I have been getting exponential curves in my negative PCR reactions. I’ve performed extraction controls (with no sample extracted) and No Template controls (on the PCR) and they are all clear. Once I add DNA from healthy samples to the PCR mix i’m getting unexpected amplification curves.
I am using new primers and probes with a promega probe kit. I also check this with SYBR Green mix and the same happened. Can anyone help? How my negative samples could get contaminated and not my negative extraction controls? Should I use UNG?
Thanks!
You should find out which reagent is contaminated. I suggest changing one by one and avoid discarding reagents without contamination. Contamination may occur on your concentrated primers or even on your Sybr
Thanks but I don't think I have my reagents contaminated because my no template control on my qPCR is clear. It seems something is happening during DNA extraction or when I add DNA to the master mix. Thanks for your time!
Hi,
How sure are you that your "negative PCR reactions" are truly negative? Perhaps, you can start looking at the melting curve and run a gel of your PCR products to check if the products correspond to the segment that you to amplify.
Personally, I don't think the signal was due to contamination because Taqman probe, which is supposed to be more specific, also gave the signal while your negative controls worked well. It is likely that your "healthy samples" may have the very same amplicon. You may want to rerun the qPCR to confirm the result. If you get the same PCR product (after checking the melting curve and with gel electrophoresis), then you may want to consider sequence it to know the identity of the PCR products.
I hope this helps.
Have you run your samples on a gel to see the size of the amplicon in the samples that are supposed to be negative. If the positive result is due to non-specific binding or any other technical reason, the size would be different.
If contamination is suspected (although probably not the case here), checking the reagents one by one is usually more expensive and time-consuming than just open a new box of reagents.
More to the point, what's the Cq value? If it's 35 or similar, while your actual samples are 25 or similar, then...well, ignore it: that's a 1000-fold drop in signal, so it's background noise.
Freak mispriming event or something.
Remember, PCR is an exponential process, so a mispriming only has to occur ONCE, and then it'll amplify exponentially.
Just to make sure if the negative samples have actually amplified the target gene, you can try sequencing the product. This gives a rough idea about the source of contamination of your negative samples. If the sequencing result turns out to be different from your target gene, you can perform a gradient (annealing temp) using your negative samples with same set of reagents just to assess the right temperature that don't produce signal anymore in your negative samples.
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