Long Range PCR - (Jun/07/2007 )

i working on long PCR recently, and i want to design a pair of primer. May i know if primer that i design with Tm 57C is it ok? Because read some reference, they are recommend 60C. My PCR product is around 18kb.

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can you add the extra base pairs to reach a little higher? Higher temperatures do generally give better specificity.
How long is your PCR product? 10kb or 10kb and above?

The most important thing is the primer sequence itself. Make sure it doesn't have any self annealing problems, multiple annealing sequence in the genome (if your are amplifying from genomic DNA). Binds to a region composed of a good mix of nucleotides (no nasty dinucleotide or mononucleotide repeats nearby)

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i think it would not be so difficult for you to add some extra base as perneseblue said. and you could try some trial, once i used primer of Tm 58c. in that case i changed my anneling and extension time

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