how to choose best annealing temp. for primers? - (May/31/2006 )
I have problems how to interpretate the results of gradient temperature for my primers. In each temperature I have different ct value. Which temperature to choose and why? Should I choose this one, that gives the smallest ct? Or should I look at melting curves and choose this one that looks best.
Or maybe change primers? For another pairs of primers (another genes) I have exactly the same ct values in a wide range of tempretures, everything loos beatiful. Only in this one case that I showed you on the picture it goes like that. What does it mean?
PS. The blue line without any peak is my NTC.
Still waiting for your opinions. Any ideas?
We never change annealing tempertures. You are not flxible at all when you like to run several Primer in one Plate. The cost of a primer is very low compared to the time and money you spend for optimization. If they don't work @60C they go to the trash! And ask for the melting curves you attached, I would say they all look bad. -Hope I could help you somehow.
I run several primers on one plate because they have exactly the same annealing temperatures (55).
And I have performed gradient temperatures for them also - they were working (the same ct values) in a range of temperatures (50-60). Only in this case, that I showed you, it is like that.
I'm new to this method so I don't know what is wrong with these primers. Why melting curves look bad - you mean primers and dimers? I'm getting frustrated cause it is already third pair of primers to this gene. According to what you say I should look for another one.
I can imagine that you are pissed when the assay doesn't run after the third Primer you designed. It seems to be a difficult sequence. What Programm are you using to design the Primer? You could also give comanys like ABI or Roche a chance. They have assays on demand (at least for common Organism like HS,MM...) and they promise that they work fine. And if this is not the case you can complain about it. What Cyler do you work with? ABI machines for example do the 2step PCR and usually the annealing at 60C. I dont know if this would change something.
I would try to titrate your primer concentration and see if that cleans up your curves
I tried the the next pair of primers and this one was ok:) Thanks for your advice.