I ran a gradient PCR and there seems to be amplification at 52 and 54 degree celcius but not at 53 degree celcius. What could this possibly mean? My primer and DNA template concentrations were the same for all reactions (1micro M for primers and 250 ng for DNA
Yes, for your straight-forward question we require a fully annotated figure at 300dpi with a legend stating the exact conditions, make and model of the PCR machine, your inside leg measurement and shoe size. Then we'll take it seriously.
I noticed a similar very weird situation one years ago with one of my primers sets. The only explanation I gave myself about the possible reason?
The target sequence intrinsic features, I mean the way the template self-arrange secondary structures and also the way primers self-interact and also heterodimerize. All these intramolecular or reciprocal interactions are subdued to melting-annealing rules in different manner depending on GC content, GC distribution, competitive interactions. And also the way primers can unspecifically interact with other regions of your DNA. In this way at different annealing temperatures you could have different situations of advantage or not for one of these molecular interactions. The situation still weird for sure.
This is only a speculation obviously. In every case you should select the annealing temperature that gave you better results and discard the others.
Without repeating it I would assume a pipetting error. Or a faulty well in the PCR machine.
You're right Michael. I assumed he already repeated the PCR at least one more time.
Indeed I had the same weird problem also after a triplicate PCR, changing cycler and vials position on cycler plate.
You're talking with me or with Vasundhra?
If you're talking with me, yes I still have gel pictures but I don't need any kind of confirmation from you.
I used the best working conditions for my PCR and discarded the other non-working conditions, I sent product to sequencing and the product was fine. End of the story.
Anyway we are here for opinion exchanges, your answer is not barely kind and useful.
Yes, for your straight-forward question we require a fully annotated figure at 300dpi with a legend stating the exact conditions, make and model of the PCR machine, your inside leg measurement and shoe size. Then we'll take it seriously.
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