I have already tried different combinations of optimized DNA Nicking Assay protocols. The plasmid DNA that I use is puc19 from a transformed E.coli. Every single time I perform my Assay, I always extract a fresh DNA from a cultured transformed E.coli. Despite that, I only keep seeing single bands. I use gel red as my stain since EtBr isn't available in the lab. The image below shows an example of my run gel. This one for example is run for 20 minutes in 100 volts using 1x TAE buffer and 1.5% Agarose gel.
Hello Butiko here
In a DNA nicking assay, you typically use an enzyme to create nicks or breaks in the DNA strands. Linear DNA, open circular DNA, and supercoiled DNA have different structures, so they might not all be visible in the same assay. Linear DNA has two ends, open circular DNA has one nick, and supercoiled DNA has twists and turns. Depending on the assay conditions and detection method, you may only be able to visualize one or a few of these forms at a time.
It looks like there is no plasmid on your gel at all. Be sure to put DNA length markers on to see how long your band is, and be sure to apply a sample with an intact plasmid or maybe a commercial one. If the isolation of the plasmid is good, then it appears in two bands on the gel: the relaxed form (or open circular DNA) - this will be the upper band, and the lower one - the supercoiled form. If the plasmid is of poorer isolation quality, then a linear form appears that goes between these two, and the length of which can be tracked using length markers.
- Badges
- Science method