DNA ligation

Dr Andrew J. Doherty doherty at bsa.bristol.ac.uk
Thu Jul 18 02:32:58 EST 1996

killer yeast wrote:
> In article <31E30ABF.383B at inserm.u-strasbg.fr>, u.381 at inserm.u-strasbg.fr wrote:
> > dear net people
> >
> > I have some problems for subcloning DNA fragments (0.2 - 6 kb) eluted
> > from agarose gels. I have already used several methods and kits without
> > optimal results (NaI solubilization, low melting point agarose, agarose
> > free of ligase inhibitors, freezing method, etc).
> Dear J-N,
> it also depends on what you call optimal results. I've been cloning a
> variety of digested PCR products, plasmid fragments and oligos isolated
> from FMC agarose gels (SeaKem for the first 2 and SeaKem/NuSieve mixes for
> the oligos). Without selfligation (different sites and/or SAP treatment)
> the efficiency is >90%. Method I use for elution is based on e/transfer to
> DE81 paper (Whatman) inserted into the gel in front of the band of
> interest, follwed by washing of the paper removed from the gel and hot
> high-salt elution of DNA, 2XPCI and ethanol precipitation. This procedure
> seem to be compatible with T4 ligase from Boehringer and Pharmacia. It's
> cheap and rather straightforward and works fairly well even with
> oligonucleotides. Let me know if you don't have this protocol.
> Regards,
> Vlad
> --
> Of what use is a philosopher who does not hurt anybody's feelings?
>                                 Diogenes
I'd also be very interested in this protocol as I've had some problems
with DNA spun out of agarose gels (Gibco UltraPure).
   Dr Andrew Doherty                          
   Department of Anatomy                   
   School of Medical Sciences             
   University Walk                               
   BS8 1TD

   e-mail  Doherty at bsa.bristol.ac.uk


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