PFGE Help: Does anyone use PMSF?

Corona90 corona90 at aol.com
Thu Aug 21 10:11:46 EST 1997


>When digesting the agarose plugs with Proteinase K, the procedure I have
>has as the next step the inactivation of the ProK with PMSF, then
extensive
>washes.  Most of the procedures I have found in the lit. do not have this
>step.  Can anyone out there tell me if this step is required?  I am
>analyzing Shigella dysenteriae strains

Sounds like you are using BioRad's GenePath system.  We use that at our
facility as well.  When they first offered the system a few years back,
they included this step.  I have discussed this with other researchers,
specifially Dr Joel Maslow [who wrote a section on PFGE in ASM's
publication "Diagnostic Molecular Microbiology: Principles and
Applications" 1993] and he felt that 3-4 washes should remove the
proteinase K sufficiently without the use of PMSF.  That was fine with us,
because we didn't want to have to deal with it if we didn't have to. 
BioRad has since revised their procedures to elimiate this step.  The gels
we have obtained without PMSF have been comparable in quality to those
with PMSF.  Hope this helps.
-gary cage
 Clinical and Reference Microbiology
 Arizona State Laboratory
 1520 W. Adams
 Phoenix, AZ  85007
 (602) 542-6131
 (602) 542-1169 FAX



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