HELP!!! In-efficient Ligations? (Long)

ladasky at my-deja.com ladasky at my-deja.com
Mon May 8 22:21:19 EST 2000


In article <38FA8726.1DFFD016 at hort.cri.nz>,
  Kimberley Snowden <ksnowden at hort.cri.nz> wrote:
> Hi Mark,
>
> I know you say in your post that the water you use is OK, because you
> use it to resuspend other things, but are you using the same purity
> of water in your resuspensions as you use to make up your TBE/TAE? We
> have been having some similar problems in the lab I work in (multiple
> people with cloning problems), and it came down to 2 things. One was
> a particular batch of agarose (not your problem as you tried different
> sorts). The other was our deionised water. People here often use
> deionised water (made in one place in the building and stored in a
> tank on the roof) to make up what they consider as non-critical
> solutions - perhaps some bacterial plates, and often used to make up
> TBE. Every thing else is ultra-pure water. It turned out our source of
> deionised water is causing problems.

I had a similar problem myself, but in my case my ultra-pure water --
wasn't.

A few weeks after we installed it, I pulled a liter of distilled water
from our lab's new ultra-pure deionized water filtration system, and
autoclaved it.   My blunt-end ligations went to hell and it took me weeks
to figure out why.  This same water was fine for bacterial work and even
long PCR.  Most of the other people in the lab had switched to a
topoisomerase-based cloning strategy and did not notice any problems.

Although I first decided to test that my water was problematic by
taking a control sample from a different laboratory (talk about
paranoid!), a second sample from our lab's water purification system
worked fine.

--
John J. Ladasky Jr., Ph.D.
Department of Structural Biology
Stanford University Medical Center
Stanford, CA 94305
Secretary, Californians for Renewable Energy <http://www.calfree.com>


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