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big differnece in my primers Tm - (Jan/19/2012 )


I have to do cloning and I have cDNA but the problem is with primers, I designed the primers and eventually one of them has Tm 66 the other is 80, I did gradient PCR with control DNA and I have very small barely seen band at 76, can i proceed my experiment though or should I redesign my primers?

any advice will be appreciateed.
Thank you so much in advance.


Tm 80 is really high. "At 76" means what? Barelly seen band would be difficult to clone. I would redesign.


As it's a gradient PCR 76 surely refers to 76°C as one of the annealing temperatures (as lowest?). Anyway I'd redesign it too, for me around 72°C is kind of limit (then having a two step PCR) and the differences anyway shouldn't be too large.