In some end point PCR, I observ a weak band in white lane (no DNA) not present in any other lane with samples to assay and not according to the expected PCR product. I exclude a contamination of any component because the band in white lane is not present in all lanes.
Someone can help me?
It might just be primer dimer if it is a diffuse band at about 50bp. PD is often worse when there is no amplification which is the case in the no template control.Can you post a picture of the gel please?
It's probably just unused primers. Some primer sets do this more often than others.
Here is the picture of my problem.
Thank you very much
Alessandra
Thank you Alessandra Panattoni .The band is not primer dimer or primer which is at 5cm. It looks real. What is a white lane? Are the samples blue and white clones that you are amplifying and if so are your primers specific to the insert or designed plasmid into insert?
Hi Alessandra Panattoni , from the picture it looks like a typical case of contamination and misamplification. Just to be on the same page, white lane means no template control i.e, water was used instead of sample. One of the reasons for the band seen in the "control/white" lane can be that the amplification conditions are not stringent for the desired amplification. If the Tm is increased by 5 or 6 degrees I would expect no band in the control lane. The rationale is, in the no template control when there is no sample, the primers are able to bind to some template (contamination) that is very low in concentration due to less stringent conditions. But when the samples are actually present then the primer is binding to its actual template and binding less to the other template. The extra band might be present in other wells also but probably needs more number of amplification cycles to visualize it.
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