We have been running qPCR to detect Fusarium in wheat grains. Suddenly out of nowhere, we kept getting crossing point signal in the blank (PCR water). We have done the following:
- ran with only PCR mix and water --> crossing point signal appeared
- ran samples with new probes, new primers, new Taq, new everything --> crossing point signal appeared
- ran samples with autoclaved tips, UV-ed pipettors etc --> cp signal still appeared
- ran blank capillaries --> no cp signal whatsoever
Any suggestions as to where else we could find the contamination? What else can we do to de-contaminate our things/method?
Hi, sometime ago there was a similar question. Here is the link, you may see there my recommendations.
https://www.researchgate.net/post/How_to_get_rid_of_contamination_by_PCR_product
Did you try to run your NTC on the gel?
But for the new primer and new probe means that you ordered a new stock or you prepared a new dilution? Because if you prepare a new dilution and the problem persist means that you have a contamination in your stock.
Did you try to change the temperature of the extension step from 72 to 76 degrees? Jean Ramon Dee Yap
Hi,
Clean surfaces with Hypo Solution multiple times and use fresh aliquots of primers for setting up PCR. If you are still getting contamination, you may need to order fresh primers.
Good Luck.
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