Gel purification ligation problem

Wolfgang Schechinger wolfsc at ibms.sinica.edu.tw
Thu Jul 19 21:29:14 EST 2001


In my experience :-(, the cause rather is wrong primers, wrong 
RE sites or DNAse contamination - or wrong design 8-(((

Wo

> From this thread it seems that everyone is pointing to UV-damage
> as the most probable culprit for failed ligations.  To avoid UV
> altogether, I've been using crystal violet to stain my gels as
> suggested in
> 
> "Crystal violet can be used to visualize DNA bands during gel
> electrophoresis and to improve cloning efficiency", K.N. Rand,
> Technical Tips Online 1996.
> 
> Using crytal violet, there is no UV at all.  You can sit and
> stare at the gel all you want and trim it as small as you want. 
> The only drawback is that it isn't as sensitive as Ethidium
> staining but for cloning you should be using enough DNA to see it
> by crystal violet anyway.
> 
> Chad Rappleye
> Aroian Lab
> UC, San Diego
> (858) 822-1397
> 
> http://www.biology.ucsd.edu/labs/aroian
> 
> On 18 Jul 2001, Michael L. Sullivan wrote:
> 
> > As somebody else suggested already, one frequent culprit is
> > over-irradiation on a short-wave UV light box.  I find that
> > using a hand held longwave (365 nm) UV lamp works very well for
> > isolating fragments.  I use it underneath the gel tray to
> > transilluminate the gel.  Even relatively long exposures to the
> > lamp (several minutes) don't seem to effect the "ligatability"
> > of fragments in my hands.
> > 
> > Hope this is useful.
> > 
> > Mike
> > 
> > >Hi folks --
> > >	In my lab we are currently having problems with gel
> > >purification of DNA from agarose.  We are using Qiagen gel
> > >purification kits with all of the extra steps, and we are
> > >getting good yields of fragments (as judged by running an
> > >aliquot of the yield on a gel), but absolutely everything we
> > >purify is unligatable (as judged by failed
> > >ligation-transformations).  We know the restriction enzymes
> > >(and ligase and competent bugs) are working because if we do
> > >not gel purify, we can get the vectors to circularise (ie
> > >non-gel purified vector + no ligase --> 14 colonies, while
> > >non-gel purified vector + ligase --> 200 colonies). We suspect
> > >there may be something coming out of our gel purification step
> > >that contaminates ligation; we currently purify from Biorad
> > >molecular biology grade normal melting point agarose.  Do you
> > >have any suggestions or ways to move forward on the gel
> > >purification front?  When you reply, please note that you have
> > >to delete "deletethis" from my email address. 	All the best,
> > >		Harry
> > 
> > Michael L. Sullivan, Ph.D
> > 
> > U.S. Dairy Forage Research Center
> > 1925 Linden Drive West
> > Madison WI, 53706
> > 
> > (608) 264-5144 Phone
> > (608) 264-5147 Fax
> > 
> > ---
> > 
> 
> 


[tiss mezzage wahs broduceRd using TYPO GENERATOR zoffwer]
-----
Dr. Wolfgang Schechinger
Lab N233 (c/o Dr. Steve Roffler)
Institute of Biomedical Sciences
Academia Sinica
128 Yen-Chio Yuan Rd. Sec.2
Taipei 115
Taiwan R.O.C.
Tel +886-2-2789-9152
Fax +886-2-2782-9142
Mobile +886-925-136893
e mail wolfsc at ibms dot sinica dot edu dot tw
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