I am currently trying to troubleshoot why my gels are not showing anything at all except for my ladder marker bands. I have attached an example of a gel that I used to run previously with no problem.
I am trying to run a 1.5% agarose gel, with a TAE buffer volume of 150 mL and 2.25 g agarose. I carefully mix the buffer and agarose and heat up until melted. I am careful to make sure the solution is cooled before adding the ethidium bromide.
I usually add 10 uL of EtBr to this solution, and I suspect this amount is what is causing problems. The EtBr stock solution my lab uses is 10mg/mL. The EtBr is kept wrapped in aluminum foil and stored in a dark place to avoid degradation.
If anyone uses a better addition of EtBr, or knows of any other mistake I am making, I would welcome any advice. Thank you!
You have good staining of your marker and it looks like you have faint bands in all wells below the lowest marker band. I think I can also see smears in most wells. If I had to guess from appearance, I would say that the tiny bands are excess PCR primer and it is a set of PCR samples that has failed to produce significant product. I don't think the stain is the culprit.
You have good staining of your marker and it looks like you have faint bands in all wells below the lowest marker band. I think I can also see smears in most wells. If I had to guess from appearance, I would say that the tiny bands are excess PCR primer and it is a set of PCR samples that has failed to produce significant product. I don't think the stain is the culprit.
I use 50% less of primers than the protocols recommend, or I tend to get pcr failures like this. And I use very little template, 5 to 10 ng only. DNTPs also go bad often, that seems to be a weak link in the reaction. DMSO at 5% seems helpful in avoiding primer-dimers. But still, my typical phusion taq pcr result is around 30 ng/ul, but on occasion (maybe 1 in 10 times) I get results like 73 ng/ul or once 201ng/ul. I wish I could isolate what makes those runs so rich! I always do the same protocol, the same way, and make a master mix beforehand, etc.
I can't recognize smears. But I agree - you have marker bands thus the ethidium bromide is just right.
Ergo: Your DNA amplification went wrong, probably.
Exchange every reactant in your PCR mix with new aliquots.
If it still doesn't work, maybe your DNA extraction didn't work, so rerun the extraction too with fresh reactants.
If this still doesn't work vary the amount of DNA in the PCR reaction and also vary the Primer concentration.
And then, if it still doesn't work, try other temperatures for the annealing step.
It is also possible that your primers don't match.
Good luck!
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