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From: MSALMINEN%FINNPHI@PUCC.PRINCETON.EDU (MIKA SALMINEN)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Restricting while ligating
Message-ID: <9008011112.AA04379@genbank.bio.net>
Date: 1 Aug 90 13:02:00 GMT
Sender: daemon@genbank.BIO.NET
Lines: 23


Hello fellow netters!

Has anyone done ligations in the presence of BamH1 restriction enzyme? I have
two fragments that both have a BamH1 and a Stu1 site in ther opposite ends. As
you know Stu1 produces blunt cuts and BamH1 recessed 3':s. I want to join these
fragments at the Stu1 site without getting ligation at BamH1 (and concatamers).

In theory this should be easy if i have BamH1 in the ligation reaction, but as
i haven't tried it before, i'm a bit concerned about getting star activity by
BamH1. Is there anyone out there who knows if BamH1 cuts unspecifically in +15
C at low salt concentrations (ligation buffer) and long incubations (24-48 h),
and if so, how i could awoid this.


Thanks already, Mika Salminen

e-mail: MSALMINEN@FINNPHI (BITNET)
Adress: Finnish Public Health Institute
        HIV-lab
        Mannerheimintie 166
        00300 Helsinki
        FINLAND