Hi,
Even if not supposed to be done, I ran a melt curve after a taqman assay. Only few wells (different samples) have a peak (6 out of 42 samples, all with same master mix and same taqman assay). These 6 wells were located on edge of the plate, coincidence? What does this peak mean?
On the attached image: the top line is a normal well and the lines below have unexpected peaks.
Hi Jeremie,
Were these 6 samples the same as positive ones for the Taqman essay? Also if you could attach a picture of the peaks that's help.
Ciao,
Anna Liza
Hi Anna, they were different samples but same master mix and taqman assay. see above for the picture of the peaks. Thx for any help.
So the peaks on the right are a bunch of wells amplifying, with the product having the same GC content (roughly 0.5 Tm either way is considered the same). That usually means that they are amplifying the same thing and points towards contamination - do you have a melt curve of a positive well? Comparing the positive Tm to these would give you an idea if it's amplifying the target.
Especially at the edge of the plate, contamination is likely. qPCR amplification is really damn finicky. I have no idea how the Taqman assay impacts the melt curve interpretation - usually if you're using SybrGreen you would look at the cycle threshold for amplification in conjunction with the melt curve and then compare them with the negative control. Even in the negatives you usually end up with amplification, late in the run (Ct 38+). However that they all have the same type of peak is more likely contamination, the single peak on the left seems to be random amplification (because it's on it's own).
Do you have melt curves of positives in this run? Sometimes looking at them together can help too - tells you if you're amplifying the same thing but also the scale of the peaks may change.
HI, after amplification in a taqman pcr , all probes are digested so melt curve analysis in not informative.
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