Hi everyone.
In our lab we genotype samples for our experiments. Apparently we see a band/bands in our control neg ( MQ water). we have changed everything, the buffers, water, primers, restriction enzyme. but still, we see this band. I am not sure if this is contamination or not.
Do you have any idea how to solve it?
Hey, Elina!
Is this band present in the positive control samples too? Is it not a primer dimer? Did you change the sequence of the primers or reorder the same?
I can also recommend to BLAST the primers to see if they can amplify something from the environment, from the skin. Some primers are just too sensitive to this kind of contamination and it's hard to get rid of it. How specific are they?
Hi Kristina, Thanks for your reply. the strange part of this story is we can see this band in 90% of our gels in very different ways. sometimes only one band, sometimes several. So sometimes we see in the positive control sometimes not! and according to the size, it is not primer dimer. the length is 21 long. and yes we reordered new ones, worked once, then after a while not working.
we have another set of genotyping in this lab and doing it constantly without any problem, almost the same reagent but of course different primer.
Do you have any idea?
What is the size of this mysterious band? Which marker do you amplify with your primers?
I suspect it can be environmental contamination. Please, don't think it's your fault, or that your work is not clean enough. It can be the specifics of your primers, to catch all the bacteria or fungi from the air or water.
Try to use filter tips to decrease this contamination.
The best way to check - cut out the band from the gel, purify it and sequence the product. The easiest - do primer BLAST on your primers and see what you can amplify from the air of water.
The next step - design more specific primers or you have to select and cut out from the gel all your products. Better to play with primers ;)
Thanks so much for your advice. The size of the band is mostly 150bp.
probably that makes sense if it comes from the environment. Shall I try it under the laminar fume hood?
Do you think if there is a puncture in the bottom of the wells in the gel electrophoresis, can mix the samples from the bottom? does it make sense? I am checking all the possibilities.
Thanks again.
Of course, you can try to reduce the chance of contamination (if you have it). I would also recommend cleaning your bench with solutions containing chloride (the ones you use to wash the floor, for example, but dilute it). It is the best way to get rid of DNA. But wash it out with the water afterwards.
I never experienced the problem with the puncture on the gel. But of course, there should not be any holes on the bottom of the gel. And this is the easiest thing you can check at first.
I still recommend BLASTing your primers.
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