It is a taqman based chemistry with FAM-MGB probe. I have changed every reagent used for the reaction and found it is the problem with enzyme specificity. First I used gotaq enzyme from promega when NTC was positive. But when I used taqman master mix, NTC was clear. Has anyone faced similar problem anytime? Or is it due to MGB probe which is more compatible with taqman master mix?
We use NDC/NTC in Q-PCR to ensure all the components of PCR are free from contamination after running the PCR. We never faced similar problem.
İf one of your buffer or your main sample contaminated with an other DNA and when your primers hybridized the range is upper than 5 kb ; qotaq polymerase can amplify it because it can amplifiy more than 5KB. But Taqman mastermix which contains ampliTaq can not amplify so taqman prob probably can't hidrolize also if enzyme can't amplify first cycle correctly you can not get 2^n value. . Could you please check your NTC band with a DNA ladder.
I never had an issue with NTC in RealTime PCR. You might have to check the reagents for contamination. Please follow Enes' advice, good suggestion.
Thanks Enes. I have checked NTC reaction with DNA ladder. But very small size band was observed, even confused with whether it is primer dimer or true amplification size band.
if you are not sure it is a primerdimer or a true amplification, I would say it is a primerdimer.
Does it comes up rather late? which cT?
gotaq from promega is a strong polymerase and can amplify really difficult and long templates.
But if you see something with one mix and not the other, I would check the MgCl2 concentration of your mixes. I assume that the one with the highest Mg conc. also shows that NTC amplification. Mg makes your enzyme work harder, which is good in several cercomstances, but also might give you higher chance in producing dimers (what is your primerconc in mM please?)
Ct for NTC is 32.. consistently same over repeat experiments with various primer concns, I used 20 pmole primers and 10 pmole probe concn.
Considering MgCl2, as both promega and universal taqman master mixes are ready to use mixes , did not mention MgCl2 concn.
Mastermixes are good, but they want to work in as broad as possible range, so they tend to have a higher conc. in MgCl2. Often you can see the same mastermix from 1 vender, 1 with MgCl already in mix, the other with MgCL provided in a separate vial with the mix so you can add it in conc you want.
but a constant Ct of 32, independent of primer conc and being able to be picked up by the probe (assuming you also see it with non sybrgreen MM), makes me rather think that your primerpair and probe can mismatch somewhere else (which is rather slim chance with MGB probes since a mismatch there leads to no attachment, they work for SNP's).
The only option still left standing is that somewhere you have template coming in that is not wanted. For that there is a whole list of things that might be contaminated. From sample to cDNA prep (if starting from RNA), to water, etc.
Basically the tip there is to change all your components with fresh ones. Fresh tips, clean pipets and desk with bleach (not ethanol since ethanol doesn't destroy DNA).
If this ct of 32 is far enough away from your normal samples (more then 5 Ct, preferably more then 10Ct) you may ignore the signal, otherwise ,...
You may be getting a signal in your NTC if the threshold is set too low, that's the only time I've has a signal in mine using TaqMan master mix.
Check NTC reaction with Evagreen. If you see an earlier Ct, it indicate your primers have strong, extensible dimers. There is a remote chance that sch extension can also cause probe cleavage and signal. Some master mixes prevent primer dimer extension - that could explain your observation.
Dear Minal ;
Could you please try to load your QRT-PCR's NTC to an agarose gel. İf you see same bands they are probably primer dimers.Please calculate your forward primer and reverse primer length if the total length is equel with your band range on agorese gel they are primer dimers.
But if you see no band on agarose gel ; I only suggest that your enyzme or one of the enyzme buffer is contaminated because only they are different in two reactions mix.
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