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PCR primer melting temperatures
I was designing my primers for real-time PCR using Primer3 program where it gives out also melting temperatures. Now, I've got my primers and the comapny saent also melting temperatures and for most of them these temperatures are different. So what is the max difference in melting T allowed for primers and which t i have to use for my PCR. Highest or the average between two?
Thank you already.
Re: PCR primer melting temperatures
WOW, I think I understand your problem. In the primer pair, they should be designed so that their melting temperature match, with a tolerance of 2-3C being OK, and 5C acceptable (these are just general rules of thumb). For the time being I suggest you test your primers with the commonly used melting temperature (95 degrees C) and whatever annealing temp that primer3 suggests, then optimize from there.
Primers are pretty robust, I remember doing diagnostic PCR assays using the wrong annealing temperature for about a year (and it worked fine), until I caught a typo in the literature and tracked it down (turns out the annealing temp was 4 degrees too high). By the way another rule of thumb is that the primer annealing temperature is usually 5-8 degrees lower than the predicted primer melting temp (not the 95C).
|melting , pcr , primer , temperatures|
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