geoffk@bio73.unsw.oz (Geoff Kornfeld) (11/24/87)
I am relaying this for David Mitchell who is on CSIROnet: ----------------------------------------------------------------------- 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