Real-time PCR - contamination or not? - (Aug/23/2005 )
Hi there!
I have a problem. I have never worked with real-time PCR before, just started. For first shot everything was fine, after it always seemed there's a contamination, at least the non-template controll gave the same results as samples. I changed everything, water, sybrgreen-mix, MgCl2, even I made new dilutions of the oligos, but nothing has changed. I did it with beta-actin, I wanted to use it as reference gene. Later I changed to cyclophylin, and everything worked well with the original water, sybrgreen, MgCl2 etc. Now again I wanted to try out the target gene, and I got contamination again, anything I do. I have read many topics here about PCR contaminations so I don't know what else can I do for it... does anyone have a good advice?
Thanks
I have a problem. I have never worked with real-time PCR before, just started. For first shot everything was fine, after it always seemed there's a contamination, at least the non-template controll gave the same results as samples. I changed everything, water, sybrgreen-mix, MgCl2, even I made new dilutions of the oligos, but nothing has changed. I did it with beta-actin, I wanted to use it as reference gene. Later I changed to cyclophylin, and everything worked well with the original water, sybrgreen, MgCl2 etc. Now again I wanted to try out the target gene, and I got contamination again, anything I do. I have read many topics here about PCR contaminations so I don't know what else can I do for it... does anyone have a good advice?
Thanks
hi I dont know much - but I do know that when you use a sybrgreen-mix (ie not a specific probe) - then the sybergreen may bind to primer dimers... best way to check is to run the samples on a gel and if the negative control does not have a band at the size that your samples do then you can assume it is your primers forming dimers that i causing the signal.
My problem is similar - but I am using a specific probe - hence it should not by effected in the same way by primer dimers... and I have proved that the negative control contains DNA of the same band size as my samples - ie it is a legitimate PCR product caused by a DNA contamination.
I probably wasnt much help as I odnt know that much eiother - but in the reading I did today the above it what I managed to fathom out.
Good luck
edwina
contamination in pcr is a real pain. To get rid of it you mostly have to toss everything out. Are you using aerosol barrier tips for everything? Is your water autoclaved and then sterile filtered? Are other people using the same pcr reagents and pipets as you. If so, this must stop.
Did other people use your primer stocks? If so order new primers and put them in several small aliquots.
Since this is syber-green, double check your negative control on a gel to verify it is a real pcr product and not just primer dimer.
I think the pervious advice of checking your products on a gel is very sound. Mind you, it would normally be much better to check for primer dimers and negative controls in standard PCR's before going on to use the primers in real time experiments.
could you have contaminated your samples?? you may want to reisolate some new samples (it's a pain in the butt, but I think it's worth the effort)
Nick
Many real-time PCR machines will also run a melting curve at the end of the sybr-green run. If you see more than one peak, then it is likely you have a dimer issue. I also think it is a good idea to aliquot the master mixes once you receive them to avoid having to toss a $500 product because of contamination.
Thank to you all for the answers. Fortunately the SyberGreen Master mix is already aliquoted. I tried to use now the same water and MgCl solutin, and now I got nice negatives. I used different oligos. The strange is that with beta actin I used freshly ordered oligos and I didn't get negative, just like with the old beta-aktin primers; then I used an old (1-2 years old) cyclophylin primer-pair with the same water etc, and the negative was ok then (and the positives were postives). These are only my trials, so I don't stop at 40 cycles, but in these nice samples I do not get peak in negatives even after 50 or more cycles while my positives have a Cp between 15-20 cycles. But with some other oligos (and what I cannot understand, a new one also) I just cannot make negative. So I guess my contamination or whatever should be in my primers which is strange for I often make new dilutions of the stock and in this case it doesn't help.