ligation of a 8kb fragment

[Marc_Champagne]

In article <belville-2410961404200001 at cherubin.ens.fr>,
belville at wotan.ens.fr (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°C).I put 5 or 20 more inserts than
> plasmid.
> Is it necessary to change of vector?  is it better to work at +4°C?
> 
> Thank you for your answers
> 
> 
> Corinne Belville
> 
> -- 
> Daniele EUSEBE(Ecole Normale Supérieure-INSERM U293-Paris-France)
> e-mail:deusebe at wotan.ens.fr

Salut Corinne,

most important thing is:  have you used Taq polymerase?  If not, using pGEM-T
will never work.  What about your control without inserts?  Have you tried
CIPing the vector?

Marc