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primer melting temperature - (Jun/21/2005 )

What is the maximum melting temperature that we can set when designing primers for PCR?

Thanks smile.gif


assuming that polmerisation occurs at 72°, i don't go over 68°


Hi biochem,

Fred33 is right as extension is generally around 72C for specificity you shouldn't really go above it. However, I've used primers with melting temperature in the high 80's and still got specific product (lucky I guess), what I ended up doing is a two step PCR where I had the denaturation (94) and then anealling and extension (72C) I added an extra 30 seconds onto this step to counteract serving two purposes. In theory the higher the melting temp above your extension temp the more chance of non-specific product.

Hope this helps