ligation problem

Vittorio Verzillo verzillo at icgeb.trieste.it
Wed Jul 25 12:35:43 EST 2001


I would keep the volume of the reaction to 10 or 15 ul, if not needed
more, also I would use a
temperature of 16-18 and check the buffer, dtt can go nuts.
You want to have an high concentrations of ends.
Regards Vittorio.

***************************************************************
Vittorio Verzillo   Ph.D.
Molecular Diversity,
Bioscience Division, BN-1
MS M888, LOS ALAMOS NATIONAL LABS,
87545, LOS ALAMOS, NEW MEXICO, USA =20
Ph:505 6673785 Fx:505 6672891
e-mail:verzillo at icgeb.trieste.it or verzillo at lanl.gov
***************************************************************



On Tue, 24 Jul 2001, Fabien Jovelin wrote:

> Dear all,
> I have to ligate two digested fragment with cohesive ends. These fragment=
s
> are 2.7 and 6 kb long. Ligation is made at 14=B0C using Sigma kit in a to=
tal
> volume of 60 =B5l. Running results on agarose gel shows a degradation pat=
tern
> with 3 very "poor" bands corresponding to the fragments and the expected
> ligated fragment; my problem is that these band of interest is too faint =
to
> be purified, and located into a degraded lane...
> How can I improve my ligation yield ? Any help welcome.
> PS : ligase is fine because it works in other experiments...
> Is the high final volume a problem ?
>=20
> Fabien
>=20
> --
> Fabien Jovelin
> ESGS-Cyberg=E8ne
>=20
>=20
>=20
>=20
>=20




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