dear researchers
I have been doing PCR with material extracted from nematodes, using different specific primers and the results have been as expected.
when using ITS primers in the same samples, the agarose gel shows contaminated bands, even in the water control.
I thought it was a contamination in the primers, so I tried a new (factory) preparation and the result was the same.
If this is not a contamination, what else can it be?
It's there something that I'm overlooking?
If your no template control amplifies then you do have contamination. It does not need to be in the primers. Any reagent, your working area,labcoat plasticware and pipettes can also carry contamination. The simple answer is to design a new set of primers with one primer outside of the original amplimer then the contamination cannot amplify.
Alternatively clean up your materials and pipettes . Meanwhile move to another workers area and use their pipettes,tubes,pcr reagents and pcr machine, You will have to use your primers but moving to a clean area while you sort out where the problem lies at least keeps you working
Can you label your gel more precise? What band contains the water control, what are your expected pcr product sizes, how do you stain your gel and which buffer are you using?
Degraded DNA bands will appear as a thin band with a short smeared tail. According to your agarose gel results, your DNA ladder is in perfect working order because the bands in the lane are clearly visible and smeared-free. However, since you are also observing small smears in other lanes with post-amplification pcr products, including DNase-free water, which indicates the presence of degraded DNA. The PCR tips and tubes or the DNase-free water could both be contaminated. Additionally, there are other elements that could play a role in contamination of the PCR products and water control, such as contaminated workspaces, equipment, and improper handling of samples while preparing master mix for PCR amplification.
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