I am doing microsatellite instability HRM on a Rotor Gene Q. I always run a negative control containing just the reagents and Nuclease Free water.
The reagents are prepared in a workstation, that is in a room just for the mix preparation. The negative control is also prepared in this room, since it only includes reagents and water, and soon after, the tube is closed and goes straight to the thermalcycler.
Then I take other tubes with the mix to another room to put the DNA samples. Finally, I close them and put them in the device too.
Even with all these precautions, for some reason the negative control has been amplifying.
I have already sanitized the entire room with diluted Hypochlorite. I diluted new primers and opened new Nuclease Free water. I tried to run only the negative control (without the samples) When I did it that way it didn't amplify. However, when running through the samples again, it amplified again. I didn't understand where this contamination comes from if the reagents never come into contact with the samples. Is it possible for contamination to occur inside the device? Because it only starts to amplify after the 35th cycle (red line in the figure sent).
Hi, are you sure that's contamination? is it just fluorescence because of satured dye in HRM? (cuased by other than target), primer dimer?
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