While there is some value in calculating TM and such, sometimes you just have to take a chance and order the primer and use it at 55-60C annealing temp. Primers are cheap!
As for your particular question, I would calculate the TM for the part of the primer that will bind to the template and go from there. Also in my experience high TMs never hurt my PCR reactions, low TMs have though.
On 11/2/09 12:32 PM, "Azam Rahimpour" <[Only registered users see links. ]> wrote:
I am new to primer design and I have a simple question: when I add extra sequences like restriction sites and epitope tags to my primer, should I calculate primer tm with these sequences or not? actually when I calculate Tm with these sequences the tm is about 80 and if I ignore them it seems that the calculation isnt complete.
is there any rule for that?
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