Genomic Library Construction

Brian Foley brianf at med.uvm.edu
Mon Dec 13 10:43:23 EST 1993


ed beaty (ed_beaty at qms1.life.uiuc.edu wrote:
: Hi!  I'm about to construct a genomic library to isolate a gene by colony
: hybridization using a related gene as a probe, and also by genetic
: complementation of an E. coli with a temp. sensitive copy of the same gene.
: I have two questions:  How can I randomly cleave my genomic DNA into pieces
: of about 8-12 kb?  I've heard of sonication, vortexing and passage through
: a syringe needle to break the DNA, but I have no references.  Also, any
: information on ligating the cleaved genomic vectors into plasmid vectors (I
: was thinking of using pBluescript) would be really useful.

	What do you mean by "Genomic vectors"?  If you want to ligate your
Lambda clones into pBS, forget it.  You can't put 50 kb of DNA into a
plasmid.  If you mean genomic DNA, the ligation method will depend on the
method of cleaving the DNA.  It will be different for sheared vs. partial
digest.   

: Also, I was trying to look on the University of Indiana Bionet archives for
: this information, but the gopher system I'm using seems really slow.  It
: shows me about a hundred files with such informative names as "RE>
: libraries" and "RE>RE>RE>RE>libraries", but it takes about a full minute to
: open up one of them.  Does anyone know of a faster way to get at the
: archives (software-wise), or should I accept the fact that the digital
: highway has a 10 mph speed limit sign on it?

	Not True!  It might not be the HIGHWAY that is slow, but your driveway
or you state's secondary roads that connect your driveway to the highway.  Are
you using a modem or a faster line?  What is the configuration of the net
between you and Indiana?  What version of Gopher are you using?  

	It could also be the IUbio server that is slow.  What time of day
did you call?  If it is trying to process 10,000 requests for info per
minute between 8 AM and 5 PM and only 500 requests per minute at 1:00 AM
you will see a tremendouse increase in performance at 1:00 AM.

	I do not know exactly where your system is slowing down, but I do
know that Gopher serches are incredibly fast for me if I do them in the
early morning hours.

: As always, grateful for any assistance!

: -Ed Beaty

--
********************************************************************
*  Brian Foley               *     If we knew what we were doing   *
*  Molecular Genetics Dept.  *     it wouldn't be called research  *
*  University of Vermont     *                                     *



More information about the Methods mailing list