Hi,
After qPCR this odd melting curve with multiple peaks within one big peak appeared in all my samples (attached below). I have performed qPCR on these cDNA samples two times before this run and those melting curves were fine (one peak with no peaks on them). I have used the same primer stocks all three times and the multiple peaks should therefore not appear due to primer-dimer.
I would love your inputs, thanks!
Best regards,
Rikke
Looks to me like you have really weak signal so you're seeing a lot of the background noise. I don't see a secondary peak or "shouldered" peak like you get when you have primer dimers so I think you can rule out that possibility.
My guesses are:
Run out some of your PCR reactions on a gel to see if it's low amplification. If you've done too many freeze/thaw cycles on your reaction mix, then it's time to throw away the tube and try again with fresh reagents.
Thanks for the quick response Katie!
Interesting. I interpreted the number of cycles in my amplification as if I had enough PCR product - would you interpret it differently? I have attached one of the amplification plots below if you would like to see it.
My dye was ordered just before this experiment and I have only been using it for about a month. Can it already have expired?
Your dye might have degraded in that time. Fluorescent dyes are extremely sensitive to light and temperature. If you've been repeatedly freezing and thawing your dye, it can quickly degrade. I'd suggest aliquoting it into small portions.
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