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Help in calculatiing Tm and GC content of primer

Shrinivas D via methods%40net.bio.net (by vasu.nanobiotech91 from gmail.com)
Sat Sep 12 14:06:08 EST 2009

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 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

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