Restriction mapping BACs or PACs

Phillip San Miguel pmiguel at bilbo.bio.purdue.edu
Wed Nov 18 11:19:48 EST 1998


I've had luck using 8 cutters (eg NotI, SfiI, PacI, PmeI, AscI, etc.)
lambda terminase (we got it from Epicenter) and a FIGE (Field Inversion
Gel Electrophoresis) inverter (a PPI-200 from CBS Scientific).  Then
it's just like doing a normal plasmid restriction map.  It is critical
to recirculate the buffer (I just use 0.5xTBE with EtBr at 200 ug/liter
in both the gel and the running buffer) and not to overload the gel.  A
CHEF gel seems to sieve big bright bands fine, but (in my hands) a FIGE
gel will not.  I like FIGE over CHEF, because I can hook a FIGE
inverter to any gel box with recirculation ports.  That means I can run
40+ slot gels.  Also, it's no big deal to take a picture of a gel and
then continue running it with FIGE--I don't think that will work with
most CHEF gel rigs. Size standards are a little tough, also.  I use the
"8-48" kb standard and the 5 kb ladder (from BioRad).  Lambda ladders
would be nice, but they always come embedded in agarose and this did
not seem compatible with FIGE.  (Note, I don't embed any of my
reactions--I just avoid shearing them as much as possible.)

Roxanne Walder wrote:

> What is the best method for restriction mapping BAC and/or PAC DNA?






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