contamination in real-time PCR - (Apr/01/2005 )
It seems you are very experienced in pcr. I've got a very tough problem and I will appreciate any answer you give. I did a DNase treatment before RT and afterwards I did the q-pcr. I am not 100% sure to rule out the gDNA contamination but I am sure there is no other source of contamination since I used everything new, even new pippetors. In my case, -RT works well when detecting some genes. However, for other genes, it works so bad than the products show up even earlier than +RT. From the melting curve, I think the products in -RT and +RT are the same, or at least quite close. Do you know any possible reason for causing of this problem?
Also, never open a real-time PCR tube in the lab where you prepare your real-time PCR reactions. If you run an agerose gel to check your real-time PCR amplification, you contaminate the lab. It is very hard to get rid of the air contamination - it usually takes months before your negative control shows negative again.
I prepared my master mix in a room and pippeting into plate in another room and then run the pcr and gel in the third room. Is that okey to rule out air contamination? And can air contamination cause the -RT show up products around 25-27 cycles?
General answers to your sybrgreen NTC peaks.
Often its poor oligos that give these "contamination", that´s some sort of dimer formation. Buy new oligos- same problem change the company that makes them. Taht is if you have done your homework constructing them right from the beginning.
Is the overall sensitivity good? And the NTC comes after your cut-off. Then the PCR is probably OK.
All the new "high flourescence sybrgreen mixes" that should be so good. Maybe they are to good for your setup, in my opinion they takes up even the smallest formation of dsDNA and gives a signal. A peak that will not be seen on a gel.