based on the definition of tm, it follows that since binding will not (or at least should not) occur between the template and your added restriction site, you should not therefore factor it in when calculating tms, gc etc. Fyi, adding flag/myc tags etc on a primer will give you crazy tms. Again, no binding so dont worry about them. Gl
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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On Mon, 2 Nov 2009, [Only registered users see links. ] wrote:
To be more precise - during the first few cycles of PCR, you will be
priming directly off your DNA template, and so the added parts will not
match. For these cycles, you need to calculate the Tm based only on the
matching parts of the primer (without your added tags). In later cycles,
you are priming off the molecules synthesised during the earlier cycles,
and the primer will thus match all the way along, including the tags.
This means that if you want, you can do something similar to a touchdown
PCR: use a lower Tm for the first few cycles, and then raise it. This
may help if you are getting a lot of background / non-specific bands.
However, I've never found this to be necessary, so I'd advise you to just
calculate the lower Tm (i.e. without tags) and use that.