In our lab, we're doing agarose electrophoresis to visualize PCR results. Recently the process started acting up. In some gels, the only thing that showed up was ladder. Sometimes the wells were lighting up, as if the DNA was too big for the gel. Other results were really fuzzy. We changed everything because we suspected contamination. Now the DNA comes through the gel but is different weight than it should be (we want 200bp, it is in 50pb). It happens also with the samples that were previously positive. We can't work out what the problem might be.
We use agarose gel, TBE buffer, Midori green.
I personaly don't do this method, I just happen to be the only one with Reseachgate account.
Thank you very much.
Dear Dr. Bechnikova,
First off I am sorry for your gel not working. I know this problem my self too.
I like to use Ethidium bromide to visualize the DNA, it is carcinogenic but works very, very well. Only times it does not work is if i forget to add it to my gel....
I have had my 10X TBE buffer go off from long term storage, this messed up my gel running buffer and thus results. I was able to just autoclave it, and the precipitates went back into solution and TBE buffer worked well again.
Dont let the temp get too warm while running the gel.
Always run Positive, Negative, and water vs template controls with ladders too.
Make sure your agarose is well dissolved, but not too much to change the gel%,
mind the volume before and after heating TBE and Agarose (via microwave, hot-stir plate, Bunsen burner).
Best Wishes
it sounds like you have a primer dimer problem giving a very small 50bp dimer and stopping the proper amplification of your amplimer, Can we see a picture of a problem gel please? What does your no template control sample look like?
I agree with Paul, if you are having issues visualizing PCR products but you are able to see the ladder there might be an issue with the amplification process. It would help if you could upload a picture of one of your gels so I and other fellow researches can see what could be happening.
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