Are you sure it's plasmid contamination? What chemistry are you using? (SYBR green, Taqman...). It could also be primer dimers...

Around which cycle do your unwanted products form? Is it possible that you get primer dimers in your samples?

I agree with Yago Pico de Coaña,If you have fluorescence in your NTC is most likely you have primer dimers, especially if you are using SYBER chemistry. In case you use Taqman that probe and primers have some homology, which is unlikely but possible if you use custom Taqman probes.

Primer dimer sounds likely. How do the melt curves look like? They should provide you with an easy way to discriminate between actual product and primer dimer. The melt curve peak of your NTC should be shifted compared to your actual sample, if the fluorescence in your NTC is a consequence of primer dimers. Good luck!

I see many good suggestions already, pay attention to all, if those are out of the list then, The contamination you speak off could very well be real, if the target is synthetically made, diluted on working area. One of the rules is dilutions of target has to be miles away from working area. It is real. One negative control is do you get target with no enzyme??? You are in trouble, no other way, Clean up good.

Good luck

Mary

I strongly suspect that the contamination is genomic DNA. Do your primers span an intron (>300bp). How was your RNA prepared? Did you treat with DNAse before doing the RT reaction. These are really the key considerations when doing RT-qPCR.

Ps: For a good clean-up phenol/chloroform extraction is the way to go. Or you could use one of the make available commercial columns.

Do a melt curve analysis; primer dimer will melt at a significantly lower temperature than a product; in the low 80's. You will be able to see two sets of peaks, with the higher temperature one being your real product.

If you're using probe-based chemistry, you can't perform the melting curve analysis that would allow you to see that you're amplifying your product of interest. I would suggest trying your primers (no probe) with a SYBR/EVA/BRYT Green dsDNA binding dye and verify that there's only a single melt peak. Alternatively, you can run your product out on a gel and verify that all of the amplification you're seeing is from a single band of the expected size (though obviously gel won't give you the same level of sensitivity). If you haven't personally validated your primers, there's a good chance that you're seeing template-independent amplification (eg. primer-dimer).

If you've cloned this product, on the other hand, then it's all bets off. We've found that once we've cloned something, it's really hard to get rid of the contamination. Ultimately, we keep our cloning bench separate from amplification areas and never do amps after working at the cloning bench. To get rid of plasmid contamination at the cloning bench, my labmate had to take apart and bleach the pipettes, bleach and UV all surfaces, handles, etc., and change every reagent and plastic container in use.

I agree with Leta Steffen and everyone above. What you're most likely seeing is primer dimers. Did you check your primers beforehand to make sure they wouldn't dimerize readily? It would be helpful to know what cycle this contamination comes up in versus what you discern may be actual product, because if the contamination comes up significantly later than product, then primer dimers is actually your best bet. And this can be checked by either looking at the melt curve or running your product out on a gel.

I find when I think something is contaminated and I can't pin down what it is (i.e. I've replaced everything and still see contamination) that I run a "contamination check" PCR where I go through each of the reagents, replace one and run a positive control, negative control and a water. For the primers, I would replace them with a primer set that I know isn't giving me problems. For the probe, I would switch to using something else. I've done this once before when I was having a particularly difficult time. The water you're diluting your primers in could be contaminated and I've actually had the rare case of the company actually sending me contaminated primers.

Good luck and tell us how everything turns out.

Yes I think that is the answer Leta. The pipettes need to be taken apart and cleaned but other than DNA away and UV I did not know what else to use. It is synthetically cloned plasmids which I use as a control in my taqman RT-PCR. I really thought the filtered tips would protect the pipettes but now I think otherwise! I have put the products on a gel and they are not primer dimers... they are my target products that are melting around the 34-38 cycle. I NEED flat lines for my negative extracts for this experiment to be valid because I can have REAL genomic product come up at 37Ct and still consider them suspects at 38ct. I have been trying to clean up this mess for 3 months now and CANNOT get rid of it! I have learned a lesson though.... and you are correct... I should never have used the cloned plasmid controls anywhere near my genomic extractions!! :( If your lab mate can give me details on the cleaning method... % of bleach ect that would be helpful.

THANK YOU EVERYONE!!

Jamille

The root of the problem lies in the target dilutions, made, this contamination is real, stubborn, difficult to deal with, the world is going to give you tons of suggestions. CLEAN, CLEAN, CLEAN.

The answer from Leta is that what you could all possibly try. You have replied that you checked it on gel and they are not primer dimers. Then it is surely contamination. I would suggest use new PCR reagents, primers and probes and decontaminate your pipettes and your work station. I hope it will help to eliminate the probelm.

Hi,

What are your standards? Commercial or home made? if commercial try to buy your primers and probes at a different supplier. Otherwise cleaning and new reagents even a new box of gloves, a new box of tubes .... Good luck.