may be an artifact of how the plasmid was prepared. Two plasmids linked together would then account for positions c and e.
I think position b is relaxed circular plasmid, and the faint band just below that one is linear plasmid. This explains why all the topoisomers in lanes 2 and 8 run between positions a and b. I think position c is supercoiled, concatenated plasmid and position e is relaxed, concatenated plasmid.
I don't know what is in position d.
Concatenated plasmids containing more than two plasmids linked together may be in positions above e. Position f could be 3 plasmids linked together and supercoiled, and position g that plasmid relaxed.
Topoisomerase 1 is not capable of decatenating concatenated plasmids because it only makes single-stranded breaks. It would be interesting to see what would happen if you treated the plasmid with topoIV, which is able to separate concatenated plasmids.
You could also have broken chromosomal DNA mixed in with your plasmid. Do you use a gel filtration column to purify the plasmid DNA? They can be made or purchased, you want one that your plasmid will migrate through the gel, while chromosomal DNA will not enter the gel and so wash away.
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