pcr site selection

serotonin1 please_post_replies at newsgroup.com
Tue Oct 16 23:03:17 EST 2001


"John Luz" <jglma at scripps.edu> wrote in message
news:3BCC99F8.22839DBE at scripps.edu...
> Hi Folks--Looking for any references or help on doing PCR site selection
> with a his-tagged protein.  I don't have an antibody.  Thanks.--john

Do you want to engineer a his tag onto your protein, or PCR a fragment of a
gene containing a his tag sequence?
What most people seem to do, AFAIK, is to use 6 alternating his codons, CAT
and CAC, then about 15 or so bp of the beginning or end of your gene,
depending on if you want a N or C terminal tag.  I heard of and have seen in
some commercial vectors(www.novagen.com) flanking the tag with arginine,
glycine, and serine or threonine.  I'm not sure why, maybe it makes the tag
stick out better or something.  Does that help?

I'm applying to scripps for grad school...do you have any opinions on it?

Grady





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