From owner-recom@hgmp.mrc.ac.uk Mon Jan 15 01:44:44 2001 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id 14C4A17AB5; Mon, 15 Jan 2001 01:44:44 +0000 (GMT) Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 60001) id F0C6017B04; Mon, 15 Jan 2001 01:44:42 +0000 (GMT) To: recom@net.bio.net Newsgroups: bionet.molbio.recombination From: "Per Holse Mygind" Subject: Tight Procaryotic Promotor Date: Thu, 11 Jan 2001 09:09:47 +0100 Message-Id: <20010115014442.F0C6017B04@mercury.hgmp.mrc.ac.uk> Sender: owner-recom@hgmp.mrc.ac.uk Precedence: bulk Dear Newsgroup I'm looking for the most tightly regulated promoter for recombinant expression in E.coli. I want to express and subsequently purify toxic proteins, so it needs to be very tight. Would the T7 promotor be the first choice ? Any comments, suggestions or references to papers dealing with the subject is greatly appreciated. Thank you in advance Per Holse Mygind, Denmark ===== Moderated bionet.genome.gene-structure __________________________________________________ Do You Yahoo!? Yahoo! Photos - Share your holiday photos online! http://photos.yahoo.com/ From owner-recom@hgmp.mrc.ac.uk Fri Jan 19 17:35:43 2001 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id 6CB9E17BF5; Fri, 19 Jan 2001 17:35:42 +0000 (GMT) Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 60001) id 9180317BF6; Fri, 19 Jan 2001 17:35:40 +0000 (GMT) To: recom@net.bio.net Newsgroups: bionet.molbio.recombination From: sripathi@home.com (Sripathi Ramadurai) Subject: question on cloning Date: 16 Jan 2001 06:12:29 -0000 Message-Id: <20010119173540.9180317BF6@mercury.hgmp.mrc.ac.uk> Sender: owner-recom@hgmp.mrc.ac.uk Precedence: bulk Hi! I am trying to clone a cDNA insert into an mammalian expression vector by directional cloning. I have tried ligating 3-4 times using a vector to insert ratio of 1:3. Everytime, the vector self ligates and I do not get any clones with the insert. This means that my Restriction Enzyme digests are not working well. I double digest the vector and insert. I check them on a gel after the first digest. Once I find that the Restriction digest worked, I go ahead with the second digest. Is there any way I can know if the 2nd digest worked? Is there anything else that I am missing out? All help is greatly appreciated. Thanks! Padmini > --- ===== Moderated bionet.genome.gene-structure __________________________________________________ Do You Yahoo!? Get email at your own domain with Yahoo! Mail. http://personal.mail.yahoo.com/