DNA recovery by phenol/chloroform extraction or GeneClean

David Hackos hackos at phy.ucsf.edu
Tue Feb 11 18:44:44 EST 1997


Wilson wrote:
> 
> Hello everybody,
>         I would like to know which method can recovery DNA from agarose
> gel is better: Phenol/chloroform extraction or GeneClean?  Which one can
> have higher yield of recovery and purity of DNA?
>         Should I vortex the mixture during phenol/chloroform since I have
> heard that if the sticky ends DNA was vortexed, they will screw up
> together.  What is the solution to this problem if DNA screw up?  Can I
> solve it by incubation of the DNA at 65C for 15 min?
> 
> Wilson
> 


I have had problems from time to time with geneclean.  This is
especially true when you are working with very large vectors (such as
baculovirus), where you tend to shear the DNA into bits.  

One technique you may want to try out is NA45 paper, available from
Schleicher & Schuell (order number = 23400).  This stuff is fantastic,
and also very cheap.  The paper is a sturdy DEAE paper that you cut into
small rectangles, which you can insert into a slit in your gel just
below the ethidium bromide stained DNA band.  Then you just turn on the
electricity, and watch your band run onto the paper.  You can then
remove the paper, wash it, and elute the DNA (in 250uL 1M NaCl 10mM
arginine) at 65C for 30min (longer for larger DNAs).  Then you just
ethanol precipitate (mabye adding a little carrier such as glycogen). 
For small DNAs, this technique is very efficient - meaning you get lots
of DNA, and the DNA is VERY clean (never giving you any troubles).  Give
it a try!

Dave Hackos
Dept. of Physiology Box 0444
University of California, San Francisco
hackos at phy.ucsf.edu



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