2 replies to this topic

[sara.r]

Hi

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?

regards

[phage434]

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.

[lsek]

Second phage434,

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.