GELase or GELace
malaine at sara.cc.utu.fi
malaine at sara.cc.utu.fi
Thu Mar 31 02:26:58 EST 1994
In article <01HAL6IZJYXU000456 at msvax.mssm.edu>, GLUX at MSVAX.MSSM.EDU (MARC J GLUCKSMAN- STRUCTURAL NEUROBIOLOGY LAB) writes:
> Dear M+Rers-
> Has anyone heard of and/or had
> experience with GELace, from a company
> [maybe in TENN.], that has an agarose
> derivative that can be 'digested' with
> a suppl;ied enzyme that is good for
> PCR subcloning.
> Rumour has it that it is better
> than the BIO 101 products.
> Thanks for the prompt replies.
I haven't heard about that agarose derivative, but my experience from the
enzyme preparations is pretty much as follows:
GELase is an enzyme preparation, which is _supposed_ to digest the long-chain
polysaccharides in molten agarose to yield a clear liquid and it is developed by
the EPICENTRE TECHNOLOGIES (Madison, WI). In principle you just melt the gel
slice containing the DNA at 70 C, equilibrate the solution to 45 C, add GELase
and incubate until agarose is digested into small oligosaccharides.
A similar product (beta-agarase) is also developed by New England Biolabs, and
they are both supposed to function at the same way. I used both enzyme
preparations for numerous times, when I was in a process of doing serious of
different clonings. When working with these enzymes, I run across with
different problems. First of all, neither of these two enzymes didn't work at
the way promised by the manufacturers. The most critical point seemed to be the
size of an agarose slice that you are trying to digest. Always when the size
of agarose slice got bigger, the amount of undigested agarose increased
remarkably even though you used more enzyme than recommended. When trying to
get rid of all of the agarose, the incubation times should have increased to at
least 4-5 hr. And often that's already enough to degrade your DNA. The best
combinations seemed to be the beta-agarase enzyme used as recommended in GELase
high activity protocol, but even that was not reproducible.
IMHO, there is much better and faster way to do this. You just mix your gel
slice (any LGT-agarose) with 300 ul of TE-buffer, melt the gel at 75 C for 5
min, add 300 ul phenol, equilibrate and spin, transfer aqueous into new tube,
add phenol/chloroform (150/150 ul), equilibrate and spin, and repeat with
chloroform. Then just precipitate your DNA with 3 M NaOAc and EtOH. Recovery
using this procedure in my hands is about 90% and it takes less than an hour to
do it, and the resulting DNA is of excellent quality. It is absolutely waste
of money to buy those enzyme preparations (in my honest opinion only).
> Marc J. Glucksman
Univ. of Turku Malaine at sara.cc.utu.fi
Dept. of Plant Physiology Malaine at polaris.utu.fi
BioCity 6th floor \\ // Fax: +358-21-633 8075
20520 Turku (-o-o-) Tel: +358-21-633 8076
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