Wed Aug 27 06:24:14 EST 1997

I recently column purified a fairly large plasmid (about 40kb) from a
column.  After elution, pption/wash, and drying, I now cannot get it
back into solution.  What I've tried:

1.  Expected yield is about 20ug.  I dried for about 5 min at RT (until
pellet was slightly translucent) and added 40ul of TLE (10mM Tris 8.0,
0.1mM EDTA).  Let set at RT for 1 hour, did not dissolve.

2.  Transfered to 65oC water bath for one hour...no dissolution.

3.  Increased volume to 100ul with more TLE, set at 65oC for one hour. 
Still there.

4.  Incubated at 37oC o/n.  Has not gone into solution.

I'm sure that others have run into this problem using these columns. 
Unfortunately, when you call the manufacturers they offer minimal
solutions (usually what's described above) and make it sound like it's
your problem, not theirs.  

"Yes I followed your protocol, yes I did that.  Huh?  Well, I never
thought of that...wait a minute...Why should I use 8.2mM Tris over
8.0?...That makes a difference?!?!...HOLD ON!...Grow it back
up?...That'd take three days...I know but...Oh, OK.  I suppose you're
the experts.  Thanks for your help."

Does anyone have any suggestions?  This DNA is very precious!!  E-mail
responses are greatly appreciated.

Lou Cantolupo
GenVec, Inc.
Dept. Molecular Virology
Rockville, MD  USA

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