contamination in real-time pcr negative control-Real-Tim

hi there,i am noew constantly getting a late peak (30+ cycles) in the negative controls for my real-time pcr (SYBR Green, iCycler). i have even bought in new nuclease free water to run as my negative contro, and still get these peaks... does this mean my primers are contaminated???? -flashboy-

Your working primer stock could well be contaminated, when you say it's great than cycle 30, how much greater? if it's high 30's ie greater than 35 then I would not worry too much about it. One other thing is to check the reaction by running it on a gel, if a distinct band comes up in your negative control(and it looks like the test lanes) well that would answer your question. Good luck! Nick

-methylnick-

hi there, am seeing Ct values of approximately 33-35 cycles. have purchased new primers, new nuclease free water which i have diluted the primers in and still get a signal!!!!!!

-flashboy-

hi i'm not in the mood with real time pcr, but i was wondering, it could be either buffer or enzyme or nucleotides, coudn't it?

-fred_33-

QUOTE(fred_33 @ Apr 1 2005, 02:52 AM) hi i'm not in the mood with real time pcr, but i was wondering, it could be either buffer or enzyme or nucleotides, coudn't it? [snapback]13333[/snapback]absolutely, could very well be. also try and setup your reactions in a clean zone (RNA and DNA free!) -methylnick-

it's primer dimer. don't worry about it. just ensure there is one peak in the melting curve.

-vetticus3-

QUOTE(vetticus3 @ Apr 3 2005, 06:59 PM) it's primer dimer.  don't worry about it.  just ensure there is one peak in the melting curve. [snapback]13442[/snapback]you can always check by running the products on a gel.Nick -methylnick-

thanks for all the advice guys. i don't think it is primer dimers as the peak on the melt curve is exactly the same as the template samples i run. also, as the only ingredients used are the Abgene master mix, primers and template/water there are no buffers or extra reagents to contaminate..... am going to run a gel to see if there is an extra product but i'm not expecting one as the melt curve data suggests it won't be there...... this is so infuriating!!!!!!

-flashboy-

[font=Arial Narrow] I have encountered this a number of times as well, and am not entirely sure on the reason why. I do know that most real time products are short and that the primer dimers may appear to have a similar melting curve as the product. Have you run triplicates and are getting the same result? Have you added a heating step (to 80 degrees) immediatly before the plate read, in order to omit primer dimer reads? The melting curve is not important if there is no registered product. Have you run a plate with only this control and no other experimental or control wells? Let us know if you find out anything more solid on this.  

-Shel-

I am having a similar problem: We are running two negative controls for every GOI. We prepare mastermixes of the Fprimer, Rprimer and water...dispense into tubes. Then add app. template. We then mix the tubes and dispense into the PCR plate in triplicate. For the negative controls (no RNA and no RT), we are getting high CTs (32+) that have the same melting peak as the experimentals. But not for all of the triplicates, just 2/3 or so. Is this contamination within the samples, or possible carryover from the pipet, or primer dimer? Thanks!!!

-medhan1012-

I had this trouble too, and sometimes still happens but after 35-40 cycles. If You get a signal in negative control under the 35-30 cycles You're having troubles. If You run triplicates and do not get results in all the three tubes I would exclude the primer working-solution contamination. More plausible is the possible carryover from the pipet. As someone already said: try to perform the reaction in a clean zone and, if possible, separete the negative control tubes from the rest [ie use a strip specifically for the blank and keep it far from the others]. I hope this could help.

-HP1-

QUOTE(HP1 @ Jun 28 2006, 01:55 PM) [snapback]57533[/snapback] I had this trouble too, and sometimes still happens but after 35-40 cycles. If You get a signal in negative control under the 35-30 cycles You're having troubles. If You run triplicates and do not get results in all the three tubes I would exclude the primer working-solution contamination. More plausible is the possible carryover from the pipet. As someone already said: try to perform the reaction in a clean zone and, if possible, separete the negative control tubes from the rest [ie use a strip specifically for the blank and keep it far from the others]. I hope this could help. After reading much on this topic, I tried using more controls to test the source...1- no RNA2- no RT3- no cDNA4- no primers, just amplicon used in curve5- just waterthe no primers and just water were totally negative, which elimnated carryover and Sybr green as sources. The others showed up late in the cycles, and not all of them amplified in all of the triplicate wells for all of the genes...some genes were clean...which leads me to think primer dimers?Any thoughts? -medhan1012-

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