From owner-cellbiol@net.bio.net Sun Jan 19 22:00:00 1997 Newsgroups: bionet.molbio.methds-reagnts,bionet.cellbiol,bionet.general Path: biosci!bloom-beacon.mit.edu!spool.mu.edu!uwm.edu!newsfeeds.sol.net!hammer.uoregon.edu!arclight.uoregon.edu!feed1.news.erols.com!news.idt.net!cdc2.cdc.net!vixen.cso.uiuc.edu!uchinews!d-gorski From: d-gorski@uchicago.edu (David H. Gorski) Subject: Re: Subcloning oligos into expression vectors X-Nntp-Posting-Host: maxreader.bsd.uchicago.edu Content-Type: text/plain; charset=ISO-8859-1 Message-ID: Sender: news@midway.uchicago.edu (News Administrator) Content-Transfer-Encoding: 8bit Organization: The University of Chicago X-Newsreader: Yet Another NewsWatcher 2.4.0 References: <5bvoro$ful@news.xs4all.nl> Mime-Version: 1.0 Date: Mon, 20 Jan 1997 17:07:45 GMT Lines: 25 Xref: biosci bionet.molbio.methds-reagnts:53683 bionet.cellbiol:6446 bionet.general:25232 > I have done the same thing without any problem. Anneal the oligos by > heating the mixture for 2 min in a boiling waterbath and slowly cool > to RT. Cut your vector, add your annealed oligos in a 100:1 ratio to > your vector, ligate i hour RT and cut your ligation mixture with Nhe I > and BglII ( I assume that the insertion of your oligo destroyes the > restriction sites) and transform. Your background is probably the > result of inefficient cutting of one or both enzymes resulting in the > selfligation of the single cut vector. Thanks for the tips. I would modify the suggestion and cut only with Nhe I, because the Bgl II site is not destroyed by ligation, although the Nhe I site is. The reason for this is so that I can cut with Bgl II again and Spe I (a site on the insert) and drop another copy of my insert into that site (Nhe I ends are compatible with Spe I ends), thus producing a doublet. I can then do it again to produce a triplet, and so on. Of course, I am getting ahead of myself. I have to produce a singlet insert, first, before going wild with making multimers.... :( -- 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 .