Introduction of restriction sites via extended primers?

Peter Ellis pjie2 at
Fri Aug 13 13:06:22 EST 2004

nospam at wrote:
>"Kyle Legate" <legatek at> skrev:
>> Most enzymes cut 90% or greater with an addition of 4 bases, but you can
>> sometimes use fewer than that. By default I always add AATT to the end of
>> an oligo that introduces a RE site because 1. It fulfills the requirement for
>> 99% of the enzymes out there (I've never had a failure, but I favour certain 
>> RE sites so the sample size is not large) and 2. It doesn't contribute as
>> much to the melting/annealing temp as using G's and C's.

>I don't understand the last bit, where you say that AATT contributes less to
>annealing that G and C? This is of cause correct, however this overhang is
>NOT going to anneal to template - so do you include it when calculating
>annealing temp?

It doesn't anneal to template in the first cycle, but it will do for 
all subsequent cycles - you want the difference to be as little as 
possible, hence using A's and T's.


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