DNA ligation

killer yeast vvsvetlov at utmem1.utmem.edu
Mon Jul 15 19:28:50 EST 1996


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



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