I am an undergraduate biochemistry student. This is my first time doing DNA agarose gel electrophoresis.
The third and fourth lanes from the right are loaded with samples obtained from E. coli while the ninth lane from the right is loaded with a HindIII digest the sample. The one in the ninth lane has also been treated with ribonuclease A.
I would assume that the bands in lanes 3 and 4 absent in lane 9 are caused by RNA contamination. The third band is probably the pBR322 plasmid (the E. coli strains are meant to be resistant to ampicillin). I have no way of testing these hypotheses.
I think a lot of people (myself included) would like to help answer your questions, but you first need to clarify a few things. Is this a 12-well gel? If so, you typically would want to refer to the lanes by their # from the left. So when you say "9th from the right", I assume you mean Lane 4? Secondly, a major problem that sticks out to me right off the bat is that your lanes are extremely saturated (overloaded with sample). For this well size, you typically only need between 100 - 500 ng of DNA, and any more will cause this saturation and smearing that you're observing. If you think some of the bands are RNA contamination, there's a pretty simple way to check that: soak the gel in a TE buffer and add little amount of RNAase, then let it gently shake on a shaker. In ~15 minutes, visualize the gel again under UV and take another image to compare with the previous one. You should see that all RNA bands have disappeared. Other noteworthy tips: linearized DNA tends to have "tails" that curve upwards on either side of a band, while circular DNA does not.
It is likely that the big bright "comets" in lanes 3-4 from the right are RNA. As suggested by Shwan Javdan you can test by treating with RNase.
In general, there are to much sources for mistakes or uncertainties, that have to be addresses, e.g. your reagents (purity/contamination of PCR buffers, dNTPs, polymerase), your targets (quality of the template), the technique itself (apparatures working well?, power supply etc.) as well as the experimentator itself (can you exclude mistakes done at any stage of the eperiment (DNA isolation, sample preparation, addition of PCR reagents, sources of contamination...)?).
Your should repeat your experiments some times to get an overview of potential outcomes of the same experiment.
- Badges
- Science topic