Why no visible bands or smears?

[Aga]

I tried to perform plasmid profiling. Plasmid DNA was isolated from several samples using commercial plasmid DNA extraction kit.

Extracted DNA was checked spectrophotometrically on nanodrop. The yield was from 60 to 80ng/ul, depending on a sample.

I prepared agarose gel electrophoresis (0.5% of agarose) according to the method presented in several publications. It was stained with EtBr.

The catch is that besides the molecular size markers I didn't have any bands or smears on the gel. Nor anything visible in the wells or throughout the lanes. No residues of chromosomal DNA either. Nothing for the tested samples.

My question is why? Has my plasmid DNA denatured to single stranded and is not visible under EtBr staining? Or maybe it's the wrong staining method I applied?

Thank you in advance for your suggestions.

[jiajia1987]

This sounds really weird.

Have you tried using other methods of gel staining? Like gelstar? or sybrgreen?

[admin]

Hi Aga, what is the size of the DNA? Also could you have mistakenly made a higher percentage gel? How long did you run the gel, any signs of ladder / picture to see?

cheers

[Aga]

I prepared new buffer solution, the gel was 0.5%, I performed the electrophoresis twice, including even different samples along those tested for the first time. I also tried to avoid the use of small pipette tips and too much pipetting cause that might degrade plasmid DNA. I expect several small plasmids and one to three large plasmids per each tested strain.

The bacteria I'm studying are known to contain many plasmids and I expected to see at least some small plasmids, so about 2000bp, even if larger plasmids were fragmented or lost. I extracted plasmid DNA from both reference and environmental strains, in both cases nanodrop showed good results - not only good concentration but quite pure DNA.

I don't expect that the strains lost their plasmids during culturing - the strains were passaged three to four times only and no small plasmids loss has been ever reported in literature (large plasmid were reported to be lost after many passages and culturing on laboratory media). I used commonly used LB medium before plasmid isolation. Everything should be fine, but it isn't.

I don't have any photo with me at the moment so I can send it later, but there's nothing on the gel except molecular mass ladders...
The most interesting thing is that even if my electrophoresis was too short, or the gel contained more agarose, the samples should not migrate but be visible in the wells under UV. However there is nothing like that, so I don't have any clues.

[jennifer.chang]

That is quite puzzling. I would say it was something with the EtBr but you can see the DNA ladders just fine.

I guess the plasmids you are trying to isolate are native plasmids that do not offer any type of selection? If you could somehow maintain selective pressure so that the cells keep their plasmids, that would definitely help matters. Whatever technique you are using to isolate the plasmids - have you tried isolating a reference plasmid from a reference strain. That way you would know that your isolation method is good. It could be possible that you are not isolating actual plasmid DNA but genomic or pure RNA (but again, you don't see any of this, correct?).

Have you tried loading increasing amounts of your DNA on the gel? Perhaps the concentration is lower than you think it is and so you can't see it on the gel. How long are you running the gels? I doubt you are running your DNA off the gel since you see the DNA markers but if you have degraded DNA that might be low MW, you might see that if you don't run it off the gel?

[Aga]

I tried to isolate the reference plasmids from reference strains. I also confirmed the presence of genes that are plasmid - encoded. Those are present in the plasmid DNA I isolated and tried to visualise on the gel.

EtBr and gels seemed to be all right. And yes - those are native plasmids, but they should be present considering the presence of plasmid - endoded genes, even if fragmented. Chromosomal DNA and RNA are not seen on the gel. I haven't run my DNAs off the gel and I will try to load more samples into the wells.

I actually wanted to know whether my DNA is degraded or not, but this I can't see. I wonder why this is so and thought that if EtBr acts with double stranded DNA I might have single stranded DNA. The affinity of EtBr to ssDNA is very low. I read that plasmid isolation is performed at higher pH conditions and maybe that's the issue. What do you thing about that?

[ramnivas]

I think you should load more amount of samples and make atleast 1% agarose gel to check
and add good amount of EtBr in gel and run it on 80-100 V for 1 Hour. It may give you better results.

[tuesunsommiel]

this identical scenario just happened to me. positive readings on the nanodrop (~20ng/uL) and ratios (260/280) all around 2.00, yet the only thing that showed up on my gel (using EtBr) was the molecular ladder. I'm at my wits end....

[butters]

what is your pasmid size? ~20ng for a large plasmid may give very very very very faint band.

If it were me I would
1. Load slightly more volume on gel (preferably use smaller well / comb)
2. Do a serial dilution of ladder or just use say 10 time dilution of ladder (this is so that you can still determine the size of your plasmid when overexposure).
3. Increase (hopefully you can control this) exposure / capture duration of gel visualization.
4. Invert image.
*Disclaimer - try at your own risk. Thank you.