37_410@uwovax.uwo.ca (07/24/90)
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@uwo.va Univ. of Western Ontario London, Ontario Canada .