Help: DNA shear using Geneclean II kit

Zainul Zain zainul at KB.USM.MY
Fri Jul 22 02:04:59 EST 1994


Dear Chao,

It may be possible that the band that was cut out from the gel actually 
contains two fragments of very similar size. The fact that you saw two 
bands after extraction may be simply due to better separation. I suggest 
you try a second round of geneclean; if you have enough DNA of course!!


On 20 Jul 1994, douglas gray wrote:

> 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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