Help in Primer designing for expression...

Dr. Hiranya S. Roychowdhury via methods%40net.bio.net (by hroychow from nmsu.edu)
Sun Jul 19 15:47:39 EST 2009


Since the ATG start codon is part of the NCO1 sequence,  we use that RE
site for in-frame ligation.  So, if your amplicon is digested and
religated at the NCO1 site, you should be fine.

However, it has always been my advise that the primary amplified product
be first cloned into a simple vector (pUC based) for obtaining ample
inserts for a ligation.  Following that, the insert may be digested out
with the compatible RE (in your case, NCO1).

> 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.
>
> waiting for suggetion from scientific community
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-- 
Hiranya S. Roychowdhury, Ph.D.
Asst. Professor,
Health & Public Services
Dona Ana Community College
New Mexico State University
Las Cruces, NM 88003



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