Jose de las Heras
josenet at tiscali.co.uk
Mon Oct 9 05:35:43 EST 2006
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Is the Tm (melting temperature) of a primer calculated on the subsequence of
the primer that binds to the template DNA? Why? because I have added
restriction sites at the 5' end of the primer, which does not bind to the
How is the Ta (annealing temperature) calcuated? from what I read, it should
be 5 deg C less than the lowest Tm of the primer pair... but why?
regarding the melting temperature when you add restriction sites... sure,
the first cycle you don't get annealing over that region... but on
subsequent cycles you will.
I normally take into account the whole oligo (typically 14-16 or so bp plus
the recognition site I want to add)
as for the annealing temperature... I'm not sure anybody does any "real"
calculations. The Tm should be calculated considering things like salt
concentration etc, and it would represent the temperature at which 50% of
the sequences are annealed. With that in mind, if you use a slightly lower
annealing temperature on the PCR, you're favouring annealing... the cost is
increasing possibility of mispriming... but if the primers are well designed
it's not normallya problem. Conversely, you can go above the calculated Tm
and still get a product. Sometimes a particular sequence is hard to amplify
cleanly, and doing a few cycles at higher Ta helps improve things a lot...
I odn't think it's an exact science, although you can calculate away your
Tms, Tas and % of annealing with your particular buffer, in practice it's
often easier use just the Tm as a guide... and optimise around it by trial
and error. Most PCRs will work fine without much fiddling, and it's
reccomeded to start witha Ta a few degrees below Tm... and see what
happens. Then change if necessary depending on the result.
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