Real Time PCR problem - different dilutions have the sam

互联网2013-12-26

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I have been working with the Real-time PCR and have run into a brick wall. My standard curve is running that all samples no matter what the dilution factor are amplifying at the same Ct. I am also getting amplification at that same Ct from a negative control (no template). Our first reaction was contamination, but I have used all new reagents, clean pipettes, different workstation and am getting the same results. We really don't think its contamination. Any suggestions as to the problem? I am getting very frustrated. Thanks for the help

Adam

-ajk16-

Unfortunately to say, this is contamination. Think about it... If all your samples are rising at the same c(t) including your NEGATIVE control, then there has to be something (i.e. template) in your mix.

Contamination is a pain to eliminate. Even thorough cleaning of all pipettes, replacement of all reagents, cleaning bench tops, etc. sometimes fails to eliminate the source.

My recommendation:
Change all reagents (again)
Setup your reaction using a different labs pipettes and workspace (different as in ANOTHER ROOM!)

Oh, are you using TaqMan or SYBR Green? What C(T) are things coming up at? Also, is it for just one assay or all assays?

-unknownphd-

Hi try to work in a hood and use 10% (v/v) bleach to wipe the workstation, instrutments, and before and after PCR experiments. It is very effective to eliminate the contimination. I often do 300-400 reactions for each experiment and seldem come across with contamination after I use this method, especially using of 10% bleach.

Aalex

-alex_osu3-

hi, i'm having a similar problem with my serial dilution of cDNA to estimate efficiency, my results are all over the place!

-flashboy-

QUOTE

My standard curve is running that all samples no matter what the dilution factor are amplifying at the same Ct. I am also getting amplification at that same Ct from a negative control (no template). Our first reaction was contamination, but I have used all new reagents, clean pipettes, different workstation and am getting the same results. We really don't think its contamination

I had a similar problem when using SYBRGreen, and it was not due to contamination, but to strong primer-dimer formation. I tried the dissociation curve method, measuring fluorescence at the higher Tm of my product, changing Mg, etc... but no improvement. I finally discarded those primers and went for TaqMan.
However, if you are using TaqMan, I guess you've still got contamination, as suggested before. The only other option, if you're sure that is not contamination, is that your probe is somehow degrading, but I can not think of a reason for this to happen. Maybe you could run your samples in a gel after the PCR, specially the blank, to determine if it is or not contamination.
Hope this helps!

-badcell-

Hey, I feel for you, having exact the same problem BUT whereas for my target gene (FAM-TAMRA) I have strong amplification (NTC, Standard...) from 3rd cycle...for Actin (VIC) in the same wells everything works just fine (zero NTC, increasing standards). Can it be contamination? Contamination only with my target gene but not actin? How?

-zuzmajka-

zuzmajka, what's the size? is it possible you have some strong primer-dimer? that would perhaps explain the great amplification in your NTC...

-aimikins-

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