ligation of a 8kb fragment

Yan Yu yuyx at BIOTEC.JOUY.INRA.FR
Fri Oct 25 03:37:10 EST 1996



On Thu, 24 Oct 1996, Corinne BELVILLE wrote:

> I would like to realize a ligation of a fragment whom the size is 8kb. My
> ligation failed in vector pGEM-T (at 15=B0C).I put 5 or 20 more inserts t=
han
> plasmid.
> Is it necessary to change of vector?  is it better to work at +4=B0C?
>=20
> Thank you for your answers
>=20
>=20
> Corinne Belville
>=20
> --=20
> Daniele EUSEBE(Ecole Normale Sup=E9rieure-INSERM U293-Paris-France)
> e-mail:deusebe at wotan.ens.fr
>=20
I have ligated similar large fragment to vector,my condition is 15=B0c,10=
=20
ul volume,T4 1ul(5u),PEG8000 1ul, for 20 hours. It is blunt end ligation=20
. The key, I think, is to treat the fragment and vector properly.

Yu Yan Xing Ph.D


Unite  Virologie and Imminologie Moliculares>of INRA
 >=20



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