primer tm - tm calculation for tagged primers (Nov/02/2009 )
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?
In general, the primers should be designed for Tm with only the initial matching region. This is complicated because in later pcr cycles, the entire primer will bind, leading to a higher Tm. People sometimes do touchdown pcr to account for this difference, but usually simply ignore it, working with the Tm of the initial binding event.
For me, the primer Tm is the sequence complementary to the target, whereas the extra sequences for RE and tagging is ignored. However, when determining the homo- and heterodimerization, and hairpin (deltaG value), I include all the sequence as query input.