TM for primers with restriction secuence tail. - Suggestion (Aug/18/2006 )
Always I have some dudes with the TM used in PCR assays with restriction tails primers.
If I calculate the TM only with the complementary sequence I will need to use 60°C, but if I put the uncomplementary 5' tail (with restriction sequence) the TM will be 72°C.
Normally I use the 72°C... but in some cases could be difficult to have a good yield.
use higher template, might also increase PCR product.
I would use 60 or 65C for annealing.
try 2-step PCR. you will get the maximum amount of specific product, i promise
your pcr machine may already have a touchdown programme within its protocol library so u'll adjust annealing temp, extention time, etc on that programme. but if not, your programme will briefly be like;
TM only with the complementary sequence 45s (for first annealing temp)
X 5-15 cycles; I usually do 10
then switch to second annealing temp, TM with the uncomplementary 5' tail (with restriction sequence) that is the entire lenght of the primer.
you will be amplifying the product you have made in the first 10 cycles.
TM of the entire lenght of the primer 45s
X 25-30 cycles; I usually do 25
then do your final extension at 72 for 5 min or whatever you like
this is not a specific programme. u need to change things according to your product length, your polymerase...
i have learned above things in this forum some time ago. but i couldn't find the link to send. i love and appreciate this circulation of info here.
We clone with primers that have 5' restriction sites + several irrelevant bases routinely (like every day ), and always use only the Tm of the complementary bases in deciding how to set up the PCR programs. We use a straight forward 30-35 cycle program.
dodosko makes a good point, though -- if you're going after maximum yield, two-step is the way to go; we just haven't found it necessary...
If you use the primers later to screen your clones (or sequence them), then you should consider the Tm of the restriction site, as it is under those circumstances it is complementary.