Yoroshiku, Masita-san,

It looks like a contamination of your primer (stock). I would try ordering it new - Sigma has nice program with 50% off on oligo synthesis; they can deliver it solved for you. You will have an answer within a few days, and could save lots of chemicals and cycling time. Also, order FRESH batch of PCR water, and ALWAYS use filter tips. You also might want to consider cleaning the pipettes (step 1 - 0.5N NaOH; step 2 - mild detergent. NO ALCOHOLS since that would cause the DNA to stick even more to pipettes). Finally, try pipetting the PCR in another room / clean bench.

Regarding the PCR mixes, my long-time favorite that saved me in dire PCR situations, is the Promega's GoTaq, I highly recommend it to everyone always. They sell it with nice colored buffer, so no loading buffer needed for gel electrophoresis. Also, they can send you samples, and you can get 50% off on the Poly after you post gel pictures on their webpage.

お幸せに。

Yoroshiku, Masita-san,

It looks like a contamination of your primer (stock). I would try ordering it new - Sigma has nice program with 50% off on oligo synthesis; they can deliver it solved for you. You will have an answer within a few days, and could save lots of chemicals and cycling time. Also, order FRESH batch of PCR water, and ALWAYS use filter tips. You also might want to consider cleaning the pipettes (step 1 - 0.5N NaOH; step 2 - mild detergent. NO ALCOHOLS since that would cause the DNA to stick even more to pipettes). Finally, try pipetting the PCR in another room / clean bench.

Regarding the PCR mixes, my long-time favorite that saved me in dire PCR situations, is the Promega's GoTaq, I highly recommend it to everyone always. They sell it with nice colored buffer, so no loading buffer needed for gel electrophoresis. Also, they can send you samples, and you can get 50% off on the Poly after you post gel pictures on their webpage.

お幸せに。

Hello Masita,

do your PCR produst focused to 16S rDNA of microbial community??

Iva Sakmaryova, I would be interested in the 16S rDNA (rRNA too?) story... 

Check your water contamination.

Hello Masita,

I totally agree with Marcin. Usually, the stock of primers gets contaminated with DNA. The best way to solve this problem is checking your pipettes, especially if you use the same pipette for preparing your PCR and getting DNA. Couple of years ago, we had a contamination in the lab when doing PCR and it turned out that pipettes were contaminated with "aerosol DNA". Aerosol DNA sticks within the tip holder of pipettes and it can contaminate your negative controls. Interestingly, when you use two negative controls it can contamicate one and not the other.

Changing or cleaning your pipettes and especially using one pipette only for DNA can solve this problem. Moreover, designating a specific area in the lab only for DNA amplification is a good idea.

Hi Masita,

I agree to Marcin too. It seems you have contamination in your primer stocks. So, try to order a new one. And also clean your pipettes or use the other ones!!

Thank you everyone for the suggestion. To Marcin, for the detailed instruction, thank you very much.

I am going to order new primer stock. I hope everything will work fine after this. Anyway, I never thought that the primer stock itself got contaminated since I add the primer into pcr mix before the DNA. But, contamination from the pipettes make sense. We can never be too careful.

@Iva No, I am not working on that. My work is transgenic mice.

Let me suggest another reason that happened with me. it was the loading dye, where i checked all the PCR components to follow the contamination source. at finally i found that the DNA loading dye was the reason. Please check its purity. thanks

Hello, I am getting the same troubles. I changed all the components of the reaction but I sill getting bands in the negative control. The worst is that the patterns of the bands are different each time. I autoclaved the pippets, the tubes and everything. I do not knoe what to do. Someone has any suggestion?? Thanks

Jailson Santos: Are you sure that your negative control is a true negative control?

Did you also change the buffer in your electrophoresis chamber? Cheers, Nadine

Hi, I also facing this problem am using 2 different primers and the i did not see any band in the negative control, but suddenly I saw a band I tried with new  PCR components and I still get this band, Then i decided to see from where this contamination comes from and I used the old and the new PCR components and I found in one of the negative control a band and the others nothing... logically my PCR components are not contaminated coz the negative control for the other gene did not show any band. I always sterile my PCR water and pipette with UV light each day before work for 10min.

Am really confused from where this contamination, if you have another probabilities please let me know I could consider?

One of the followings:

1. contaminated sample.

2. primer dimer, and this is resulted from different things!

-I also have the same condition, I think the most probable cause is the loading dye get contaminated form something whatever it is.

-When you prepare your pcr tubes start with negative control tube and close soon to avoid DNA contamination.

I am also facing similar problem, the lab I am using no work on my template have been carried out there. yet repeated bands on my negative controls. any further suggestions ?