(none)

[Klaus.Matthaei at anu.edu.au]

+  David L. Haviland, Ph.D.	     Internet:"haviland at kids.wustl.edu" 
Writes

"With interest I read your reply to question on sequencing gels.  You 
suggested adding 1/3 vol of NaAc (1M) to the bottom buffer just before 
running.  It sounds like an interesting idea and I intend to try it with my

next gel.  However, I have a question(s):  is it just a 1M solution of
NaAc, 
or is it made up to any particuar starting pH?  Is it made up in water, 
thus diluting the 1X TBE running buffer?

BTW - we don't dry our gels directly, the urea has caused us far too many 
problems, especially in the humid Mid-west.  We eliminated the Acetic Acid
fix, but we do soak and lift the gel off the plate in 10% EtOH, 90% water.
We let it soak there for about 20 mins, then we don't need to leave saran
wrap on for the exposure." 



I have had quite a bit of E-mail about my comment on the Na acetate method.
 I make the solution up in water and don't bother to Ph it.  Most comments
to me say that they also use the acetate but use a final concentration of
1M in the bottom tank.  I found my addition so good that I didn't bother to
try anything else.  It is possible of course that other concentrations are
better.  Maybe someone else out there has direct comparisons?

I also soak my gels for 20 minutes in 10% acetic,10% methanol before drying
for 1 hour.  I might add that we no longer run a lot of gels ourselves
since it is far cheaper if you don't have a lot of templates (and you have
to count  a technicians time) to use the ABI automated sequencer
(~$A20/template). (Unless of course you are a big cheese and have lots of
PhD students whose time costs nothing).

Cheers, Klaus

----------------------------------------------------------------------------
--Klaus Matthaei
Gene Targeting
The John Curtin School of Medical Research
The Australian National University
E-mail: Klaus.Matthaei at anu.edu.au

"Don't try and fix it if it ain't broke".
----------------------------------------------------------------------------
--