Inclusion of restriction sites in PCR primers

Kevin Mulcahy, by way of "David L. Haviland, Ph.D." <dhavilan at imm2.imm.uth.tmc.edu> K.Mulcahy at sheffield.ac.uk
Thu Oct 31 10:39:37 EST 1996


Hi:

>I am trying to clone a cDNA using RT-PCR and have designed a primer pair 
>through the help of a computer programme (MacVector). This programme 

** SOME EDITED**

>However, I added a further 10 bp onto the 5' ends of each primer so that 
....

>Therefore, does anyone know what rules should be followed in the design 
>of primers when adding restriction sites? Also, are there any general 
>rules or suggestions for determining the cycling parameters for PCR when 
>using these modified primers?

What I have done with a good rate of success (some failures, yes) is to pick
the location of the primers and then start designing the primer with 10 G's
and or C's and I keep adding bases until I reach a projected Tm of about
65'C.  If that means it is a 30-mer, then it is a 30-mer.   THEN I add the
bases necessary to engineer the restriction site of choice, but it doesn't
stop there.  Enzymes need to see DNA on the other side of the site so some
folk here use the table in the NEN catalog which correlates base composition
with cutting effiency.  I simply add 6-8 more bases past the restriction
site that also matches the target sequence.  Take your pick.  But the point
is to  design your oligio first for the conditions you intend to use, *then*
add your restriction site, then the extra 6+ bases...

Hope this helps,
David




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