DNA ligation help

Sailesh Surapureddi Sailesh.Surapureddi at mcb.liu.se
Sat Jun 1 18:11:47 EST 1996


>Hi,there,
> Could anyone help me solve the DNA ligation problem? THe Case below:
>       Insert: 16.3kb SalI cut
>       Vector: Bscript KS or other expression vector SalI cut
>       Want to transfer to E.Coli DH5a cell
> I could not success ligate and transfer them no matter using chemical
>or electroparation method,
>  Did anyone have ever met the same case? any suggestion will be
>appreciated!


Relax man!

        This problem was repeatedly discussed here and its not new.  Sal1
is a lousy cutter and it needs atleast 4-5 extra nucleotides on the ends
and upon that it needs a lot of enzyme for cutting. NEB catalog should give
you the amount of enzyme and time required for complete digestion and the
no of over hangs required.

I bet in your case, Sal1 is the one and only culprit.

Be sure to clean up your cut DNA too by gel and you shouldnt be having any
more of these  problems.

Good luck

Sailesh.





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