problems with NTC - (Oct/04/2005 )
Recently i have started realtime PCR work. i'm using Stratagene machine and sybergreen detection system. when i'm performing the serial dilutions for standard curve, first 2-3 CT values are fine but then onwards all the CT values are same and i'm finding some signal in the NTC as well. for NTC i'm using just water as template. when i load this on agarose gel, i'm finding a band in the NTC at my expected fragment size. initially i thought this is contamination with water or contamination of the primers. but i ordered fresh set of primers again and i found this strange phenomena. When i checked the melting curve, there is no primer dimer.
anybody has answer for this or have such an experience.
thanks in advance
Did you change any of the other reagents you were using???
Is your standard a plasmid per chance???
Sounds like contamination.
pipets, tips, tubes...water is not the only culprit
good luck! PCR contamination sucks
you don't analyze any products near where your set-up happens, do you?
what about the airflow over your bench?
i have ruled out the possible contramination chances of all the components as the NTC has no amplification in some other reactions. to rule out the primer contamination, i ordered new set of primers. this is unbelievable but at the moment i don't have any clue.
i don't think I am understanding you correctly?
Your NTC (with water as the template) sometimes gives product and sometimes does not. is this right? because that is pretty much a classic example of what you would see if there is contamination....but not necessarily contamination of one particular reagent.
I would assume that some mechanical part of the process is inducing contamination, or that it is due to the location of the set-up. that is why I suggested checking your pipettors and the airflow, and asked if products are analyzed anywhere near your setup. this would explain the 'now-I-see-it, now-I-don't' appearance of contamination, which would not necessarily be the same in every NTC.
Am I misunderstanding you, about seeing the problem with some NTC but not all?
Check your primers again. You have to be very careful with the primers in sybr green, even very short extensions may look like amplification because of the extreme primer concentrations and the way the sybr dye binds