From owner-cellbiol@net.bio.net Wed Jan 22 22:00:00 1997 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!news.sgi.com!howland.erols.net!EU.net!uknet!usenet1.news.uk.psi.net!uknet!uknet!newsfeed.ed.ac.uk!cardhu!chrisb From: chrisb@hgu.mrc.ac.uk (Chris Boyd) Newsgroups: bionet.molbio.methds-reagnts,bionet.cellbiol,bionet.general Subject: Re: Subcloning oligos into expression vectors Followup-To: bionet.molbio.methds-reagnts,bionet.cellbiol,bionet.general Date: 23 Jan 1997 14:32:32 GMT Organization: MRC Human Genetics Unit Lines: 33 Message-ID: <5c7sq0$cft@scotsman.ed.ac.uk> References: <32E382DF.2DD9@sheffield.ac.uk> <32E484AC.31D2@sheffield.ac.uk> NNTP-Posting-Host: cardhu.hgu.mrc.ac.uk X-Newsreader: TIN [version 1.2 PL2] Xref: biosci bionet.molbio.methds-reagnts:53830 bionet.cellbiol:6486 bionet.general:25279 Kevin Mulcahy (K.Mulcahy@sheffield.ac.uk) wrote: : 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. Ah, but have you looked in the latest (96/97) NEB catalogue pp 238-239? Now NEB show cleavage for a selection of enzymes not only of short duplexes, but of sites near the ends of large duplexes (the normal situation). As a general rule, it seems enzymes will cut efficiently near to the end of a long duplex even if they won't cut a short duplex with a site equally near to an end. Eg, for NheI (as David asserts), the table indicates 82-100% cleavage of a site 1 bp from the end of a linearized vector. This makes intuitive sense, and is somewhat reassuring. There is reference for the new table: Moreira, R and Noren, C (1995) Biotechniques, 19, 56-59. Best wishes, -- Chris Boyd | from, | MRC Human Genetics Unit chrisb@hgu.mrc.ac.uk | not | Western General Hospital http://www.hgu.mrc.ac.uk/~chrisb | for | Edinburgh EH4 2XU, SCOTLAND .