primer designing - (Sep/01/2008 )

Hi,
I designed primers for my gene with this online service http://www.basic.northwestern.edu/biotools/oligocalc.html. I didnot find any dimers,hairpins and 3 prime mismatch. the problem i put same sequence of primer in net primer primer analysis software,in this i got dimer with g vales minus 10 kcal,hairpins minus 2 kcals etc. i came to found that designing is entirely different than northwestern.
Is north western biotools service is good in designing primers.
can any one worked with primers designed by this tools,are they working good or not,please help me in this issue,because i want to isolate ORF of gene.
Thnaking you very much,
VRREDDY,
Ph.D STUDENT,
ITALY

I use Northwestern as part of my primer design process. I think you should just stick to Northwestern and not over think the problem. All software are unfortunately predictions. They are not 100% accurate.

If the Northwestern oligocalculator says it is okay.. The primer is ~okay. Aim for a primer tm of around 58 Celsius to 60 Celsius. If there are slight problems, the high tm will melt any secondary structure that might arise.

Assuming that you are amplifying from a genomic DNA template, make sure your primer does not bind to any other region within the genome. Do a BLAST search. Avoid amplifying from regions containing nucleotide repeats (mon, di and trinucleotides repeats).