HELP!!! In-efficient Ligations? (Long)

Mark Youles my at mole.bio.cam.ac.uk
Thu Apr 13 11:37:28 EST 2000


Dear all,

While currently working at the MRC centre some members of our lab have
encountered a few problems with the efficiency of ligation, for which we
can find NO immediately apparent reason.

While attempting to sub-clone a 4.0kb fragment into an 8kb expression
vector, via a blunt-end (Sma 1) restriction site it was noted that post
purification of the relevant DNA fragments, O/N ligation @ 16°C and
Electroporation, the efficiency of ligation appeared to be virtually nil.

Subsequent tests on vector self ligation revealed this unbelievable story
to which we still have no clear answer(s).

The results from the tests we have conducted are as follows:-

If we digest any of the relevant vector, P/C extract and Etoh precipitate,
Ligate O/N @ 16°C and Electroporate the vector will quite happily self
ligate.

However, if the digested DNA is run on a gel (1.0% agarose) and
subsequently purified by any of the methods listed below, before ligation
and transformation, the vector DNA fails to ligate.

a) Electro-Elution, P/C extraction, Etoh precipitation
b) Electro-Elution, Column purification, Etoh precipitation
c) Low Melting Point Agarose Extraction, Etoh Precipitation
d) Zymoclean Gel purification
e) QIAGEX II Gel purification

Subsequent to this, an aliquot of the vector DNA was digested.  Post
digestion, the sample was split into two equal parts.  One half was
purified directly using a zymolcean column while the other was run on a
gel and subsequently purified from the gel using the zymoclean gel
purification kit.

N/B the columns and the majority of the solutions within these 2 kits are
virtually identical.

The obtained results (post ligation and transformation) revealed that the
directly purified DNA ligated fine while the gel purified DNA again failed
to ligate.

Further tests on the running buffer being used (TBE) revealed, that
although slightly better results were obtained with TAE than TBE the
efficiency of ligation was still EXTREMELY low when the relevant DNA is
purified by any means which involve gel extortion.

Just to confuse matters further, ligation is fine for smaller fragments
when they are purified from a gel by the QIAquick method and different
vectors also yield the same results as described above.

To re-cap therefore.

We have controlled the cells used as a standard plasmid transformation
gives efficiency of >10 to the 8.

We have controlled the ligases used as 4 different high and low
concentration enzymes ALL give the same result.  In addition to which the
un-purified vector quite happily ligates.  This has also been controlled
using a transformed aliquot of the digested DNA which was ligated without
ligase and gave minimal background undigested plasmid carry-over.

All other ligation factors (including resuspension water/T.E.) are
controlled via the fact that any DNA un-purified from the gels ligate
fine.

We have controlled the agarose used as the DNA fails to ligate when
purified from 2 different kinds (standard and LMP).  In addition to which
DNA purified via the QIAquick kit appears fine.

We have controlled the electrophoresis buffers, all which give virtually
identical results.

We have kind-of controlled the methods of purification used as I fail to
believe that ALL of the methods tested above (some of which I know from
past experience and have worked fine) suddenly now do not work.

We have controlled the vector DNA used as we have obtained the same result
from 3 different vectors irrespective of the fact of they are blunt-end
and sticky-end digestion.

Once loaded onto the gel some-thing somewhere is subsequently effecting
the efficiency of ligation.

If anybody has idea's on what this something might be, then I'd love to
hear from you.



Mark




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