Good idea would be to start using UNG glycosylase in the reaction mix and dU instead of dT in nucleotide mix. This work very well in my lab. Here you can find example:

http://www.biotechniques.com/BiotechniquesJournal/2005/April/Minimizing-DNA-contamination-by-using-UNG-coupled-quantitative-real-time-PCR-on-degraded-DNA-samples-application-to-ancient-DNA-studies/biotechniques-45431.html

Some more comprehensive description how to manage contamination problems you will find here:

http://www.gene-quantification.de/mifflin-optimisation-report.pdf

However, as long you do have your PCR product in air, as long you will have troubles. You can try to wash all surfaces in the lab with 5-10% bleach or (more expensive) DNA removing reagent sold by many companies (DNA-off reagents).

As Ruslan wrote at this point designing primers amplifying the same gene, but not targeting regions amplified by old primer set could be the best thing to do.

i am getting the same size of bands as IL2, I use filter tips and i am not getting to the root of this problem. I'd changed primers. What else should I try?

What is your ´no template control´? Is it a water control?

1) One possibility is contaminated water.

2) Second possibility is contamination with human DNA.

Are IL2 primers mouse specific? or rather recognize some conservative region which is present in both mouse and human DNA?

Good idea would be to start using UNG glycosylase in the reaction mix and dU instead of dT in nucleotide mix. This work very well in my lab. Here you can find example:

http://www.biotechniques.com/BiotechniquesJournal/2005/April/Minimizing-DNA-contamination-by-using-UNG-coupled-quantitative-real-time-PCR-on-degraded-DNA-samples-application-to-ancient-DNA-studies/biotechniques-45431.html

Some more comprehensive description how to manage contamination problems you will find here:

http://www.gene-quantification.de/mifflin-optimisation-report.pdf

However, as long you do have your PCR product in air, as long you will have troubles. You can try to wash all surfaces in the lab with 5-10% bleach or (more expensive) DNA removing reagent sold by many companies (DNA-off reagents).

As Ruslan wrote at this point designing primers amplifying the same gene, but not targeting regions amplified by old primer set could be the best thing to do.

For bleach check this:

http://bitesizebio.com/3473/pcr-the-right-way-to-decontaminate-and-eliminate-false-positives/

@Agnieszka: I had water as no template control. I used the same for control primers and they don't amplify. The problem only happens with IL2 primers. These primers are specific for mouse. So I am really freaked out if I should change to another set of primers or do something else.

Hi Mr. Raza, we had a similar problem detecting hepatitis B virus using RT-PCR with taqman. The problem here is that RT-PCR is so sensitive that sometimes it picks up anything, from a trace of plasmid used in your standards. Dr. Treder is right recommending a nuclease spray to remove possible contaminating template from any surface. I would additionally recommend to use filter/barrier tips while loading the samples and to load the negative controls first. Additionally, try to use optical strips instead of the sticker, in that way wells are loaded per separately while the others remain closed. Good luck.

Yes, filter tips is a MUST and they should be used on every step starting from DNA preparation and ending on loading to gel final reactions. Also proper lab organization is very important but all of that (and more) can be found in links I provided above.

this paper can be useful too:

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0013042

In my lab we also experienced the same problem and we started to wash all lab in bleach (especially room dedicated to set the reaction and room dedicated for electrophoresis). At the same time we added UNG glycosylase and dU instead of dT to the reaction mix. However I still was forced to design new primers, which were targeting the same gene but were not amplifying any portion of former PCR product. From time to time we were checking old primers and it turned out that after few months the old PCR product which was contaminating our lab vanished.

Of course if tubes are not opened, the risk is much lower - it can only be caused by accident or poor quality tubes or plates.

First i would diagree with loading the no template control first. The no template control should always be the last tube loaded and closed. You may want to try reducing your assay sensitivity -less cycles, reducing the magnesium concentration, increasing annealing temperature or decreasing primer concentration.

Hi Abbas

If the mouse IL2 gene is in fact something you have floating around in the lab, I agree with most of the other comments here that getting rid of false positive signals would require some effort (I do not believe in any kind of nuclease spray though). Using the UNG glycosylase is very effective but is better suited for qPCR where you are interested in absolute quantification. For normal cloning work, you should be able to solve the problem as Roy suggests by simply running fewer cycles of PCR. I realize this doesn't solve the problem, but it is much cheaper and since false positives seems to be restricted to you IL2 gene, I think this would be a good first approach.

Good luck

Jesper

of course Roy is right - negative controls should be closed as the last during setting up reaction. Idk if loading is so crucial but better be too cautious in every step of PCR to save possible frustrations and time/cost consumption.

I have changed the primers to see if the previous batch was amplifying something fishy, i've cleaned the bench and now running a new PCR with all controls and also the previous set of primers as a comparison with new ones. Let's see how it goes.

It amplied the negative controls again this time. Now my primers were different from the previous region used.

Hi Abbas

same problem appear with me when work on CMV primers but when change the primers the problem is solved. I agree with the changing the annealing temperature and reduce the cycle number. Good Luck

I changed the primers as well. didn't work. i changed to a different taq as well. still amplificaiton in negative controls. i reduced cycle numbers. it just wont' go.

You are experiencing the -ve control amplification/product formation for only the IL2 primers and none for other primer -ve controls, so I doubt its an air contamination issue.

If I may know what size product you are getting and preferably you can post the image with the -ve control amplicon/product.

Are new primers targeting region amplified be4? Or they target outside region?

@Krzysztof: they amplify a different region. @Somanna: i am getting the same sized product in negative control as my positive control. its not a dimer as my amplicon is 500bp and it shows up at the same size.

so it has to be contamination, but maybe not with PCR product but with DNA fragment coding for entire gen you're trying to amplify.

Question, do you have human control? If your gene is highly similar to human gene, you yourself can be constant source of this contamination...