Mis-priming is known to occur...  e.g., even though Ribo 18S has no poly A tail, it somehow gets amplified to an extent - even when adding only poly[dT]n for primer.... As well, recall that RNA fragments actually prime some of eukaryotic DNA replication.  Ribozyme formations, unexpected priming, is a reality - although we can brush it under the rug on most occasions due to thermodynamic preference for the most favored interactions - but only due to introducing the most favorable reactants in the first place. Something that 'commercial company products' excel more and more at over time.  Mother nature does not arrive human-customized for the tube:  but PCR does.

PCR is an enzymatic reaction in which substrate convert to product. Primer is substrate so ur reaction mixture contaminated with primer.

• Thanks for your reply. But there is no primer dimer in that lane. Lanes from the sample and NC have primer dimer. So how can I assure that this is the problem from primer contamination?

contamination or droplet spraying between tubes? Did you use aerosol barrier pipette tips? 

Thanks for your writing. I don't think that any contamination or droplet spraying between tubes. I only used filter pipette tip. 

I agree with the earlier answers. Contamination is the most probable reason.

Did the PCR worked fine earlier? Especially, did the water control worked well earlier?

Use complete new reagents to test if a solution was contaminated. To identify the one, replace the new solutions step by step by the old ones. (Infact, in extreme case, also the primer solutions and even the primer stock solutions have to be changed.)

And sometimes, it's because of the material. I experienced contaminated PCR-tube from factory earlier one time. (Is this problem appearing after changing something in your procedure? e.g. using a new bag of PCR tubes?)

Cheers.

I agree with others its a common problem in labs and it might be due to the contamination which is more likely to happen when many people use same materials for PCR. It would be best to setup ur PCR reactions inside laminar airflow. 

Sterilize your pipette and use fresh batch of PCR materials. Make sure water used is sterile and autoclaved.  

Mis-priming is known to occur...  e.g., even though Ribo 18S has no poly A tail, it somehow gets amplified to an extent - even when adding only poly[dT]n for primer.... As well, recall that RNA fragments actually prime some of eukaryotic DNA replication.  Ribozyme formations, unexpected priming, is a reality - although we can brush it under the rug on most occasions due to thermodynamic preference for the most favored interactions - but only due to introducing the most favorable reactants in the first place. Something that 'commercial company products' excel more and more at over time.  Mother nature does not arrive human-customized for the tube:  but PCR does.

Self priming of genomic DNA could also be a possible problem. This may occur when the template DNA is used in high amounts, typically >100 ng/reaction and when the template DNA is fragmented. Small DNA fragments originating from repetitive fraction of genome (satellite, rDNA etc) may serve as primers for Taq polymerase. As a result, banding pattern is seen in a negative control i.e. a reaction without the primers.

Suggestion: (i) Keep the DNA template amount low (

Thanks everybody for your answer and suggestion as well.

You may not have added the primer, but the DNA is still there (may be with UNTP) and that is giving you the band.

Above discussion is very nice and the reasons may be autopriming as well as contamination.

Sometimes the deionised water or tips we are using somehow get cross contamination and the result produces.

One can use entire new set and give your PCR reaction to someone other in your lab to perform it may help you.