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Help: DNA shear using Geneclean II kit

Tracy Aquilla aquilla at salus.med.uvm.edu
Fri Jul 15 11:50:11 EST 1994


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



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