Annealing temp using Quikchange SDM kit

Tom Knight via methods%40net.bio.net (by tk from mit.edu)
Sat Mar 22 06:43:09 EST 2008


"Clarisa Bejar" <cbejar from berkeley.edu> writes:

> I'm designing my Quikchange-SDM and have a question regarding the
> annealing temperature. What is the best way to determine the annealing
> temperature since, in this case, primers should be designed with a
> minimum Tm= 78ºC? (so the annealing temperature= Tm- 5ºC wouldn't
> apply here)

For Quikchange, both the 3' and 5' ends of the primer must bind well.
The easiest way to assure this is to pretend you are designing two
different primers, one 5' of the mutation, and a second 3' of the
mutation, and get the Tm of these primers roughtly correct.  The
manual tells you what you need to know.  The Tm's that any program
calculates will just confuse you, and you should ignore them.  In
general, design 16-20 bp matching, surrounding the mutated region,
more if there is high GC content.  You might try for high GC at the
extreme 5' and 3' ends.



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