diaz@aecom.YU.EDU (Dizzy Dan Diaz) (11/13/87)
I am interested in putting a gene of interest in front of an inducible
promoter. The polylinker on the vector has lots of sites, but getting
my gene right in front of the ribosome-binding sequence will either
require using oligonucleotide engineering of my gene (yuck! the time,
the expense, what a pain!), or a controlled digestion of my gene's
promoter and subsequent ligation to the vector (lots of screening, but
not as much work as messing with oligos).
Bal 31 nuclease as prepared is much too difficult to control for my
purposes. As sold by most firms, Bal 31 is a mixture of two distinct
polypeptides, the so-called "fast" and "slow" forms. IBI is the only
company I know of that will sell you either form, or a mixture of the
two. It may be that this is the sort of enzyme I have been looking for,
but I am uncertain, since I know of no one who is experienced with this
"slow" form of Bal 31.
If anyone can be of assistance with this matter, I would appreciate some
helpful email. Thanks.
--
dn/dx Dept Molecular Biology diaz@aecom.yu.edu
Dan Diaz Albert Finkelstein College of Quackery werner@aecom.YU.EDU (Craig Werner) (11/13/87)
In article <1445@aecom.YU.EDU>, diaz@aecom.YU.EDU (Dizzy Dan Diaz) writes: > Bal 31 nuclease as prepared is much too difficult to control for my > purposes. In certain more relaxed labs on the western spiral arm of the Galaxy, the double stranded chewing of Bal31 has been completely superceded by exoIII followed by single-stranded exonuclease. The exoIII chews one strand 3'->5' under reasonable control, and the resulting frayed end is subsequently blunt ended. Quite clever, actually. -- Craig Werner (future MD/PhD, 3 years down, 4 to go) werner@aecom.YU.EDU -- Albert Einstein College of Medicine (1935-14E Eastchester Rd., Bronx NY 10461, 212-931-2517) "... Has Determined That Cigarette Smoking Is Dangerous To Your Health"
diaz@aecom.YU.EDU (Dizzy Dan Diaz) (11/13/87)
In article <1446@aecom.YU.EDU>, werner@aecom.YU.EDU (Craig Werner) writes: > In certain more relaxed labs on the western spiral arm of the > Galaxy, the double stranded chewing of Bal31 has been completely > superceded by exoIII followed by single-stranded exonuclease. The > exoIII chews one strand 3'->5' under reasonable control, and the > resulting frayed end is subsequently blunt ended. Quite clever, > actually. Fine Werner, that's just great, but how's about some NUMBERS, eh? Under reasonable control could mean 400 nt min^-1 or 2 nt min ^-1. If you're going to be of help, you must be complete about it. Besides, I don(t want to look it up myself. -- dn/dx Dept Molecular Biology diaz@aecom.yu.edu Dan Diaz Albert Grubelstein College of Osteopathy