From owner-cellbiol@net.bio.net Fri Jan 17 22:00:00 1997 Date: Fri, 17 Jan 1997 20:26:14 -0600 From: d-gorski@uchicago.edu (David H. Gorski) Newsgroups: bionet.molbio.methds-reagnts,bionet.cellbiol,bionet.general Distribution: world Subject: Subcloning oligos into expression vectors Message-ID: Organization: The University of Chicago Mime-Version: 1.0 Content-Type: text/plain; charset=ISO-8859-1 Content-transfer-encoding: 8bit X-Newsreader: Yet Another NewsWatcher 2.4.0 NNTP-Posting-Host: xs2-04.xsite.net Lines: 30 Path: biosci!fcs280s.ncifcrf.gov!cpk-news-feed1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!su-news-hub1.bbnplanet.com!newsfeed.internetmci.com!fremen.xsite.net!d-gorski Xref: biosci bionet.molbio.methds-reagnts:53620 bionet.cellbiol:6431 bionet.general:25193 I've been trying to make several of constructs with various promoter elements placed in front of a luciferase reporter gene (in a PGL3-based vector) in order to test them for inducibility in my system. Unfortunately, I've been having a lot of problems getting my subcloning to work. For each construct, we've designed two complementary oligos set up so that when they anneal, there should be an Nhe I overhang on the upstream end and a Bgl II half-site on the downstream end. The plan was to clone these oligos directionally into the Bgl II and Nhe I sites in the multiple cloning site. Two questions: First, does anyone have any hints on what's the best way to anneal two complementary oligos prior to a ligation reaction? The oligos range from 36mers to 80mers. Second, in my previous cloning attempts, after cutting the vector with Bgl II and Nhe I serially and gel-purifying the fragment, I find that I'm getting a lot of self-ligation, as evidenced by lots and lots of colonies on my vector-only control plate. At least according to every restriction site list I read, Bgl II and Nhe I should not be compatible, but tell that to the vector. I've already verified that my enzymes are good by test digestions with each enzyme (I can't see the fragment that a double digestion should produce because it's only about a dozen nucleotides between the sites). Has anyone ever seen this or heard of it before? It looks like we're going to have to change our entire subcloning strategy if I'm going to get self-ligation between supposedly "incompatible" sites. Thanks. -- David H. Gorski, M.D., Ph.D. |"What is man, when you come to think Department of Surgery | upon him, but a minutely set, ingenious University of Chicago | machine for turning, with infinite | artfulness, the red wine of Shiraz E-Mail: d-gorski@uchicago.edu| into urine?"--Isaak Dinesen .