Electroelution/Centricon Problem

Barry Moore barrym at stella.med.utah.edu
Wed Aug 30 11:28:46 EST 1995


I have been doing a number of gel purifications lately.  I am moving clones
from one vector to another.  I have had several attempts that worked and
several that were terrible.  Following is my basic methodology, can anyone
find a problem that might account for my inconsistent results?

I NotI cut the insert out of the old vector, run a wide lane agarose gel, and
cut out my band of intrest (the insert).  Then I use Schleicher & Schuell
electroelution apparatus and membranes to retrive the DNA from the gel, and
Centricon 50 from Amicon to concentrate and wash the DNA.  I do the
electroelution in 0.5 X TBE at 100 V overnight, collect the fluid from the
trap, reverse electrodes and do run 100 V for 20 sec. collect the fluid from
the trap, and repeate for a total of three collections from the trap.  I
refill the trap with buffer between each collection.  I load the ~600 ul of
collected DNA onto a Centricon 50 and bring to 2 ml with H2O.  I spin at 6000
rpms for 20 min., fill with 2 ml of H2O, and spin again repeating for a total
of four spins (three washes).  Then I add 100 ul of H2O, pipette it against
the membrane several times, and invert the coulmn and spin off the DNA at 3000
rpms for 3 min.  Sometimes (mabye 50% or less) I get great recovery.  Other
times I get absolutely nothing.  There must be some terribly obvious reason
why this happens, but it has so far eluded me.  Has anyone else had similar
troubles, or can anyone see from my protocol what I might be doing wrong?

Any help would be greatly appreciated.

Sincerely,

Barry Moore


--

***************************************************
*                  Barry Moore                    *
*               Univeristy of Utah                *
*           Department of Human Genetics          *
*            Salt Lake City, UT 84123             *
*                     USA                         *
*                (801) 281-4636                   *
*            barrym at corona.med.utah.edu           *
*  http://www.genetics.utah.edu/~barrym/home.html *
***************************************************



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