alls not well....

37_410 at uwovax.uwo.ca 37_410 at uwovax.uwo.ca
Tue Jul 24 10:06:34 EST 1990


	I don't really have a direct answer to your problems but maybe this
note about our experiences may help you ralize you are not alone.  We too have
purchased a "Vacugene" system from LKB/Pharmacia. The primary use of the
instrument in our hands has been to do southern blotting of bacterial genomic
DNA in undergraduate student labs although we do plan to make use of this
system for our own (procaryotic) work.  We have had no real problem with the
system thus far however others in our department have.  In our system I have
found that simple solutions such as increasing the time during the
depurination, denaturation and transfer steps helps in the transfer of DNA
fragments as large as 48 kb. (The largest we've had to do yet.  Furthermore we
did not purchase the vacuum pump for the system.  Instead we have constructed
our own by building a basic manometer and hooking it up to the in house vacuum
lines. I wouldn't think that this would make a difference except in the
pocketbook. We also are using a non-radioactive DNA detection kit available
from Boeringher-Mannheim to visualize our bands.  Again, I don't see why this
would be any more effective than using a radioisotope??
	Those in our deparatment who have encountered problems are working with
eucaryotic DNA.  In fact, they have had a very difficult time just getting the
DNA to transfer to the membrane whether it be nitrocellulose or nylon. I had
suggested increasing the time during the depurination and denaturation steps as
well as the transfer time but none of these seems to have worked.  In their
discussions with the Pharmacia representative the suggestions given were of no
valid use since it ended up being no faster to use the vacuum system than the
good old capillary system.  Needless to say they are not very impressed with
the system or the technical support they have recieved to date.  So far nothing
they have tried has been very successful.  If t hey make some big discovery to
unlock the mysteries of the system I'll let you know.  If you have any ideas or
tips please pass them along as I'm sure they will be much appreciated.

		
			Sincerely, 
					
				Lou Passador
				
				c/o T.LINN at uwo.va
				Univ. of Western Ontario
				London, Ontario
				Canada

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