positive NTC in real time reaction - (Sep/03/2012 )

Hi all,

In last RT reactions i get a positive NTC (~cycle 30). The strange thing is that I ussually load two NTC samples in each reaction, and one NTC gives no signal, but the second one, which is following my samples on plata, gives a signal. My lowest samples ended on cycle 26-27.

Is this mean I have a NTC contamination? Please help me!!!

Thank you

Natali

Did you mix NTC before taking on first one. It may be due to primer dimer. Are you getting siting curve like one in positive, if yes then might be contamination

If you're sure it's not a dimer, the most probable cause of a single positive NTC adjacent to sample wells on plate is cross-contamination from template pipetting.
If the contamination was in the reaction premix, both NTCs would be possitive. This however means that you have improper handling of a pipette and you may have cross contaminated other sampes as well.

Make a very strict rules about the movement of the pippete above adjacent wells. Try to move between wells or completely outside when possible. Don't release the knob unless you put the tip directly above the bin. Don't put new tip boxes in the trajectory that leads to bin. Don't splash or pipette up and down furiously.

It can still be normal contamination though, from the air, or the pipette, so generaly stick to GLP.

Run the reaction again, this time very carefully. If that still happen, clean everything and run only NTCs on a plate to check your reagents are OK. Putting NTC in wells far away from your samples would help NTC cross contamination from samples, but that would only hide the primary problem with the improper pipetting so I don't recomend that.

Close cap of NTC tubes before handling template.

prabhubct on Tue Sep 4 04:41:38 2012 said:

If that's a plate, it's not possible. Also it still only hides the fact, that other samples may be cross contaminated too.

Hi! Thank you for reply.

I don't think it's an improper handling of a pipette, because I have this NTC read only resently (in last experimental set), before I had no NTC read at all. And, I do put my NTC in wells far away from the samples. Moreover, I close the tubes with NTC after adding the water to the mix even before I begin to add my samples. Maybe I have the contamination among the samples, but it not suppose to be in NTC.

So, are you sure it's not a dimer? Is it SYBR assay? Is the melting curve same for samples and NTC?

Yes, it is SYBR assay, and the melting curves are the same. As I know, dimer gives two picks, but my reaction has one. Maybe I need to replace my primers anyway.

Did you check running your Realtime PCR product both NTC and positive by running on agarose gel? whether it shows primer dimer. Desired product lengths? If it is giving desired product in positive I don't think you need to reinvent wheel by designing primers again.

No way, if you are having signal in a NTC and this signal is giving you the same melting temperature that your amplicon of interest your NTC is contaminated. The Melting temperature of non-specific amplifications or dimer primers should be different (usually one pick) and much lower , than the melting temperature of your product. Try to know if this signal is coming from dimers/non-specific amplification or contamination in this way. If you are having non-specific amplifications you should set up your reading step at a temperature 2°C higher that the melting temperature of the non-specific product, so you only will have signal from your specific amplicon.