Sequencing problem. Is it the GeneClean?
ds1539 at u.cc.utah.edu
Sun Feb 13 02:13:14 EST 1994
NORVALC at QUCDN.QueensU.CA wrote:
: My question is straightforward: Have anybody else noticed that there are
: critical steps in the GeneClean protocol? If so, what should I be focusing on?
After trying unsuccessfully for many months to clone fragments purified by
GeneClean, my lab now uses anything BUT GeneClean! It is felt by our lab
that GeneClean trashes the ends of the DNA, and that's what interfears
with ligation. I have also done a test using GeneClean to purify
supercoiled plasmids from a 1% agarose TAE gel; I got a LOT of nicked
circle and also some linear. I did not see linear fragments when I used
low melting point agarose instead. Anyway, my point is that there is some
evidence that GeneClean tends to trash the DNA (maybe it's nicked by those
sharp glass beads when you resuspend the pellet in NEW, maybe one
molecules gets stuck to two glass beads and gets pulled and damaged, who
knows...) and that may be causing you problems in sequencing.
As far as critical parameters, I must admit I haven't used GeneClean
enough. BUT, I have heard that if you put the glassmilk in the NaI
solution as you melt the gel slice, the glass beads are at a low enough
concentration that they for sure only bind one molecule of DNA. You might
IMHO, I think the damage is done during the NEW washes. Maybe if you are
super sweet to your pellet (like not trying to resuspend it but just
putting in the NEW, let it set, and then suck it out) there would be less
But then again, it may not be damage at all that's your problem.
- Dave Segal
- Biochemistry, U. of Utah
- dave.segal at m.cc.utah.edu
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