positive NTC in real time PCR - (Sep/06/2005 )
Hi u all. I've been setting Real time PCR for 3 years but I've never seen a problem like that. I have resuspended my oligo and my probe (dual labbeled FAM-TAMRA) in a 1 mM Tris-HCl pH=8/EDTA 0.01 mM EDTA as recommanded by APPLERA. I've perfomed my PCR reaction but the NTC was positiveve (the Ct value was greater than the samples). I'm not new in PCR practise but please give some light to me!!!
I've read the paper concerning Contaminations Occuring in Fungal PCR Assays. Journal of Clinical Microbiology 37(4): 1200-1202 and I think this could a possibility. I'm now auticlaving (sterilizing) the water before set-up a new PCR.
All normal procedure to setting up PCR have been followed.
Looking forward to some hearing from u
regards
federico
PS H12 is the NTC (attached figure)
Do you have a picture of an agarose gel of these samples?
So that you could exclude a Probe degradation or probe/primerdimer etc.
So that you could exclude a Probe degradation or probe/primerdimer etc.
I got a picture but I do not agree with u cause working with dual labelled probe unables primer dimers detection.
Only if i work with syber green i can see primer dimer amplification in real time PCR. No probe degradation has been detected from agarose gel ananlysis.
Anyway thank u for your replay
co@fede
I got a picture but I do not agree with u cause working with dual labelled probe unables primer dimers detection.
Only if i work with syber green i can see primer dimer amplification in real time PCR. No probe degradation has been detected from agarose gel ananlysis.
Anyway thank u for your replay
co@fede
[/quote]
I did not mean, that you would detect possible primerdimers. But when the probe is binding to a sequence within the primerdimer, that could be.
Degradation of your Probe could give a positive signal, cause the reporter and the quencher are seperated, as you said already.
Ok, so the gel shows the specific band size, then it is obviously a contamination.
With taqman, primers dimers can be ruled out as a source of signal. This seems like a standard contamination issue. The high cycle number in which your amplification is occurring suggests that whatever is there is at a real low copy number. I would consider trying new primers, h20, master mix, ect. to prevent this. Degradation of the probe usually does not produce a sigmoidal curve, thus I do not think that is an issue either. What are you trying to amplify?
[quote=Bigmac,Sep 7 2005, 02:25 PM]
I got a picture but I do not agree with u cause working with dual labelled probe unables primer dimers detection.
Only if i work with syber green i can see primer dimer amplification in real time PCR. No probe degradation has been detected from agarose gel ananlysis.
Anyway thank u for your replay
co@fede
[/quote]
I did not mean, that you would detect possible primerdimers. But when the probe is binding to a sequence within the primerdimer, that could be.
Degradation of your Probe could give a positive signal, cause the reporter and the quencher are seperated, as you said already.
Ok, so the gel shows the specific band size, then it is obviously a contamination.
[/quote]
OK today I've done another reaction with 2 differents set of primers and probe to test reagent purity. As i thougth I don't find any kind of contamination in the new one set (for another gebe) while NTC is again positive with the first set of primers and probe. What can I do now?
How can I check the purity of a couple of primers and probe without perform another real time PCR. Is it possible to affect oligo purity during resuspendig procedure (the solution used were all autoclaved and made with H20 molecular grade)
I'm amplifying human somatostatin receptor 1.
Dear co@fede,
This is a typical contamination problem. Either your primer (stock solution) is contiminated or your pipette is contaminated by DNA template. I assume that you are using filter tips for doing PCR work.
Sometime I am tired troubleshooting for my own contamination problem, so I will purchase everything new (including primer), autoclaving my water and pipette and strat all over again.
This will save you more time.
Best regards
We experience this often using isothermal techniques. with a high CT or in our case a long incucbation time then a primer dimer will form that incorporates the complement to the probe sequence. The molecule includes both primers and the 5' terminus of the probe. It is a multigeneration event and is quite comlex but it can hapen. Change the sequences that you are using.