cloning basic

[Kyle Legate]

rocky wrote:
> Hi
>
> I  like to know some tips on "sure win" protocol for cloning.
>
Wouldn't we all.

> I wonder what is the best ligation condition for such a PCR cloning.
> Is it possible to use only 0.1U T4 ligase (Invitrogen) in 25'C for 1
> hour? Of course most of the time I use 3U T4 ligase (Promega) for O/N
> reaction in 4'C.
>
Your ligation reaction is too cold. I use 16C overnight for blunt ligations
and a few hours on my bechtop for sticky ligations.