geoffk@bio73.unsw.oz (Geoff Kornfeld) (11/24/87)
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