DNA ligation

killer yeast vvsvetlov at utmem1.utmem.edu
Wed Jul 17 19:42:00 EST 1996


In article <31EDE8AA.72A0 at bsa.bristol.ac.uk>, "Dr Andrew J. Doherty"
<doherty at bsa.bristol.ac.uk> wrote:


> I'd also be very interested in this protocol as I've had some problems
> with DNA spun out of agarose gels (Gibco UltraPure).

We have done enough of attempts to mechanically separate agarose and DNA
solution (spinning trough filters, punctured Eppendorfs etc.) to realize
that we are not meant for each other. Reproducibility is the first to go.
Using anion exchanger (DEAE cellulose) DE81 paper is very straightforward,
paper itself and salt are cheap as dirt and the method is not at all
labor-intensive. It goes like that
Purification of DNA from the agarose gels using DE81 paper

1. After satisfactory separation of DNA fragments gel is placed on UV
transilluminator and incisions/slits are made in front (1-2 mm from the
band) and if desired behind the band of interest with a scalpel blade
(paper inserted in the back slit will protect from accidental transferring
of larger fragments if the transfer is run longer - e.g. in case when
Terry wants to tell you again how he published 8 papers in 2 years of his
graduate carreer - alternatively part of the gel behind the band can be
completely removed).
2. A piece of dry DE81 paper is inserted (use forceps) in the slit on the
entire depth of the gel.
3. Return the gel in the chamber and resume e/phoresis until the band is
transferred onto the paper (UV monitor) - usually 5-10 min.
4. Place paper into Epp. tube and wash it once with 100-400 mcl 10 mM
Tris-HCl, 7.5, 1 mM EDTA, 7.5, 100 mM NaCl (aspirate as much wash buffer
as possible). 
5. Add 100-400 mcl of elution buffer (1M NaCl, 0.1 mM EDTA, 7.5) and
incubate at 68-70C for 15-60 min.
6. Transfer eluate into a new Epp. tube, PCI twice and add 2 volumes of
100(96)% ethanol (there is enough salt already).  
Expected recovery 75-90%. Recommended for fragments from 60 to 3000 bp,
used without modifications for isolation of 11 kb plasmid backbone.

Instead of precipitation I've used Chromaspin and variety of other gel
sizing spin columns to change the buffer (1M NaCl to smth. like TE) -
works fine if the DNA is in high concentration (plasmid backbone or PCR
isolations), otherwise precipiation also helps to concentrate. 

Regards,
Vlad

-- 
Of what use is a philosopher who does not hurt anybody's feelings?
                                Diogenes



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