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                               
   Bristol                                            
   UK
   BS8 1TD

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

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