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Path: utzoo!mnetor!uunet!munnari!otc!metro!basser!elecvax!bio73!geoffk
From: geoffk@bio73.unsw.oz (Geoff Kornfeld)
Newsgroups: sci.bio
Subject: Re: BAL31
Message-ID: <962@bio73.unsw.oz>
Date: Mon, 23-Nov-87 17:55:52 EST
Article-I.D.: bio73.962
Posted: Mon Nov 23 17:55:52 1987
Date-Received: Fri, 27-Nov-87 05:42:05 EST
Organization: Uni of NSW, Sydney, Australia
Lines: 40

I am relaying this for David Mitchell who is on CSIROnet:


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From:	SXR::MITCHELL     23-NOV-1987 16:07
Subj:	Reply to diaz@aecom.YU.EDU


diaz@eacom.YU.EDU Dear DDD, 
	Read with interest your problem about engineering gene behind ribosome
binding site.

Oligonucleotide engineering Vs ExoIII Deletions

One unit ExoIII removes 50 bases/min at 37 C or for the layman, about 
one base/second. Assuming you can muck around with the conditions you might 
almost get it slow enough to remove an aliquot with a window of 10 bases 
either side of your rbs. Then you have to transform and sequence a 1000 colonies
to geta bang on deletion. In your own words yuck, what a pain!

ON THE OTHER HAND........

Biorad now have a nifty kit that even I can get to work for oligonucleotide
engineering. Better than 70% mutants.

Day 1  Make deletion oligo
       Transform plasmid into Biorad special strain No. 1

Day 2  Purify oligo
       Isolate single strand DNA for mutagenesis

Day 3  Do mutagenesis and transform into Biorad special strain No. 2

Day 4/5 Pick six transformants, grow up and sequence. 

Day 6  Engineered gene ready to use.


                                     DAVID MITCHELL