How to add restriction enzyme sites to the primer - (Aug/03/2006 )

I have designed primers by the software of primer premier 5,now i want to add the restriction enzyme sites ,but i do not know how to do.

Start the primer with a few A's or T's to give some bases for the enzyme to digest properly follow it with the restriction site and continue with the corresponding bases from the template.

eg.
5 prime- with PspomI site (underlined) followed by start codon
AAAAAA GGGCCC ATG ... ...

3 primer - with xhoI site followed by stop codon
AAAAAA CTCGAG TTA ... ...

I prefer something like GGG CCC instead of A/T, but it really doesn't matter. Give four to six base overhang, at least.

-Matt

check out the neb catalogue, very helpful things in there...

greetz

tobi

in addition to what scolix and MisticMatt said, i recommend you to check your primers with some software to see whether they generate secondary structures which may lead you not having your specific PCR product. that's because your primers will be generating hairpins or self-dimers during PCR instead of priming your template .
i am using IDT oligo analyzer. below is the link:

http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/

as mentioned by tobikenobi, this page is very instructive for adding new sites.
Cleavage Close to the End of DNA Fragments