Help in calculating the Tm and GC content of primer..
 

Hi friends..

I am working with pET vector system. for expression of the enzme in
E.coli BL21.
I have to design primer with Nco1 site in forward primer.
so can anybody will suggest me about the placing of ATG site of
ORF...whether it should be in the primer's R.E site or else again i
have to add in the primer aftre the R.E site.

and one more thing
for calculating the Tm and GC..in this primer designing.
whethet i should consider the R.E site sequence along with the primer
sequence..for the calculation or else i should exclude it...


waiting for suggestion from scientific community