RT-PCR contamination in negative control. - (Mar/28/2012 )
So I have been working on these RT-PCR experiments for a couple of months now and with each experiment except for one I am getting a band that is the same as my product size in my negative control which is the -RT control. I take all procautions necessary that I can do given our limited acess to fume hoods. So I do all my PCR experiments in a seperate place in the lab. I have changed all my primers, DNTPS, water and yet I still get contamination in some of my samples but not in all my samples. I do use the tubes that are connected but I also have acess to tubes that aren't connected. I really don't know how I am getting contamination in these controls? We don't have access to filter tips in the lab of UV light. So I am somewhat pretty limited in controling some aspects.
Your negative control is RT- control? Do you also have NTC (non-template control) having the same band?
RT- can be contaminated with DNA giving a positive signal, if your primers are within one exon.