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Help: DNA shear using Geneclean II kit

douglas gray cancer research gray at labsun1.med.uottawa.ca
Wed Jul 20 14:26:37 EST 1994

Tracy Aquilla (aquilla at salus.med.uvm.edu) wrote:
: In Article <wumolbio.1.0 at mail.ncku.edu.tw>, wumolbio at mail.ncku.edu.tw
: (Chao-Liang Wu) wrote:
: >Dear Netter,
: >        Recently, two persons working in my lab have experienced some 
: >trouble in using Geneclean II kit.  When they checked yields of DNA on gel 
: >purified from the Geneclean procedure, an extra band with smaller size than 
: >the expected one often appeared.  As I have used the kit for four 
: >years without any problem when I was in Britain, I tried the procedure 
: >myself, but still I got an extra faint band.  We did not handle longer DNA, 
: >which is more apt to shear, and we avoided use of votrex mixing, I do not 
: >know what's wrong during the procedure.  I have discussed with one of my 
: >colleagues who have had the same experience before.  He thought it may be 
: >due to the kit itself as batch-to-batch difference happened.  Any suggestion 
: >and advice is appreciated.  As far as the cost is concerned, if anyone has a 
: >better procedure to isolate DNA from agarose gel, please share your 
: >experience with us.
: >******************************************************************************
: >   Chao-Liang Wu, Ph.D.                 EMail: wumolbio at mail.ncku.edu.tw
: >   Department of Biochemistry                  t17015 at dec2.ncku.edu.tw
: >   Medical College                      Phone: +886-6-2353535 ext 5536
: >   National Cheng Kung University       Fax  : +886-6-2741694 
: >   Tainan, Taiwan
: >******************************************************************************
: >
: >
: Dear Chao-Liang Wu,
:     Based on your message, I do not think the DNA is being sheared, since
: the extra DNA is forming a distinct band in the gel. It is possible some
: portion has been deleted. What host strain and vector combination are you using?
:     As far as eluting DNA from gels, I use NA45 membranes from S&S. This is
: a DEAE membrane that binds DNA up to 7kb quantitatively. It is very fast and
: easy to get 100% recovery from gel bands using this method. If you need more
: info, you can contact me by email.
:                                 Tracy
: Tracy Aquilla, Post-doctoral Research Associate
: Department of Molecular Physiology and Biophysics
: University of Vermont, College of Medicine
: aquilla at salus.med.uvm.edu

We have seen the extra band as well.  It may be a denatured  form of
your DNA, in that it did not appear if the DNA was eluted at room
temperature rather than at 50 or whatever.  

Doug Gray

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