deleting URA3

Michael Lichten lichten at HELIX.NIH.GOV
Tue Oct 8 08:59:33 EST 1996


A couple of days ago I posted a request for a plasmid to make a ura3
deletion.  Ed Davis sent me this message, which I am posting with his
permission.

>Date: Mon, 7 Oct 1996 9:53:58 -0400 (EDT)
>From: DAVISE at FCRFV1.NCIFCRF.GOV
>To: lichten at helix.NIH.gov
>Subject: RE: deleting URA3
>
>Hey, Mike, I think I can help you out.  Joachim Li, when he was in Jef Boeke's
>lab, made URA3 deletion plasmids.  One deletes the HindIII fragment, the other
>deletes from the 5' Hind III to the Sma I site, 60 bases upstream of the 3'
>Hind III site.
>These truncated URA3s are in pRS305 (LEU2);  one cuts at a unique Stu I site,
>transform & select Leu +, then pop out with FOA.  However, the Hind
>III-Hind IIIdeletion does not work.  You don't get FOAs (from deletion of
>URA3).  Only the
>Hind III-Sma I deletion gives FOAs.  This is my experience, & I talked with
>Boeke & he confirmed that that is indeed the case.  There is something
>esential
>about that 60 bases.  I have used the Hind III-SmaI deletion version with suc-
>cess, and the Hind III-Sma I fragment does make cells Ura + when integrated at
>MAT & at ARG4.  I would be more than happy to send the relevant plasmids
>to you
>if you can use them.
>
>Ed
>
>=============================================================
>Ed Davis, Ph.D., B.A., M.Phil, H.S., K.9., *.69., D.dt., M.Sg.
>
>ABL-Basic Research Program
>NCI-FCRDC
>P.O Box B
>Frederick, MD 21702-1201
>

Michael Lichten
lichten at helix.nih.gov





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