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real time contamination - (Jan/05/2007 )

Dear all,

getting better with efficiencies of my primers. However now I have another problem. I cloned one gene and looks like I have the plasmid everywhere. I changed everything, when I say everything is everything, believe me. Even I make the PCR in another room where I leave the day before the UK lamp on.
The only thing that maybe, maybe, is that pippets are contaminated, although I use filter tips and everything, pippets, tips, eppendorf, water, are under UV long time. Well, I am thinking to change the room and change again everything, but maybe I do not have I really big problem, the amplification in the negative control start around Ct35, that's mean that is OK?
I read/listen that is normal to get this signal around these cycles.
That's all. Thanks a lot


Theoretically you should not have any amplification, but it can happen. If you have changes all your reagents, ect., you maybe getting non-specific amplification. You maybe able to play around with the annealing temp and imporve this.


Thanks a lot. Very useful information. I though that this kind of contamination is not really worried. I am totally sure that there is plasmid everywhere, sure, althouhg I changed everything.
I amtrying today a new experiment. I went to another room, I used other pipets, etc.
I'll tell you what's going on. If I make different dilutions I can see different Ct values. That's mean that I have contamination but as you say, is a small percent of the template.

Thanks again, see you later