From owner-cellbiol@net.bio.net Mon Jan 20 22:00:00 1997 Path: biosci!bcm.tmc.edu!cs.utexas.edu!howland.erols.net!newsxfer3.itd.umich.edu!news.bbnplanet.com!su-news-hub1.bbnplanet.com!arclight.uoregon.edu!usenet.eel.ufl.edu!warwick!bignews.shef.ac.uk!usenet From: Kevin Mulcahy Newsgroups: bionet.molbio.methds-reagnts,bionet.cellbiol,bionet.general Subject: Re: Subcloning oligos into expression vectors Date: Tue, 21 Jan 1997 08:56:12 +0000 Organization: Institute for Cancer Studies, University of Sheffield, UK Lines: 22 Message-ID: <32E484AC.31D2@sheffield.ac.uk> References: <32E382DF.2DD9@sheffield.ac.uk> NNTP-Posting-Host: mac107020.shef.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=iso-8859-1 Content-Transfer-Encoding: 8bit X-Mailer: Mozilla 2.02 (Macintosh; I; 68K) To: "David H. Gorski" Xref: biosci bionet.molbio.methds-reagnts:53717 bionet.cellbiol:6459 bionet.general:25238 David H. Gorski wrote: > Thanks for the tip. Oddly enough, though, the NEB catalog doesn't state > that you need that much flanking sequence for Nhe I to work. I'm not sure if I agree with you on this. The NEB catalogue states that Nhe I digestion of a ds-oligo comprised of the Nhe I site and 3 flanking base pairs on either side results in only 10% of the oligos becoming cut after 2 hours or 50% after 20 hours. No data is given for oligos with longer flanking sequences, but I assume they would be cut more efficiently. Thus, you would definitely require more flanking sequence than what is stated in the NEB catalogue (just 3 bp) in order to get efficient cutting by Nhe I. >I'm going to try to digest with Nhe I and Bgl II for several hours >and see if that makes a difference. I'm not sure how heat sensitive the Nhe I enzyme is. If it is inactivated after a few hours at 37°C you may also need to add another aliquot of Nhe I midway through the digestion. Best wishes, Kevin. .