Posted 02 November 2009 - 03:57 AM
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?
Posted 02 November 2009 - 04:32 AM
Posted 02 November 2009 - 11:11 PM
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.